i
ISOLATION OF LOCAL BACTERIAL CAPABLE OF DEGRADING
HALOGENATED COMPOUNDS AND ANALYSIS OF PUTATIVE
HALOACID PERMEASE GENE
NG HONG JING
A thesis submitted in fulfillment of the requirement for the
award of the degree of Master of Science
(Bioscience)
Faculty of Science
Universiti Teknologi Malaysia
FEBRUARY 2007
iii
For Science
iv
ACKNOWLEDGMENTS
I am deeply indebted to my advisor, Dr. Fahrul Zaman Huyop for his
guidance and continuous encouragement throughout this project moving. He has
been a mentor for my professional life and has helped make my stay at UTM an
enjoyable one.
I would also like to express my sincere gratitude to my lab mates, lab
assistants and fellow graduate student for their advice, encouragement, humor and
friendship. Their professionalism made it a pleasure to work with them and without
their help, it would be impossible for me to overcome the problems that occurred
during this project.
My sincere appreciation especially extends to Ms. Wong Yun Yun for her
valuable advices in statistical analysis. In addition, I would like to thanks all my
colleagues and others who have provided assistance at various occasions. Their
views and tips are useful indeed. Unfortunately, it is not possible to list all of them
in this limited space. I am grateful to my entire family member as well.
v
ABSTRACT
3-chloropropionic acid and 2,2-dichloropropionic acid are synthetic
halogenated compounds used in herbicide. A bacterium isolated from a soil sample
and characterised as Rhodococcus sp. by 16S rRNA analysis, was able to degrade
and utilised 3-chloropropionate as the sole source of carbon and energy. This was
supported by the ability of the bacterium to grow on 20 mM 3-chloropropionate with
a doubling time of 11.72 hours. The utilisation of 3-chloropropionate was also
confirmed by detection of 3-chloropropionate depletion in the medium using HPLC.
Cell free extract of Rhodococcus sp. had an enzyme specific activity of 0.013 μmol
Cl-/min/mg protein towards 3-chloropropionate. Another bacterium isolated from
the same soil sample and identified as Methylobacterium sp. by 16S rRNA analysis
was found to be able to degrade 2,2-dichloropropionate. The bacterium grew in
20mM 2,2-dichloropropionate minimal medium with a doubling time of 20.32 hours.
Degradation of 2,2-dichloropropionate was further confirmed by detection of 2,2dichloropropionate depletion in growth medium by HPLC. Cell free extract
prepared from the cell showed 0.039 μmol Cl-/min/mg protein specific activity
towards 2,2-dichloropropionate. A putative haloacid permease gene (dehrP) from
Rhizobium sp. was subcloned into Novagen pET 43.1a plasmid. The newly
constructed plasmid was designated as pHJ. The cloned gene was sequenced and
analysed using various online analysis tools. DehrP has a calculated molecular
weight of 45 kDa and an isoelectric point of 9.78. The nucleotide sequence of dehrP
showed significant homology (86%) with the putative mono-chloropropionic acid
permease from Agrobacterium sp. NHG3 and 62 % homology with the haloacid
specific transferase from Burkholderia sp.
vi
ABSTRAK
Asid 3-kloropropionik dan asid 2,2-dikloropropionik ialah sebatian halogen
sintetik yang terkandung dalam racun rumpai. Bakteria yang disaring dari sampel
tanah dikenal pasti sebagai Rhodococcus sp. melalui kaedah analisa 16S rRNA
mampu mengurai 3-kloropropionat sebagai sumber karbon dan tenaga. Ini disokong
oleh kemampuan bakteria untuk tumbuh dalam 20 mM media 3-kloropropionat
dengan masa penggandaan 11.72 jam. Penguraian 3-kloropropionat juga disokong
oleh analisa kultur menggunakan HPLC. Ekstrak dari Rhodococcus sp.
menunjukkan aktiviti enzim spesifik sebanyak 0.013 μmol Cl-/min/mg protein
terhadap 3-kloropropionat. Bakteria lain yang disaring dari sampel tanah yang sama
telah dikenalpasti sebagai Methylobacterium sp. dengan menggunakan analisa 16S
rRNA. Bakteria tersebut mempunyai keupayaan untuk mengurai 2,2dikloropropionat. Bakteria itu tumbuh dalam 20 mM media minimal 2,2dikloropropionat dengan masa penggandaan dua sebanyak 20.32 jam. Kebolehan
Methylobacterium sp. untuk mengurai 2,2-dikloropropionat juga disokong oleh
analisa media kultur dengan menggunakan HPLC. Ekstrak sel menunjukkan aktiviti
spesifik enzim sebanyak 0.039 μmol Cl-/min/mg protein terhadap 2,2dikloropropionat. Gen putatif haloasid permease dari Rhizobium sp. telah disubklon
ke dalam plasmid pET 43.1a Novagen. Plasmid itu dinamakan pHJ. Penjujukan gen
yang telah diklonkan itu telah dikenalpasti dan dianalisa dengan menggunakan
pelbagai perisian komputer secara terus menerus. Protein DehrP mempunyai
pengiraan berat molekul 45 kDa dan tahap isoelektrik 9.78. Jujukan nukleotida gen
dehrP telah menunjukkan persamaan homolog yang ketara (86%) dengan gen
putatif asid monokloropropionik permease dari Agrobacterium sp. NHG3 dan 62%
jujukan homologi dengan gen haloasid spesifik transferase dari Burkholderia sp.
vii
TABLE OF CONTENTS
CHAPTER
1
TITLE
PAGE
DECLARATION
ii
DEDICATION
iii
ACKNOWLEDGMENTS
iv
ABSTRACT
v
ABSTRAK
vi
TABLE OF CONTENTS
vii
LIST OF TABLES
xiii
LIST OF FIGURES
xv
LIST OF ABBREVIATIONS
xvii
LIST OF APPENDICES
xix
INTRODUCTION
1.1
Xenobiotics as Pollutants
1
1.2
Halogenated Compounds in the Biosphere
5
1.3
Usage of Halogenated Compound in Malaysia as
7
Pesticide
1.4
Pollution Caused by Halogenated Compound in Malaysia
8
1.5
Microbial Degradation of Halogenated Compound
14
1.6
Microbial Degradation of Halogenated Compound in
17
Mixed Population
1.7
The Chemistry of Halogenated Compound
23
1.8
The Biochemistry of Dehalogenation
28
1.8.1
30
Dehalogenation of Haloalkanoic Acid
viii
1.8.1.1 Dehalogenation of α-Halocarboxylic Acids (2,2dichloropropionate)
30
1.8.1.2 Dehalogenation of β-Halocarboxylic Acids (3chloropropionate)
33
1.9
The Genetics of Haloalkanoic Acids Dehalogenase
38
1.10
Role of Transport Protein in Transporting
39
Halogenated Compound
1.10.1 The Presence of A Putative Rhizobial dehrP gene
in pSC1
1.11
2
Objectives
40
41
MATERIAL AND METHODS
2.1
Bacteria Strains and Plasmids
42
2.2
Growth Media
46
2.3
Glycerol Stocks
47
2.4
Measurement of Microbial Growth
47
2.5
Plasmid Cloning
47
2.6
Preparation of Chromosomal DNA
48
2.7
Measurement of DNA Concentration
49
2.8
Restriction Enzyme Digestion
49
2.9
Agarose Gel Electrophoresis
49
2.10
Isolation of DNA Fragment from Agarose Gels
50
2.11
Ligation of DNA
51
2.12
Polymerase Chain Reaction (PCR) for Amplification
of DNA
51
2.12.1 PCR Amplification of 16S rRNA Gene for
Bacteria Identification
52
2.12.2 PCR Amplification of Putative dehrP Gene
For Cloning
2.13
2.14
52
Preparation of Competent Cells and Transformation
of Plasmid DNA
54
Screening for Recombinants
54
ix
2.15
Synthesis
55
Differential Staining for Bacterial
55
2.16.1 Gram Staining
55
2.16.2 Acid-Fast Staining (Ziehl-Neelsen Method)
56
2.16.3 Spore Staining
56
Biochemical Tests
57
2.17.1 Oxidase Test
57
2.17.2 Catalase Test
57
2.17.3 Citrate Utilisation Test
58
2.17.4 Gelatine Liquefaction Test
58
2.17.5 Lactose Utilisation Test
58
2.17.6 Motility Test
59
2.18
HPLC Analysis of Growth Medium
59
2.19
Preparation of Cell Free Extracts
60
2.20
Measurement of Protein Concentration
60
2.21
Assay for Dehalogenase Activity
61
2.22
Assay for Halide Ion
61
2.23
Standard Curve for Chloride Ions
62
2.24
Technique for Protein Analysis
62
2.25
Computer Analysis
64
2.16
2.17
3
DNA Sequencing and Oligodeoxyribonucleotide
ISOLATION AND CHARACTERISATION OF
3-CHLOROPROPIONATE DEGRADING BACTERIA
3.1
Introduction
65
3.2
Results
67
3.2.1
Isolation of 3-chloropropionate Degrading Bacteria 67
3.2.2
Identification of Bacterium A by 16S rRNA Gene
Sequencing
67
3.2.2.1 DNA Analysis of Bacterium A
67
x
3.2.3
3.2.2.2 Sequencing of the 16S rRNA Gene
70
Bacteria Morphology, Staining and Biochemical
74
Characterisation
3.2.4
Growth of Rhodococcus sp. in 3-chloropropionate 76
Minimal Medium
3.2.5
Growth of Rhodococcus sp. in Other Halogenated
78
Compounds
3.2.6
HPLC Analysis of Growth Medium
80
3.2.6.1 3-chloropropionate Calibration Curve
80
3.2.6.2 Detection of 3-chloroproprionate in
80
Growth Medium
3.2.7
Dehalogenase Activity in Cell Free Extract of
3-chloropropionate Grown Bacteria
4
84
3.3
Discussion
85
3.4
Conclusion
89
ISOLATION AND CHARACTERISATION OF
2,2-DICHLOROPROPIONATE DEGRADING BACTERIA
4.1
Introduction
90
4.2
Results
92
4.2.1
Isolation of 2,2-dichloropropionate Degrading
Bacteria
4.2.2
4.2.3
92
Identification of Bacterium B by 16S rRNA Gene
Analysis
92
Bacteria Morphology, Staining and Biochemical
97
Characterisation
4.2.4
Growth of Methylobacterium sp. in
2,2-dichloropropionate Minimal Medium
4.2.5
4.2.6
99
Growth of Methylobacterium sp. in Other
Halogenated Compounds
102
HPLC Analysis of Growth Medium
105
4.2.6.1 2,2-dichloropropionate Calibration Curve 105
xi
4.2.6.2 Detection of 2,2-dichloroproprionate in
Growth Medium
4.2.7
Dehalogenase Activity in Cell Free Extract of
2,2-dichloropropionate Grown Bacteria
5
106
109
4.3
Discussion
110
4.4
Conclusion
114
ANALYSIS AND CLONING OF RHIZOBIUM SP. dehrP
GENE
5.1
Introduction
116
5.2
Results
119
5.2.1
Analysis of Putative dehrP Gene
119
5.2.1.1 Protein Translation and Sequencing Comparison
119
5.2.1.2 Analysis of Putative Conserved Domain
125
5.2.1.3 Hydrophatic Character: Analysis of
Transmembrane Segments
126
5.2.1.4 Further Analysis of Putative dehrP Gene and
5.2.2
Protein Sequence
127
Cloning of the Putative dehrP Gene
132
5.2.2.1 Restriction Map Analysis of Haloalkanoic Acid
Permease (dehrP) Gene
5.2.2.2 Analysis of pSC1 Plasmid
132
138
5.2.2.3 PCR Analysis of Haloalkanoic Acid Permease
(dehrP) Gene
5.2.2.4 Engineering of Plasmid pet 43.1a
140
142
5.2.2.5 Ligation of Haloalkanoic Acid Permease (dehrP)
Gene with pET 43.1a
144
5.2.2.6 Nucleotide Sequencing of pHJ
147
5.3
Discussion
149
5.4
Conclusion
152
xii
6
CONCLUDING REMARKS & SUGGESTIONS
153
REFERENCES
156
Appendices A - J
171 - 186
xiii
LIST OF TABLES
TABLE NO.
TITLE
PAGE
1.1
World and U.S. pesticide expenditures by pesticide type
3
1.2
Most commonly used conventional pesticide active ingredients in
4
agricultural market
1.3
Annual industrial production volumes and the use of industrially
6
important chlorinated hydrocarbons
1.4
Common pesticides in Malaysia
9
1.5
Halogenated pesticides detected in Selangor river
12
1.6
Various form of microbial interaction
18
1.7
Electronegativity, bond length and dipole moment
27
2.1
Bacteria strains used in this course of study
44
2.2
Plasmids used in this course of study
45
2.3
PCR cycle profile for 16s rRNA gene
53
2.4
PCR cycle profile for putative dehrp gene
53
2.5
Preparation of acrylamide gel for SDS-PAGE
63
2.6
Computing utilities used in this study
64
3.1
Top 5 entries in the database that shows highest identity to bacterium A
73
3.2
Colony characteristic on 3-chloropropionate minimal medium
75
3.3
Morphological and biochemical characteristics of Rhodococcus sp.
75
3.4
Growth properties of Rhodococcus sp. in different 3-chloropropionate
concentration
77
3.5
Growth properties of Rhodococcus sp. in different substrate
79
3.6
Data used to construct the 3-chlropropionate calibration curve
81
3.7
Comparison between the 3-chloropropionate consumed and turbidity
of Rhodococcus growth medium
82
xiv
4.1
Top 5 entries in database that showed highest identity to bacterium B
96
4.2
Colony characteristic on 2,2-dichloropropionate minimal medium
98
4.3
Morphological and biochemical characteristics of Methylobacterium sp.
98
4.4
Growth properties of Methylobacterium sp. in different
2,2-dichloropropionate concentration
101
4.5
Growth properties of Methylobacterium sp. in different substrate
103
4.6
Data used to construct the 2,2-dichloropropionate calibration curve
107
4.7
Comparison between the 2,2-dichloropropionate consumed and turbidity
of Methylobacterium growth medium
107
5.1
Amino acid sequence composition of DehrP
121
5.2
Top 6 entries obtained from blast search of DehrP
124
5.3
Endonucleases that would not restrict haloalkanoic acid permease gene 137
xv
LIST OF FIGURES
FIGURE NO.
TITLE
PAGE
1.1
A three-member microbial community growing on TCA
21
1.2
A seven-member microbial community growing on
22
2,2-dichloropropionate (Dalapon) continuous culture
1.3
Calculation of pKa
24
1.4
Substituent effects on acidity
25
1.5
Polarised carbon-halogen bond
25
1.6
Schematic representation of the proposed reaction mechanism of
29
DCM dehalogenase (Kayser, 2001)
1.7
Proposed pathways for catabolism of trans-3-chlorocrotonate and
36
3-chlorobutyrate by Alcaligenes sp. strain CC1
1.8
Nucleophilic substitution in degradation of 3-chloropropionic acid
37
1.9
The formation of acrylic acid from 3-chloropropionic acid
37
2.1
Restriction map of the cloning vector pET 43.1a
43
3.1
Structure of 3-chloropropionic acid
66
3.2 Agarose gel electrophoresis of undigested genomic DNA.
68
3.3 The PCR amplified 16S rRNA gene fragment
69
3.4 Bacterium A 16S rRNA partial sequence
71
3.5 Sequence comparison of bacterium A 16S ribosomal RNA gene
72
3.6 Growth curves on 5, 10, 20 and 40 mM 3-chloropropionate for
77
Rhodococcus sp.
3.7 Growth curve on 20 mM of various halogenated compound for
79
Rhodococcus sp.
3.8 Calibration curve for 3-chloropropionate
81
xvi
3.9
HPLC elution profile of medium from cells utilising
83
3-chloropropionate as source of carbon
4.1 The molecular structure of 2,2-dichloropropionic acid (Dalapon)
91
4.2 Agarose gel electrophoresis of undigested genomic DNA and 16S rRNA 93
gene fragment
4.3 Bacterium B 16S rRNA partial sequence
94
4.4 Sequence comparison for bacterium B 16S rRNA gene
95
4.5 Growth curves on 5, 10, 20 and 40 mM 2,2-dichloropropionate for
100
Methylobacterium sp.
4.6 Growth curve of Methylobacterium sp. when grown on different
104
halogenated substrate
4.7 Calibration curve for 2,2-dichloropropionate
107
4.8 HPLC elution profile of medium from cells utilising
108
2,2-dichloropropionate as source of carbon
5.1
Plasmid used in this course of study
117
5.2 The full nucleotide sequence of the upstream of dehD gene
118
5.3 The deduced amino acid sequence of dehrP
120
5.4 Amino acid sequence comparison between DehrP and DehP
122
5.5 Amino acid sequence comparison between DehrP and Deh4P
123
5.6 Results of the conserved domain search
125
5.7 Plot of probabilities of TM helix found in DehrP
126
5.8 Multiple Alignment of mutated DehrP with DehP and Deh4P
128
5.9 DNA alignment of putative dehrP gene sequence
129
5.10 Multiple Alignment of original DehrP with DehP and Deh4P
131
5.11 The predicted restriction enzyme map of dehrP
133
5.12 Agarose gel electrophoresis of pSC1
139
5.13 Sequencing strategy
140
5.14 Isolation of the haloacid permease (dehrP) gene
141
5.15 Plasmid map of pET 43.1a
142
5.16 Restriction enzyme digest of pET 43.1a
143
5.17 The newly constructed plasmid (designated as pHJ)
145
5.18 Restriction digestion of pHJ using EcoRI and NdeI
146
5.19 Sequencing of haloacid permease (dehrP) gene
148
xvii
LIST OF ABBREVIATIONS
2,2-DCP
-
2,2-dichloropropionic acid
3-CP
-
3-chloropropionic acid
A
-
Absorbance
BLAST
-
Basic local alignment search tool
DDBJ
-
DNA Data Bank of Japan
DNA
-
Deoxyribonucleic acid
E.coli
-
Escherichia coli
EDTA
-
Ethylenediaminetetraaceticacid,
(HOOCCH2)2N(CH2)2N(CH2COOH)2
EMBL
-
European Molecular Biology Laboratory
EtBr
-
Ethidium Bromide
H
-
Hour
kb
-
Kilo base
kDA
-
Kilo Dalton
min
-
Minutes
NCBI
-
National Center for Biotechnology Information
OD
-
Optical Density
PCR
-
Polymerase chain reaction
RDP
-
Ribosomal database project
RNA
-
Ribonucleic acid
rDNA
-
Ribosomal DNA
rRNA
-
Ribosomal RNA
s
-
Second
TAE
-
Tris-Acetate-EDTA
xviii
TMHMM
-
Transmembrane helices Markov model
UV
-
Ultraviole
A
-
Adenine
C
-
Cytosine
G
-
Guanine
T
-
Thymine
N
-
Any base; A or C or G or T
M
-
Amino; represented by either A or C
Y
-
Pyrimidine; represented by either C or T
W
-
Molecule with weak interaction; represented by
DNA BASES:
either A or T
R
-
Purine; represented by either G or A
xix
LIST OF APPENDICES
APPENDIX
TITLE
A
Rhodococcus sp. HN2006A 16S rRNA gene (AM231909)
B
Growth curve of Rhodococcus in various concentration
PAGE
171
of 3-chloropropionate
172
C
Calculation of doubling time
174
D
Growth curve of Rhodococcus in 20 mM of other
177
halogenated compound
E
Enzyme activity calculation for Rhodococcus sp. HN2006A
F
Methylobacterium sp. HN2006B 16S rRNA gene
G
178
(AM231910)
179
Methylobacterium growth in various concentrations of
180
2,2-dichloropopionate
H
Methylobacterium growth in other halogenated compound
182
I
Enzyme activity calculation for Methylobacterium sp. HN2006B
183
J
Rhizobium sp. dehrP gene (AM 260971)
184
K
Standard curve for Bradford assay
185
L
Standard curve for chloride ion assay
186
1
CHAPTER I
INTRODUCTION
1.1
Xenobiotics as Pollutants
Environmental pollution, caused by increased levels of industrial chemicals,
manufacturing wastes and biocides in water and on land, poses a considerable
problem for society. Consequently, this has led to major research programs, which
have studied the fate of these chemicals in the natural environment (Ejlertsson et al.,
1996; Golovleva et al., 1990; Higson, 1991; Javorekova et al., 2001; Rozgaj, 1994)
and in bioreactor (Baumann et al., 2005; Daugulis and MacCracken, 2003).
Chemical compounds found in the environment can arbitrarily be classified
into groups, according to those which are natural products and those that are foreign
to the biosphere (Hutzinger and Veerkamp, 1981). Xenobiotic (Greek, xenos
“foreign”; bios “life”) compounds may be defined as those having structural
moieties or groups, which are not found in natural products, and normally arise as a
direct result of man’s industrial activities.
Transient or permanent contamination by xenobiotic disrupts the normal
functioning of the biosphere, which is typically controlled by equilibrated cycles of
organic and inorganic matter by plants, animals, and microorganisms. Modern
2
agriculture methods result in widespread release of novel chemical particularly
herbicides and pesticides. Such biocides are directly and deliberately applied into
the environment for crop protection, while other industrial lipopholic chemicals are
locked for tens of years in closed systems, are susceptible to biomagnifications
through food chains, resulting in increased environmental distribution by certain
organisms (Atlas, 1996). Table 1.1 provides some general information on world and
U.S. pesticide expenditures in year 2000. Table 1.2 shows the most commonly used
conventional pesticide active ingredients. Most of them contain halogenated
compound.
Pollutants might enter the environment by numerous routes such as illegal
dumping from industrial waste, effluent leakages, leaching and from chemical
evaporation. The quantity of release is not always sufficient to gauge pollution
problems, and properties such as toxicity, carcinogenicity, biomagnifications and
persistence must all be considered to appreciate fully the extent of environmental
damage.
3
Table 1.1: World and U.S. pesticide expenditures by pesticide type, year 2000
(Kiely et al., 2004)
Year
Type
World Market
U.S. Market
Mil $
Mil $
%
U.S. Percentage of
World Market
%
%
Herbicides
14,319
44
6,365
57
44
Insecticides
9,102
28
3,129
28
34
Fungicides
6,384
19
860
8
13
Other
2,964
9
811
7
27
Total
32,769
100
11,165
100
34
Note:
•
Totals may not add due to rounding. Table does not cover wood
preservatives and specialty biocides.
•
“Herbicides” include herbicides and plant growth regulators.
•
“Insecticides” and “fungicides” exclude sulfur and petroleum oil.
•
“Other” includes nematicides, fumigants, rodenticides, molluscicides,
aquatic and fish/bird pesticides, other miscellaneous conventional pesticides,
plus other chemicals used as pesticides (e.g., sulfur and petroleum oil).
4
Table 1.2: Most commonly used pesticide active ingredients in agricultural market
(Ranked by range in millions of pounds of active ingredient) (Kiely et al., 2004)
2001
Active
Ingredient
1999
1997
1987
Rank Range Rank Range Rank Range Rank Range
Glyphosate
1
85-90
2
67-73
5
34-38
17
6-8
Atrazine
2
74-80
1
74-80
1
75-82
1
71-76
Metam Sodium
3
57-62
3
60-64
3
53-58
15
5-8
Acetochlor
4
30-35
4
30-35
7
31-36
NA
NA
2,4-D
5
28-33
6
28-33
8
29-33
5
29-33
Malathion
6
20-25
7
28-32
NA
NA
NA
NA
Methyl Bromide
7
20-25
5
28-33
4
38-45
NA
NA
Dichloropropene
8
20-25
11
17-20
6
32-37
4 30-35
Metolachlor-s
9
20-24
12
16-19
NA
NA
NA NA
Metolachlor
10 15-22
8
26-30
2
63-69
3
Pendimethalin
11 15-19
10
17-22
9
24-28
10 10-13
Trifluralin
12 12-16
9
18-23
10
21-25
6
25-30
Chlorothalonil
13
8-11
13
9-11
15
7-10
19
5-7
14 8-10
15
8-10
13
10-13
19
5-7
Chlorpyrifos
15
8-10
16
8-10
14
9-13
14
6-9
Alachlor
16
6-9
17
7-10
12 13-16
2 55-60
Propanil
17
6-9
18
7-10
22
6-8
13 7-10
Chloropicrin
18
5-9
14
8-10
25
5-6
NA
NA
Dimethenamid
19
6-8
20 6-8
20
6-9
NA
NA
Mancozeb
20
6-8
21 6-8
17
7-10
21
4-6
Ethephon
21
5-8
24 5-6
NA
NA
EPTC
22
5-8
19
18
7-10
Simazine
23
5-7
NA NA
NA
NA
NA NA
Dicamba
24
5-7
22
16
7-10
23 4-6
Sulfosate
25
3-7
NA NA
NA
NA
NA NA
Copper
Hydroxide
7-9
6-8
45-50
NA NA
8 17-21
5
1.2
Halogenated Compounds in the Biosphere
Halo-aliphatic compounds such as 2,2-dichloropropionic acid and
trichloroacetic acid are commonly used as herbicides. Halo-aromatic compounds
can also be considered important for this purpose. 2,4,-dichlorophenoxyacetate (2,4D) and 2,4,5-trichlorophenoxyacetate (2,4,5-T), having generated much interest with
respect to their degradation, and consequently have initiated many researchers to
study their fate in the environment (Ghosal et al., 1985; Han & New, 1994) and the
mechanism of degradation at molecular level (Farhana et al., 1998; Kitagawa et al.,
2002). Nevertheless, halo-organic compounds such as polychlorinated phenols,
polychlorinated biphenyls (PCB’s), chlorinated benzoates and various insecticides,
e.g. methoxychlor, aldrin, lindane and 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane
(DDT), also contribute to environmental contamination (Ritter et al., 1995).
This leads us to believe that synthetic halo-organic compounds are
undoubtedly a problem of major concern as listed in Table 1.3. However, naturally
occurring halogenated compounds are not uncommon. This is demonstrated by the
relative abundances of halogens as inorganic salts or minerals in soil and fresh water
environments: CL > F > Br > I, with the reversal of F and Br in sea water, where
organic fluorine is more limited, and associated with certain plants (Fetzner, 1998).
Gribble (1994) have listed more than 2000 halo-organic compounds as natural
products, which are released into biosphere by various marine organisms, higher
plants and ferns, insects, bacteria, fungi, and mammals. In addition, some bromo
and iodo chloralkane analogues have been reported as occurring naturally
(Gschwend et al., 1985), and due to their chemical and analytical simplicity can be
regarded as models for biodegradation studies of more highly halogenated alkanes.
6
Table 1.3: Annual industrial production volumes and the use of industrially
important chlorinated hydrocarbons (Fetzner, 1998)
Hydrocarbon
U.S
Year Some of its major uses
production
(x 103 tones)
Monochloromethane
390
1992
Production of silicones,
tetramethyllead, methycellulose
Dichloromethane
162
1992
Degreasing agent; paint remover,
extraction technology
Trichloromethane
229
1991
Production of
monochlorodifluorimethane
Tetrachloromethane
143
1991
Act as solvent. production of
trichloromonofluoromethane
Monochloroethane
67
1990
Production of tetramethyllead,
ethycellulose
1,1-dichloroethane
220-250
1985
Feedstock for production of 1,1,1trichloroethane
1,2-dichloroethane
7230
1992
Production of vinyl chloride,
ethylenediamines
1,1,1-Trichloroethane
327
1992
Dry cleaning; capor degreasing
1,1,2-Trichloroethane
200-220
1984
Intermediate for production of
1,1,1- trichloroethane
Monochloroethene
6000
1992
Production of PVC
150-200
1986
Basic material for poly(vinylidane
(vinyl chloride)
1,1-dichloroethene
(vinylidane chloride)
chloride)
Trichloroethene
110
1984
Dry cleaning, metal degreasing
Tetrachloroethene
110
1992
Dry cleaning, metal degreasing
2-chloro-1,3-butadiene
648
1983
Basic material for
(chloroprene)
poly(chloroprene) rubber
7
1.3
Usage of Halogenated Compound in Malaysia as Pesticide
Organochlorine pesticides are known for their environmental persistence and
global concerns. The half-life of most organochlorine pesticides can range from
several years to more than 10 years (Ritter et al., 1995). There are also toxic due to
their high capacities for bioaccumulation that poses a threat to the ecosystem and
human health. However, in Malaysia, the legislation, monitoring and control of
halogenated compound usage are still lacking. Not much information concerning
halogenated compound in Malaysia had been gathered.
Some countries promote monitoring programs and stepwise evaluation
process that seek to identify and minimise risk caused by accumulation of pesticide
in general and halogenated compound in particular to human, wildlife and the
environment. In the United States of America, environmental pollution in aquatic
ecosystems is still monitored today. Some highly toxic organochloride pesticides
are still detected due to their persistency in the environment even after almost 30
years of banning. Contamination in aquatic ecosystems that causes various harmful
effects on human and wildlife have been well studied especially in North America,
Japan and many parts of Europe.
In Malaysia, the input pathways of organochlorine compound into the river
environment originate from domestic sewage discharge, industry wastewater, runoff
from non-point sources such as from agricultural area and direct dumping of wastes
into the river (Leong et al., 2002). Owning to the persistency and bioaccumulation
of organochlorine pesticides; a different group of pesticide namely organophosphate
is being widely used in agriculture to replace the previous group of pesticide because
organophosphate pesticides are less toxic and easily degradable. The transformation
of organophosphate group in the environment takes place by conversion of the
phosphorothioate (P=S) group to their oxon (P=O) analogues. Both of these
compounds have strong inhibition of acetylcholonesterase activity, an enzyme that is
involved in neural function. The long-term application of these pesticides will pose
a chronic risk to health (Leong et al., 2002).
8
In view of the hazardous characteristics of these compounds, a number of
them had been listed by the UNEP as persistent organic pollutants (POPs). In
Malaysia, the usage of organochlorine pesticides listed as POPs are either prohibited
or restricted. For example, aldrin, dieldrin, DDT and chlordane were all restricted in
usage but after 1998, their usage had been discontinued. Heptachlor,
hexachlorobenzene, mirex, toxaphene and endrin are never registered for use. Even
though the usage of most POPs-listed organochlorine pesticides are prohibited,
studies on rivers and sediments throughout Malaysia demonstrated that most of these
compounds are present in the aquatic environment (Cheah and Lum, 1994; Leong et
al., 2002). However, detailed information is not available since not much study in
this area was conducted in Malaysia. In most of these studies, the sources of the
contamination were unknown. Table 1.4 shows some status of common pesticide in
Malaysia.
1.4
Pollution Caused by Halogenated Compound in Malaysia
The monitoring of several different pesticides in the Selangor River,
Malaysia had been done under UNU Project on EDC Pollution in the East Asian
Coastal Hydrosphere (Leong et al., 2002). According to the author, Selangor River
was chosen as a water sampling area for the UNU hydrosphere project for many
reasons. Firstly, it is a main source of water supply to many agricultural activities
such as vegetable farms, oil palm and rubber plantation. Furthermore it is also used
to support aquaculture activities such as the cultivation of fresh water fishes for
human consumption. Further down stream of this river is an ecotourism area for the
observation and study of fireflies which is somehow dwindling with time and human
interference. Selangor River is also a source of water for most of the residents living
along and will soon be a major source of water to part of Kuala Lumpur city after
building of a dam is being carried out at the upper stream of the river.
9
Table 1.4: Common pesticides in Malaysia
CAS Number
Chemical
93-76-5
2,4,5-T
309-00-2
Aldrin
Registered
Banned or
for Use
Restricted
No
Banned
1336-36-3, 11097-69-1 Aroclor
1332-21-4, 12001-28-4,
77536-66-4, 77536-67- Asbestos
5, 77536-68-6
485-31-4
Binapacryl
Banned
2939-80-2, 2425-06-1
Captafol (cis isomer)
Banned
Chlordane
Banned
6164-98-3
Chlordimeform
Banned
510-15-6
Chlorobenzilate
50-29-3, 789-02-6
DDT
Banned
60-57-1
Dieldrin
Banned
x88-85-7
Dinoseb and dinoseb salts
Banned
106-93-4
Ethylene dibromide
Banned
107-06-2
Ethylene dichloride
Banned
75-21-8
Ethylene oxide
Banned
640-19-7
Fluoroacetamide
Banned
133-07-3
Folpet
76-44-8
Heptachlor
118-74-1
Hexachlorobenzene
608-73-1, 319-86-8
Hexachlorocyclohexane
58-89-9
Lindane
57-74-9, 12789-03-6,
5103-71-9, 5103-74-2
No
No
Banned
Banned
No
Banned
Yes
10
x7439-97-6
Mercury and mercury
Banned
compounds
10265-92-6
Methamidophos
Yes
298-00-0
Methyl parathion
No
6923-22-4
Monocrotophos
Yes
56-38-2
Parathion
No
87-86-5
PCP
13171-21-6
Phosphamidon
61788-33-8
Polychlorinated terphenyls
8001-35-2
Toxaphene
Restricted
Restricted
Banned
No
Banned
* The only data available for Malaysia is the information on banned and severely
restricted chemicals provided by the United Nations Prior Informed Consent (PIC)
Circulars. (PIC Circular XII, 2000; PIC Circular XIV, 2001)
11
Pesticides that were listed in Table 1.5 are halogenated pesticides, for
example lindane, chlorpyrifos, DDE, endosulfan, endosulfan sulfate, o,p’-DDT and
heptachlor whereas fenitrothion, malathion and diazinon are organophosphate. All
of the halogenated pesticides listed here could be detected in Selangor River.
Lindane is most heavily used by the plantation operators and farmers around the
Selangor District. The presences of halogenated pesticides in river waters may
impair the river beneficial uses and their biological resources. In view of that,
knowledge in degradation of halogenated pesticide is important to protect the
environment.
12
Table 1.5: Halogenated pesticides detected in Selangor River (Leong et al., 2002)
Structure Drawing
Compound
IUPAC Name
Name
Lindane
1,2,3,4,5,6hexachlorocyclohexane
chloropyrifos
O,O-diethyl O-3,5,6trichloro-2-pyridyl
phosphorothioate
p,p'-DDE
1,1-dichloro-2,2-bis(4chlorophenyl)ethylene
endosulfan
6,7,8,9,10,10hexachloro1,5,5a,6,9,9ahexahydro-6,9methano-2,4,3benzodioxathiepin-3oxide
endosulfan
6,7,8,9,10,10-
sulfate
hexachloro-6,9methano-2,4,3benzodioxathiepin-3
o,p'-DDT
2-(2-chlorophenyl)-2(4chlorophenyl)-1,1,1trichloroethane
13
heptachlor
1,4,5,6,7,8,8heptachloro-3a,4,7,7atetrahydro-4,7methanoinden
14
1.5
Microbial Degradation of Halogenated Compound
Microorganisms are the primary agents of biological recycling, and have
evolved an extensive range of enzymes, pathways and control mechanisms in order
to degrade and utilise pollutants as an energy sources (Madigan et al., 2000, Talaro
and Talaro, 2002). No single organism possesses the mechanisms for the
biodegradation of every compound, and evidence from laboratory based studies
clearly shows that the involvement of microorganisms in the transformation of
halogenated xenobiotics is an important factor in determining the fate of these
compounds in the environment (Talaro and Talaro, 2002). Microorganisms are not
solely responsible for degradation of organic compounds, as many other organisms
do participate albeit to a smaller extent. Photochemical decomposition has been
suggested as a significant route by which some compounds are degraded (Zabik et
al., 1976; Konstantinou et al., 1999; Hirahara et al., 2001).
All organic compounds in existence are thought to be thermodynamically
unstable to varying degrees, and in principle, can be mineralised by microorganisms
to generate CO2 and energy for microbial growth. In the kinetic sense however,
most organic compounds are perfectly stable, and under physiological conditions in
the absence of catalysis, they will not be degraded or mineralised at significant rates.
Such catalysis using enzymes are common in aerobic soil microorganisms seeking to
obtain energy for growth.
The general requirement for biodegradation may be summarised as follow:
1. Accessibility of a compound to microorganisms.
Organic compounds may be adsorbed to particular matter in the soil (e.g.
clays), thereby preventing potential microbial attack. Similarly, chemical
complexing to other molecules leads to the same consequence.
15
2. Entry of a compound into an organism.
The lack of penetration, coupled with the absence of suitable extra-cellular
enzymes may result in a compound being resistant to biological attack. Specific
transport mechanisms have evolved to facilitate the entry of naturally occurring
compounds. However, on initial exposure to many unnatural halogenated molecules
the uptake mechanisms are unlikely to function.
3. Induction of catabolic enzymes.
The compound must induce the synthesis of, and act as substrate for
degradative enzymes (assuming that the enzymes are not produced constitutively).
Xenobiotic halo-organic compounds are quite frequently unable to be utilised as
substrates for microbial growth, as they are too far removed from the main stream of
catabolic pathways. Consequently, a xenobiotic compound is unable to be converted
to an intermediate that can be mineralised further in an existing biochemical
pathway. Halogen substitution sufficiently alters a molecule’s structure so as to
reduce the rate of its transformation, and possible prevent its metabolism. In the
instance, the fate of xenobiotics is to some extent determined by the degree of
structural analogy between the synthetic compound and a natural compound for
which catabolic function exist. Such structural analogies include comparable
reactivities, together with analogous size and polarity of functional groups (Hughes,
1988).
4. Aerobic or anaerobic environments.
Anoxic/aerobic environments can accumulate some compounds which would
otherwise be degraded under different condition (Alexander, 1981).
Three main biodegradative mechanisms have been reported for halo-organo
compounds. Ultimately, complete degradation or mineralisation of a compound is
required, so as to prevent or reduce its persistence in the environment. These
involve complete breakdown of an organic compound into an inorganic state, and
subsequently the conversion of the carbon-skeleton into intermediary metabolites.
A second mechanism constitutes partial degradation, which is clearly
demonstrated by halo-aromatic compounds. For example, PCB’s are composed of
16
aromatic ring pairs which may possess one unsubstituted and a chloro-substituted
ring. A single organism can sometimes use the unsubstituted nucleus as the growth
substrate, while the halo-substituted ring will be excreted into the culture fluid as an
organic end-product.
The third mechanism type is co-metabolism (Alexander, 1981; Baggi et al.,
2005) as the microbial action that modifies the structure of a chemical, without
deriving energy from the catabolism for microbial growth. The population involved
in co-metabolism is assumed to grow on other substrate while performing the
transformation and the lack of increase in population biomass is reflected by the
inability of the co-metabolising microorganisms to utilise the chemical for
biosynthetic purposes.
Co-metabolism of halo-organic compounds does not result in complete
mineralisation to inorganic halide, CO2 and H2O, but it does seemingly reduce
toxicity in the environment, which indicates the ecological importance of this
phenomenon. It has been suggested that co-metabolism may account for the
degradation of many pesticides which do not sustain microbial growth (Alexander,
1981). Co-metabolism and degradation of compound such as 1,1,1-trichloro-2,2-bis
(p-chlorophenyl)ethane (DDT) and related molecules have been studied extensively
(Bumpus and Aust, 1987; Aislabie et al., 1997; Quensen et al., 1998). In some cases,
although complete mineralisation did not occur, the toxic effect of the insecticide in
the environment was reduced. The chemical alteration were however unable to
guarantee its complete detoxification.
In view of the large number of synthetic chemicals that are apparently cometabolised, establishing a physiological explanation is quite important. The most
likely hypothesis is related to enzyme specificity. Many enzymes present in
microbial cells catalyse reactions involving several different but chemically related
substrates and are enzymes of broad substrate specificities. Products that are formed
from such chemically related compounds may not subsequently be altered by these
enzymes, and consequently accumulated, preventing further mineralisation and
generation of energy for microbial growth. This suggests that the toxicity of a
compound is somewhat reduced by co-metabolism but not eliminated.
17
Very often the term recalcitrance is used for the many xenobiotics that
endure for long periods in natural ecosystems in chemically unchanged states, owing
to the inability of microorganisms to degrade them. Halogenated compound,
especially polychlorinated types are generally degraded slowly and regarded as
recalcitrant molecules. This is a phenomenon discussed by many authors since
considerations about the fates of these recalcitrant compounds by biological,
chemical and physical agents are extremely important in understanding persisting
toxological effects in the biosphere (Barriault et al., 1998; Kim and Picapdal 2001;
Araoz and Viale, 2004; Xu et al., 2004,).
1.6
Microbial Degradation of Halogenated Compound in Mixed Population
Xenobiotics are supplied as growth-limiting carbon sources in chemostat
enrichments, and it is not uncommon to isolate mixed populations. Situations arise
where primary utilisers obtain energy directly from the growth-limiting carbonsource and the secondary utilisers benefit from the breakdown products of the
primary utilisers. The struggle for existence within a growth-limiting system is
therefore dependent on competition, a phenomenon that may explain the enormous
diversity of the microbial forms and species in the case of evolution. Competition
however is not the only interaction possible between organisms, particularly in the
case of xenobiotics, where various other forms have been observed. The
interactions listed in Table 1.6 should be considered as simplified definitions, as it is
likely that many microbial interactions in nature are mixtures of two or more of
these extremes that contribute to the establishment of integrated microbial
communities (Hughes, 1988).
.
18
Table 1.6: Various form of microbial interactions (Talaro and Talaro, 2002)
Interaction
Definition
Neutralism
Lack of interaction
Competition
A race for nutrients and space
Mutualism
Organisms live in an obligatory but mutually
beneficial relationship.
Commensalisms
The commensal benefits; other member not
harmed.
Parasitism
Parasite is dependent and benefits, host
harmed.
Synergism
Members cooperate and share nutrients.
Antagonism
Some members are inhibited or destroyed by
others.
19
In halogenated substrate degradation, mutualism and commensalisms are the
2 main interacting systems to be considered, where a dependent community
population benefits from the presence of a second population. For example,
mutualism was observed in a soil microbial community isolated by Jensen (1957a)
growing on trichloroacetate. The community consisted of a primary trichloroacetate
-utilising bacterium, designated strain 3-Cl, and two Streptomyces species. The
latter organisms provided Vitamin B12 which was essential for bacterium strain 3-Cl
(Figure 1.1). In this case the Streptomyces sp. did not appear to benefit directly from
the presence of the bacterium strain 3-Cl. On the basis of these observations a stable
community was formed which was capable of metabolising trichloroacetate.
A complex microbial community, utilising 2,2-dichloropropionate (Dalapon)
as the growth-limiting substrate was isolated and studied by Senior et al. (1976),
who suggested that commensalisms was responsible for the success of the stable
community. Seven organisms were originally characterised, three of which were
primary utilisers able to grow on 2,2-dichloropropionate in pure culture, while the
remainder were secondary utilisers, unable to utilise the herbicide as a carbon and
energy source (Figure 1.2). The community was very stable and was maintained in
continuous-flow culture over 18,000 h (25 months). Initially, the three primary
organisms, Pseudomonas sp. strain P1; Bacterium strain P2 and Trichoderma viride
strain P4, co-existed under conditions where they were competing for the limiting
carbon-source, indicating that they were interacting in such a way so as to stabilise
the community, and thus exhibit a form of concerted metabolism.
However, the mixed culture showed a change in structure which was
possibly very significant in natural environments. After prolonged continuous
cultivation the carbon-limited nature of the culture conditions thus exerted a strong
selective pressure for evolving the capabilities of one of the secondary organisms,
PP1, to utilise the growth-limiting carbon source (Figure 1.2). From subsequent
studies, it was revealed that elevated dehalogenase activity of this new primary
utiliser, PP3 evolved under varying growth conditions (Weightman et al., 1979).
Senior et al. (1976) showed that a community structure produces a far more
stable catabolising population because environmental fluctuations appeared to have
20
little effect on substrate metabolism. They disclosed that changes in pH of the
culture caused marked changes in the dominant 2,2-dichloropropionate utilising
population, reflecting their varying capabilities to grow at different pH values. As a
multi specific unit however, all the primary population were retained within the
growth vessel, whatever the pH of the culture medium.
21
TCA
Bacterium 3-Cl
Vitamin B 12
Figure 1.1:
1957a)
TCA
Two streptomyces sp.
A three-member microbial community growing on TCA (Jensen,
22
Figure 1.2: A seven member microbial community growing on 2,2dichloropropionate (Dalapon) continuous culture (Senior et al., 1976)
23
1.7
The Chemistry of Halogenated Compound
The relationship between chemical structure and biological activity is very
complex and may be one contributory factor as to why certain halogenated
compounds remain recalcitrant. As discussed by Slater et al. (1995), the removal of
halogens, particularly fluorine and chlorine, from organic molecules has fascinated
chemists and microbiologists for many years since such mechanisms relieve
inhibitory effect and provide alternative carbon and energy sources for growth.
Generally, the greater the number of halogens per organic molecule, the more
difficult it is to be degraded by microbes either alone or in consort as part of a
microbial community (Commandeur and Parsons, 1990).
Halogenated compounds have been shown to function as ideal pesticides.
Leasure (1964) notes how chlorinated substrates are best suited for herbicidal
activity over the brominated, fluorinated and iodinated compounds. Within the
propionic acid and butyric acid homologous, α-chlorination results in the highest
herbicidal activity, trichloromethane, monochloroacetate (MCA) and dichloroacetate
(DCA) do not however result in activity, and in combination with α-chlorinated
compounds may weaken the desired effect hoped to be achieved. Increased chain
length also seems to reduce activity of even highly biocidal α-substituted
compounds, this is demonstrated by a weakly active 2,2-dichloropentanoic acid and
an inactive 2,2-dichlorohexanoic acid.
An additional factor which may render halo-organo compounds desirable for
biocidal action is the acidic properties associated with halogen when it is bound to
the parent molecule. The relative strength of an acid is indicated by aciddissociation constant (Ka). The stronger the acid, its dissociation constant are greater.
Due to a wide range of spanning, Ka is often expressed on a logarithmic scale
(Figure 1.3). Therefore, stronger acids would have smaller values of pKa (Wade,
2003; Solomons, 1994).
24
pKa = - log Ka
Figure 1.3: Calculation of pKa
The more highly substituted analogous aliphatic and aromatic compounds
proving the stronger acids, which are thought to be attributable to the strong
electronegativity of halogen substituents. Acidity of halo-organo compounds is also
affected by substitution position, and as indicated by pKa values, the further the
halogen substituent is placed away from the –COOH group, the weaker the acid
(Figure 1.4). This is a phenomenon termed “inductive effect”. The inductive effect
is obvious if one or more strongly electron-withdrawing groups are present on the
carbon atom. The magnitude of a substituent effect depends on its distance from the
carboxylic acid, whereas more distant substituents have smaller effects on acidity,
showing that inductive effects decrease rapidly with distance (Wade, 2003).
In a halogenated carboxylic acid, the halogen atom is bonded to a sp3 hybrid
carbon atom (Wade, 2003; Solomons, 1994). The arrangement of groups around the
carbon atom is generally tetrahedral (Solomons, 1994). The halogen atoms are more
electronegative than carbon; the carbon-halogen bond is polarised with a partial
positive charge on carbon and a partial negative charge on the halogen, as shown in
Figure 1.5 (Wade, 2003; Solomons, 1994).
25
H
H
H
O
C
C
C
H
H
OH
H
Cl
H
O
C
C
C
H
H
OH
propionic acid
3-chloropropionic acid
pKa = 4.87
pKa = 3.98
Stronger acids
H
Cl
H
O
C
C
C
H
H
OH
H
H
Cl
O
C
C
C
H
H
OH
3-chloropropionic acid
2-chloropropionic acid
pKa = 3.98
pKa = 2.83
Figure 1.4: Substituent effects on acidity
μ
C
δ+
X
δ-
μ = 4.8 x δ x d
where μ is the dipole moment
δ is the charge
d is the bond length
Figure 1.5: Polarised carbon-halogen bond
26
As go down the periodic table, the size of the halogen atom increases. Due
to the increases in size, the electron affinity of the atom decreases (Umland and
Bellama, 1999) thus causes the electronegativity of the halogen decrease as going
down the periodic table. When the atomic radii of halogen atoms become larger, the
carbon-halogen bond lengths increase. These two effects oppose each other, with
the larger halogens having longer bonds but weaker electronegativities (Table 1.7).
Therefore, the bond dipole moments decrease as go down the periodic table (Wade,
2003; Solomons, 1994).
The strength of the carbon-halogen bond is the important factor in determine
the acidity (Wade, 2003; Solomons, 1994). A more electronegative element gives a
stronger carbon-halogen bond molecule. The stronger the bond results in the weaker
the acid (Solomons, 1994). The acidic properties of such halo-organo compounds
may also be one factor which contributes to the prevention of microbial degradation,
as acidic environments do not characteristically support extensive growth of
microorganisms and are as a result associated with a chemical’s persistence.
27
Table 1.7: Electronegativity, bond length and dipole moment
I
electronegativity:
bond length
<
2.7
Br
<
3.0
Cl
<
3.2
F
4.0
CF
<
C  Cl
<
C  Br <
CI
: 1.38 Å
<
1.78 Å
<
1.94 Å
<
2.14 Å
<
C  Br <
CF
<
C  Cl
<
1.48 D
1.51 D
<
1.56 D
CI
dipole moment : 1.29 D
<
28
1.8
The Biochemistry of Dehalogenation
It was well accepted that the utilisation of halogenated substrate by
microorganisms require enzymes of broad substrate specificities that can adapt to
catalyse conversion of many substrates with the same basic structure. Halogenated
substituents therefore, are very often removed by fortuitous reactions, the enzymes
responsible being primarily associated with the metabolism of unsubstituted
substrate analogues. Alternatively, specific dehalogenase enzymes are responsible
for catalysing the removal of the halogen substituent from the substrate preferably at
an early stage in catabolism.
The effect of halogen substitutions on the biodegradation of halo-aromatics
have been discussed by Reineke (1984). Aerobic catabolism of halo-aromatics
appears to closely resemble the pathways described for unsubstituted analogous. In
addition, the biodegradation of halo-alkanes such as dichloromethane by bacteria is
also well documented. The best investigated dichloromethane degrader at the
molecular level is the pink pigmented Methylobacterium dichloromethanicum DM4
(Galli and Leisinger, 1985). Degradation of dichloromethane depends solely on the
reaction catalysed by dichloromethane dehalogenase / glutathione S-transferase, a
glutathione-dependent enzyme converting dichloromethane to formaldehyde and
two molecules of hydrochloric acid. The postulated conversion pathway of
dichloromethane to formaldehyde is shown in Figure 1.6. The latter compound can
be oxidised to CO2 for energy or assimilated into cellular organic molecules.
29
Figure 1.6:
Schematic representation of the proposed reaction mechanism of
DCM dehalogenase (Kayser, 2001)
30
1.8.1 Dehalogenation of Haloalkanoic Acids
Halo-alkanoates dehalogenases are responsible for the dehalogenation of
both mono- and di-halogenated substrates yielding hydroxyl- and keto- acids
respectively as products (Jensen, 1963). Early work by Jensen (1957a, 1957b and
1960) demonstrated that soil microorganisms able to metabolise chlorinated
alkanoates (mainly acetates and propionates) as sole source of carbon and energy
can be readily isolated. However, others have studied a broader range of substrates
which fall within the scope of this study, and have suggested that a greater number
of different bacterial species with dehalogenating capabilities can be isolated from
environment which have previously been exposed to the halo-alkanoates. To date,
only α-halo-alkanoates dehalogenases/halidohydrolases, responsible for
dehalogenation reaction at the α-substituted position have been well characterised.
1.8.1.1 Dehalogenation of α-Halocarboxylic Acids (2,2-dichloropropionate)
Since 2,2-dichloropropionate is an active ingredient in herbicides, many
reports has focused on the isolation of 2,2-dichloropropionate degrading bacteria.
Jensen (1957a), using soil perfusion and enrichment technique, isolated five strains
of Pseudomonas sp. which able to degrade 2,2-dichloropropionate and other
halogenated substrate such as dichloroacetate and 2-chloropropionate.
Magee and Colmer (1959) isolated from soil six 2,2-dichloropropionate
degrading bacteria with very similar properties and tentatively assigned them to
either of both the genera Agrobacterium and Alcaligens. Using an enrichment
technique, Hirsch and Alexander (1960) isolated five strains of Norcadia and three
strains of Pseudomonas from soil which had been incubated with 2,2dichloropropionate. All eight organisms readily decomposed the latter compound at
a concentration of 0.1% (w/v) in basal medium, liberating free chloride ion.
31
Senior et al. (1976) used a continuous-flow culture enrichment procedure to
isolate from soil a microbial community which could grow on 2,2dichloropropionate as the sole source of carbon and energy. After three weeks of
growth in the chemostat, seven different microorganisms were isolated from the
community. These could be divided into primary and secondary 2,2dichloropropionate utilisers (as describe in section 1.6 and Figure 1.2).
The dehalogenase system of Pseudomonas putida strain PP3, that was
originally isolated from chemostat culture following selection on 2,2dichloropropionate (Senior et al., 1976) is now well studied. Strain PP3 produced
two dehalogenases, DehI and DehII (Weightman et al., 1979), which were encoded
by genes of the group I and group II deh families, respectively (Hill et al., 1999).
Similar to P.putida strain PP3, a Rhizobium sp. isolated by Allison (1981),
produced two dehalogenase activities when grown in the presence of 2,2dicholorpopionate. However, further investigation indicated that Rhizobium sp.
produced three dehalogenases, collectively known as DehD, DehL and DehE. DehD
and DehL are stereospecific for D-2-chloropropinate and L-2-chloropropionate
respectively, whereas DehE can act on both D,L-2-chloropropionate and 2,2dichloropropionate (Cairns et al., 1996; Stringfellow et al.,1997).
.
In addition, four bacterial strains identified as Agrobacterium tumefaciens
RS4, Agrobacterium tumefaciens RS5, Comamonas acidovarans and Alcaligenes
xylosoxidans were isolated from 2,2-dichlropropionate polluted soils (Schwarze et
al., 1997). All bacterial strains expressed a single dehalogenase. Upon further
biochemical characterisation, two different D,L-specific 2-haloalkanoic acid
dehalogenase were described. The dehalogenases of these strains have been shown
to be inducible and catalyse halide hydrolysis with the inversion of the product
configuration.
Dehalogenation of 2,2-dichloropropionic acid is catalysed by hydrolytic
dehalogenases (Janssen et al., 2001). There are two distinct evolutionary families of
these enzymes, the group I and group II dehalogenases. Group I dehalogenases
dechlorinate D-2-chloropropionate, whereas all group II dehalogenases dechlorinate
32
L-2-chloropropionate. As described by Hill et al. (1999), group I dehalogenases do
not share any obvious feature with group II dehalogenase in terms of nucleotide or
deduced amino acid sequences, suggesting that they are not evolutionarily related.
They also seem to be functionally distinct.
Group II dehalogenase enzymes catalyse the hydrolytic dehalogenation of L2-haloalkanoic acids to yield the corresponding D-2-hydroxyalkanoic acids (van der
Ploeg et al., 1991). They belong to the Haloacid Dehalogenase (HAD) superfamily
of aspartate-nucleophile hydrolases, class (subfamily) I.
Group II dehalogenases share a conserved nucleophilic aspartate residue that
is located close to the N terminus and is involved in formation of the covalent
intermediate. The Asp-10 residue is the active-site nucleophile, attacking the αcarbon of the substrate, forming an ester intermediate which is subsequently
hydrolysed by a water molecule (Kurihara et al., 1995; Liu et al., 1995). The
aspartate is positioned by interaction with a conserved lysine. An arginine, an
asparagine and a phenylalanine residue are also conserved and are involved in
binding of the halogen/halide.
Group I dehalogenase enzymes have yet to be fully characterised. Although
no structural information is available as yet, biochemical studies have indicated that
group I haloacid dehalogenases (Ridder et al., 1999) do not use a covalent
mechanism for catalysis. Nardi-Dei et al. (1999) proposed that the dehalogenase
directly activates a water molecule to attack the α-carbon of 2-haloalkanoic acid,
thereby displacing the halogen atom. It has not yet been established which amino
acids are involved in the dehalogenation, although a number of candidates have been
highlighted by mutagenesis experiments.
Almost all of the α-halocarboxylic acids dehalogenases isolated and described
in literature was now assigned to either the group I or group II dehalogenase.
However, the DehL from Rhizobium sp. (Cairns et al., 1996) showed the same
stereospecificity as group II dehalogenase (dechlorination of L but not D-2chloropropionate) was not a member of this family. From amino acid sequences of
various L-2-haloacid dehalogenase compared, DehL from Rhizobium sp. was
suggested to constitute a separate homology group of L-specific enzymes.
33
1.8.1.2 Dehalogenation of β-halocarboxylic Acids (3-chloropropionate)
Microbial degradation of halogenated aliphatic compounds has been studied
to an increasing extent within the last five decades. However, most reports mainly
described aerobic and anaerobic bacterial degradation of various haloalkanes, αhalocarboxylic acids and aromatic halogenated compounds. Except for an early
study by Bollag and Alexander (1971) and Yokata et al. (1986) which deals with
bacterial dehalogenation of β-chlorinated aliphatic acids (3-chloropropionate) and a
recent study by McGrath and Harfoot (1997), which describe utilisation of 3chloropropionate by a group of phototrophic organisms under anaerobic condition,
all investigations concerning bacterial degradation of haloacids have focused on
saturated α -halogenated acids especially 2,2-dichloropropionate (Section 1.8.2.1).
Using an enrichment technique, Bollag and Alexander (1971) isolated from
soil a strain of Micrococcus denitrificans capable of utilising 3-chloropropionate as a
sole source of carbon and energy. Bacterium suspensions derived from cultures
grown in a medium containing 3-chloropropionate readily metabolised this
compound without a lag phase. Crude cell-free extracts of 3-chloropropionategrown bacteria liberated chloride ion from many β-substituted acids, but aliphatic
acids halogenated only in the α-position were not dechlorinated.
Yokata et al. (1986) described seven bacterial species isolated using a soil
enrichment technique which were capable of utilising β-chlorinated butyrates and
propionates as sole source of carbon and energy. Two of the organisms were
identified as Corynebacteria sp. Cell free extract of Corynebacteria sp. did not
liberate chloride ions from 3-chloropropionates although resting cells growns on 1chlorobutane were reported to dehalogenate 3-chloropropionate.
McGrath and Harfoot (1997) demonstrated that 3-chloropropionate can be
degraded under anaerobic condition by a group of phototrophic organisms from the
purple nonsulfur bacteria group. Under illuminated anaerobic conditions, strains
from the genera Rhodospirillum and Rhodopseudomonas were shown to grow
34
phototrophically on chlorinated and brominated acetic and propionic acids by the
reductive removal of the halogens and subsequent utilisation of the acids for growth.
In all cases, the degradation of each of the substrates was accompanied by the
release of the associated halogen, as halide, and the formation of the corresponding
nonhalogenated acid. Thus, the dehalogenation of haloacetic acid yielded acetic
acid and the dehalogenation of the halopropionic acids yielded propionic acid. Both
of these acids are readily photometabolised by each of the strains. Nevertheless, all
strains have slow growth rates with generation times of between 36 to 72 hours
when growing on 2mM of these substrates.
Very few reports have been published on 3-chloropropionate degradation and
dehalogenases from these organisms were not characterised and the pathways were
not elucidated. An Alcaligenes sp. that is able to degrade α -chlorinated aliphatic
acids (2-chlorobutyrate, 2-chloropropionate) as well as on the β -chlorinated fourcarbon aliphatic acids (trans-3-chlorocrotonate, cis-3-chlorocrotonate, and 3chlorobutyrate) as sole carbon and energy sources may give some idea about the
pathway of 3-chloropropionate degradation. However, the 3-chloropropionate
proved not to be a substrate for growth for this bacterium (Staub and Kohler, 1989).
As described by Staub and Kohler (1989), the proposed pathways for
catabolism of trans-3 chlorocrotonate and 3 chlorobutyrate by Alcaligenes sp. strain
CC1 was showed in Figure 1.7. The dechlorination of β-chlorinated four-carbon
fatty acids by Alcaligenes sp. strain CC1 is dependent on a prior reaction of the
aliphatic acids with CoA, which presumably results in the formation of the
corresponding CoA esters. This esterification is followed by the removal of the
chlorine substitute, allowing the four-carbon acids to be further catabolised using a β
–oxidation pathway.
The proposed reaction for dehalogenation of 3-chloropropionate is by
hydrolytic dehalogenation similar to Group II dehalogenase enzyme described in
section 1.8.1.1. During nucleophilic substitution, the hydroxide ion derived from
water (van Pée and Unversucht, 2003) attacks the electrophilic carbon atom that
attached to the chloride group. The electron pairs from the electron-rich nucleophile
moved to the electron-poor carbon atom of the electrophile. Since carbon can
35
accommodate only eight electrons in its valence shell, the carbon-chlorine bond
must begin to break as the carbon-oxygen bond begins to form. The chloride ion
leaves with the pair of electrons that once bonded it to the carbon atom (Figure 1.8).
Besides, there is a possibility that the removal of chloride from 3chloropropionic acid was catalysed by an enzyme system involved in the β-oxidation
of fatty acids (Yokota et al., 1986). Thus, it will resulting in formation of α,βunsaturated acids, which were also known as acrylic acids. The formation of acrylic
acid was showed in Figure 1.9.
36
Figure 1.7: Proposed pathways for catabolism of trans-3 chlorocrotonate and 3
chlorobutyrate by Alcaligenes sp. strain CC1 (Staub and Kohler, 1988)
37
H
H
HOOCH2C
nucleophile
electrophile
H
..
Cl
.. ¯
+
C
H
CH2COOH
3-hydroxypropionic acid
..
..
C
..
HO
..
..
..
Cl
..
..
..
HO ¯
..
chloride ion
Figure 1.8: Nucleophilic substitution in degradation of 3-chloropropionic acid
O
OH
C
O
OH
C
H
C
H
Cl
C
H
H
3-chloropropionic acid
C
H
H
C
H
2-propenoic acid
(acrylic acid)
Figure 1.9: The formation of acrylic acid from 3-chloropropionic acid
38
1.9
The Genetics of Haloalkanoic Acids Dehalogenase
A dechlorination reaction often requires only a single protein that can
recognise and convert a xenobiotic substrate. However, regulated expression by
means of binding of a halogenated substrate and interaction with the transcription
machinery requires a second protein. Therefore, if the synthesis of a dehalogenation
enzyme is subject to regulation, the pathway must be more evolved than in the case
of constitutive protein expression (Poelarends et al., 2000).
A number of regulatory genes that influence dehalogenase expression have
been characterised in dehalogenating organisms. The classical halo acid
dehalogenase are usually regulated, which is not surprising because they are natural
compounds (Janssen et al., 2001). In contrast, dehalogenase from some organisms
that utilise xenobiotic haloalkanes might be expressed constitutively. For example,
Janssen et al. (1985) described the degradation of 1,2-dichloroethane by
Xanthobacter autotrophicus strain GJ10. The bacterium constitutively produces a
hydrolytic haloalkane dehalogenase (DhaA) that converts dichloroethane to 2chloroethanol.
In addition, gene transfer is an important process during the evolution of
novel catabolic pathways. Acquisition of foreign DNA by horizontal gene transfer
requires integration into a replicon that is well maintained in the recipient
microorganism. Transposition and gene integration are key mechanisms for the
formation of stable new constructs. An classic example was showed by
Pseudomonas putida PP3 isolated by Senior et al. (1975), which grown on 2,2-
dichloropropionate and produced dehalogenase I (dehI) and dehalogenase II (dehII).
Thomas et al. (1992a, 1992b) showed that the dehI gene was carried on a
mobile genetic element and gave it the general designation DEH, since it was found
to vary in size following transposition into different plasmid targets. Thomas et al.
(1992a) identified a hot spot for insertion of DEH into the TOL plasmid pWW0
(Worsey et al., 1975) and one such DEH element insertion was cloned and
characterised (Thomas et al., 1992b) following its transposition from the
39
Pseudomonas putida PP3 genome to pWW0 and conjugal transfer of pWW0::DEH
to another strain of P. putida. Thus, the DEH element was shown to carry the dehI
gene immediately adjacent to dehRI, which encoded a
54
-dependent activator
(Thomas et al., 1992b; Topping et al., 1995).
1.10
Role of Transport Protein in Transporting Halogenated Compound
Natural organisms capable of metabolising a wide range of haloaromatic and
aliphatic substances have been isolated, and ultimately these are important factors in
determining the fate of halogenated substances in the environment. Inside the cell,
the carbon-halogen bond in halogenated substances is cleaved and this is essential
for yielding products that may be readily metabolised by the organism (Hardman
and Slater, 1981). Nevertheless, in order for this event to take place, the halogenic
compounds must first be translocated into the cell.
Active uptake of halogenated carboxylic acids has been observed in
Pseudomonas putida PP3 (Slater et al., 1985), which utilises halogenated alkanoic
acids such as monochloroacetate as its sole carbon and energy sources.
Pseudomonas putida PP3 evolved the ability to utilise halogenated alkanoic acids as
the consequence of an event that occurred during chemostat selection with the 2,2dichloropropionate as the growth substrate (Senior et al., 1976). This event led to
the inducible expression of two dehalogenases and associated membrane
transporters that permitted growth on several halogenated alkanoic acids. It is
interesting to note that none of these proteins were used by its parental strain P.
putida PPl. (Senior et al., 1976; Slater et al. 1979; Weightman et al., 1979, 1982).
Subsequent analysis showed that two permease proteins were discovered
within transposable elements in Pseudomonas putida PP3 (Slater et al., 1985).
These permeases were found closely associated with the dehalogenases, and
40
contributed an essential role of enabling strain PP3 grow on D,L-2-chloropropionate
compounds. Additionally, the permeases were reported to have broad specificity in
transporting both metabolisable substrates and the toxic analog MCA in strain PP3
(Slater et al., 1985).
Similar to Pseudomonas putida PP3, Xanthobacter autotrophicus GJ10 is a
microorganism capable of dehalogenating haloalkanoic acids. It was noted that the
growth rate of strain GJ10 with monochloroacetate as a substrate was poor
compared to that with 2-chloropropionate (Janssen et al., 1985), despite the fact that
haloalkanoic acid dehalogenase activity was sufficient and that monochloroacetate
was proven non-toxic to the cell (van der Ploeg and Janssen, 1995). Therefore, the
suggestion was that the reduction in growth rate could be caused by the transport
protein of strain G10 having a difference affinity for the substrates being used.
In addition, van der Ploeg and Janssen (1995) also reported that the DNA
sequence upstream of the dhlB gene encoding the haloalkanoic acid dehalogenase of
Xanthobacter autotropicus GJ10 contained an open reading frame (ORF),
designated dhlC. The ORF was subjected to database sequence comparison and
encoded for a protein with high similarity to the family of Na+-dependent symport
proteins. This suggests that dhlC encodes a protein with an uptake function and
further analysis proposed that it is specifically involved in growth with haloalkanoic
acids.
1.10.1 The Presence of a Putative Rhizobial dehrP Gene in pSC1
Escherichia coli K-12 strain NM522 transformed with plasmid pSC1 was
endowed with the novel ability to grow at the expense of 2-chloropropionic acid.
However, Escherichia coli without the plasmid was unable to grow on 2chloropropionic acid (Cairns et al., 1996). Therefore, it was hypothesised that the
41
pSC1 plasmid encodes gene for dehalogenase enzyme and a dehalogenase
associated permease that enable the E.coli to uptake the haloacid into the cell.
1.11
Objectives
I
Isolate and characterise a local novel bacteria strain which able to
degrade 3-chloropropionate.
II
Isolate and characterise a local 2,2-dichloropropionate degrading
bacteria as well.
III
Analysis of a putative haloacid permease gene (dehrP) located
upstream of dehD in pSC1.
42
CHAPTER II
MATERIAL AND METHODS
2.1 Bacteria Strains and Plasmids
Haloacid utilising bacteria were isolated from a soil sample taken from
Universiti Teknologi Malaysia (UTM) agriculture area. Other bacteria strains and
plasmids used in this study are listed in Table 2.1 and Table 2.2, respectively. The
plasmid pET 43.1a was used for general cloning (Figure 2.1). The T7 lac promoterdriven expression vector was designed for cloning and high-level expression of
peptide sequences fused with the 491 amino acid Nus•Tag™ protein. The Nus•Tag
sequence can be removed by double digest using NdeI plus a restriction enzyme
from multiple cloning site (MCS).
43
Figure 2.1:
Restriction map of the cloning vector pET 43.1a
44
45
Table 2.2: Plasmids used in this course of study
Plasmid
Original Vector
Relevant Features
Reference
pSC1
pUC19
amp R, dehD+,
Cairns et al., 1996
dehL
pJS771
pET 43.1a-c
pHJ
T7 promoter vector
T7 promoter vector
T7 promoter vector
+
dehE+, amp R
R
amp ,T7 lac
R
amp ,T7 lac,
dehrP
+
Stringfellow et al.,1997
Novagen
This study
46
2.2
Growth Media
Luria broth (LB) media was as described by Miller (1972) and contained
yeast extract 10 g/l, tryptone 5.0 g/l and NaCl 10 g/l. Ampicillin (Sigma) stock of
100mg/ml was prepared with sterile distilled water and stored as aliquots of 1.5 mL
at -20 °C. The working concentration of ampicillin was 100 μg/ml. IPTG
(Isopropyl β-D-1-thiogalactopyranoside, Promega) stock of 100mM was prepared
with sterile distilled water, filter sterilised and stored in a 0 °C freezer.
PJC chloride-free minimal media was prepared as 10x concentration of basal
salts containing K2HPO4.3H2O (42.5 g/l), NaH2PO4.2H2O (10.0 g/l) and (NH4)2SO4
(25.0 g/l). The trace metal salts solution was a 10x concentrated stock that
contained nitriloacetic acid C6H9NO6 (1.0 g/l), MgSO4 (2.0 g/l), FeSO4.7H2O (120.0
mg/l), MnSO4.4H2O (30.0 mg/l). ZnSO4.H2O (30 mg/l) and CoCl2.6H2O (10 mg/l)
in distilled water (Hareland et al., 1975).
Minimal media for growing bacteria contained 10 ml of 10x basal salts and
10 ml of 10x trace metal salts per 100 ml of distilled water and were autoclaved (121
°C, for 15 minutes at 15 psi). Carbon sources (1M 3-chloropropionate or 1M 2,2dichloropropionate) were sterilised separately and added aseptically to the media to
the desired final concentration. Liquid minimal cultures were supplemented with
yeast extract to a final concentration of 0.05% (w/v). In order to prepare solid
medium, Oxoid bacteriological agar (1.5% w/v) was added prior to sterilisation.
47
2.3
Glycerol Stock Culture
Glycerol stock culture of organisms was prepared by adding 0.3 ml of sterile
50 % glycerol to 0.7 ml of bacterial culture. The sample was then mixed thoroughly
and frozen by standing in dry ice. Stock cultures were stored at -80 °C.
2.4
Measurement of Microbial Growth
Microbial growth was determined by measuring the absorbance at 680nm
(A680 nm). For measurement of A680nm, 2 mL culture sample was pipetted out and
absorbance was taken with Jenway 6300 series spectrophotometer at 680 nm
wavelength. Absorbance readings for the turbidity of the broth mediums were taken
at appropriate intervals. Specific growth rate was determined by plotting a graph of
log A680nm against time of exponential phase. μ was then determined from the slope
of the graph (Stanbury and Whitaker, 1984). Doubling time (td) was calculated by
the equation td = 0.301 / μ (Shuler and Kargi, 2002).
2.5
Plasmid Cloning
E. coli carrying the desired plasmid was inoculated in LB broth (10 ml)
containing ampicillin 100 µg/ml. The culture was grown at 37 °C with agitation for
16 hours and the plasmid was extracted using QIAprep Spin Miniprep Kit (Qiagen)
according to the manufacturer’s instruction.
48
2.6
Preparation of Chromosomal DNA
The genomic DNA was isolated using Wizard® Genomic DNA Purification
Kit (Promega) according to the manufacturer’s instructions. Bacteria culture (1 ml)
in late exponential phase was added into a microcentrifuge tube. Cells were
harvested by centrifugation (13,000-16,000 g, 2 min). The supernatant was then
discarded. Nuclei lysis solution (600 μl) was added and gently pipeted until the cells
were fully resuspended. After that, the solution was incubated at 80°C for 5 minutes
to lyse the cell fully. The solution was then cooled to room temperature.
RNase solution (3 μl) was then added to the lysate. RNAse was used in
order to get rid of the RNA by degrading RNA into tiny fragments. The sample was
incubated for 45 minutes at 37 0C to ensure complete RNA digestion.
After cooling to room temperature, protein precipitation solution (200 μl)
was added to the RNAase treated cell lysate and vortex vigorously for 20 seconds.
The protein precipitation solution contains proteinase K which functioned as a
proteolytic enzyme. The sample was incubated on ice for 5 minutes and centrifuged
at 13,000-16,000 g for 3 minutes.
The supernatant containing the DNA was transferred to a clean 1.5 ml
microcentrifuge tube containing 600 μl isopropanol at room temperature. The
sample was mixed gently and centrifuged at 13,000-16,000 g for 2 minutes. The
supernatant was removed, and 600 μl of 70 % ethanol at room temperature was
added gently. The tube was inverted several times to wash the DNA pellet. Finally,
the tube was centrifuged again at 13,000-16,000 g for 2 minutes and the ethanol was
carefully aspirated.
The DNA pellet was air dried for 15 - 45 minutes. Distilled water (50 μl)
was added into the tube and the DNA was rehydrated by incubating at 65 °C for 1
hour. The DNA solution was stored at -4°C for long term storage and 4 °C for short
term storage.
49
2.7 Measurement of DNA Concentration
DNA concentration was estimated by ultraviolet spectrophotometry. An
A260nm of 1.0 corresponds to 50 μg of double stranded DNA per ml. The DNA
purity was estimated from the A260nm / A280nm = ratio. Ratios of less than 1.8
indicated that the preparation was contaminated with protein.
2.8
Restriction Enzyme Digestion
Digestion with restriction endonucleases was carried out according to the
instructions of the manufacturer using the buffer provided. Enzymes were
purchased from Promega and All England Biolabs. Routine digests of plasmids
included 500 ng- 1 μg of DNA in a total reaction volume of 15 μl. Digestion was
carried out for one to two hours at 37 °C.
2.9
Agarose Gel Electrophoresis
Restriction enzyme digests were analysed using a Mini Ready Sub-Cell GT
Cell (Bio-Rad) agarose gel. Generally, 0.8% agarose gel prepared in TAE buffer
(40 mM Tris-acetate pH 7.6, 1 mM EDTA and ethidium bromide at 0.5 μg/ml) was
used. Samples (between 2 to 5 μl) were mixed with appropriate amount of 6x gel
loading buffer (Fermentas) before loading. Gels were run at a constant 90 volts for
1 to 2 hours.
50
DNA fragments on the gel were visualised using a UV transilluminator. The
sizes were estimated by comparison with a 1 kb ladder (Promega, Invitrogen)
standard DNA marker (1 μg of DNA marker was used each time). The DNA ladder
from Invitrogen could also be used as a means of estimating the amount of DNA
present in a sample as the band at 1636 base pairs makes up 10% (0.1 μg) of total
DNA present in the marker used.
2.10
Isolation of DNA Fragments from Agarose Gels
Fragments generated from restriction digests were extracted from agarose gel
using the Qiagen gel extraction kit. The desired fragments were identified by
illumination with long wavelength UV light and corresponding region of the gel
were excised with a sterile scalpel blade and placed into a 1.5 ml Eppendorf tube.
The DNA purification was carried out according to the instructions of the
manufacturer.
51
2.11
Ligation of DNA
The ligation reaction contained vector and insert at a ratio of 1:3. Quick T4
DNA ligase (New England Biolabs, 1 μl) and supplied buffer was also added into
the ligation reaction. The reaction was carried out in a total volume of 20 μl and
incubated at 25 °C for 25 minutes. The reaction was summarised as below:
Insert : Vector ratio 3:1
1-9 µl
2 x Ligase Buffer
10 µl
Quick T4 DNA Ligase
Sterile deionised water to a final volume of
2.12
1 µl
20 µl
Polymerase Chain Reaction (PCR) for Amplification of DNA
PCR reactions were generally carried out in 50μl reaction using a thermal
cycle (Perkin Elmer GeneAmp PCR System 9700). The components in the PCR
reaction were:
2X PCR master mix (Promega)
25.0 μl
Forward primer (20 pmol/μl)
4.0 μl
Reverse primer (20 pmol/ul)
1.0 μl
DNA template
variable ( e.g > 0.5 μg)
Sterile distilled water to final volume of
50 μl
Exact parameters for PCR cycle profiles were decided to base on a number
of factors including the estimated length of the final product and the prediction of
annealing temperature for PCR primers. After completion the reaction mixture was
electrophoresed on 0.8% agarose gel. PCR products were purified using QIAquick
PCR purification kit (Qiagen) according to the manufacturer’s instructions.
52
2.12.1 PCR Amplification of 16S rRNA Gene for Bacteria Identification
The primers used to amplify 16S rRNA were forward primer, FD1 (5’-aga
gtt tga tcc tgg ctc ag-3’) and reverse primer, rP1 (5’-acg gtc ata cct tgt tac gac tt-3’).
The program used for amplification of 16S rRNA gene was shown in Table 2.3.
2.12.2 PCR Amplification of Putative dehrP Gene for Cloning
Primers specific to putative dehrP gene was designed to enable the insertion
of an NdeI site at the start of the putative dehrP gene and an EcoRI site at the end of
the gene. The program used for amplification of putative dehrP gene was shown in
Table 2.4.
PCR primer with an NdeI site that
5’ GGA ACA CCA TAT GAC TAC GAC
includes the atg initiation codon (in
TCT AG
3'
bold) at the start of the gene
PCR primer to include EcoRI site (in
5’ GGG AAT TCA AAT CAA AGG CAT
bold) after the end of the dehrP gene
GCG TCA TAT
3’
53
Table 2.3: PCR cycle for 16S rRNA gene amplification (25 cycles)
Steps
Temperature (°C)
Time (Min)
Initial Denaturation
94
5
Denaturation
94
1
Annealing
55
1
Extension
74
4
Final Extension
74
10
Table 2.4: PCR cycle for putative dehrP gene amplification (25 cycles)
Steps
Temperature (°C)
Time (s)
Initial Denaturation
94
180
Denaturation
94
30
Annealing
55
30
Extension
72
30
Final Extension
72
480
54
2.13
Preparation of Competent Cells and Transformation of Plasmid DNA
The required strains was grown overnight in 5 ml LB cultures. The next day
100 μl of the overnight culture was subcultured into fresh LB and grown for 2 hours
at 37 °C to A680nm of approximately 0.4. Aliquots of cells (1.5 ml) were harvested by
centrifugation for 30 seconds at 12,000 g and the drained pellets resuspended in 0.5
ml of sterile solution A (10 mM MOPS pH 7, 10 mM RbCl). The cells were then
pelleted with a further centrifugation for 20 seconds at the same speed and
resuspended in 0.5 ml solution B (100 mM MOPS pH 6.5, 10 mM RbCl, 500 mM
CaCl2) and left on ice for 60-90 minutes. Cells were then collected by a 10 second
spin at the same speed and resuspended in 150 μl of solution B with 0.2% v/v
dimethyl sulphoxide. DNA was added (100-200 ng) and the mixture left on ice for 1
hour. The cells were heat-shocked at 55°C for 30 seconds and chilled on ice for 1
minute.
Then 1 ml of prewarmed SOC (2% Bacto tryptone, 0.5% yeast extract, 10
mM NaCl, 2.5 mM KCl, 10 mM MgCl2,10mM MgSO4, 20 mM glucose) medium
was added. The cells were then allowed to recover at 37°C for 1 hour then spread
(100 μl) on a prewarmed LB/amp plate.
2.14
Screening for Recombinants
The LB/amp plates were screened for transformants. Single, white and
distinct colonies were picked and restreaked onto fresh LB/amp plates to avoid
satellite colonies. E.coli cells were grown at 37 °C in Luria Broth supplemented
with 100 μg of ampicillin per 100 ml culture. Plasmids were then harvested and
restriction analysis was carried out to determine the restriction pattern. Alternatively,
PCR amplification was used to detect the presence of the inserted gene.
55
2.15
DNA Sequencing and Oligodeoxyribonucleotide Synthesis
Sequencing was performed by the 1st Base Laboratory, Malaysia. Initial
sequencing of both strands was carried out by using ABI PRISM ® 377 DNA
sequencer by employing forward and reverse PCR primers. Sequences were
extended by designing downstream primers based on the available determined
sequence. These oligodeoxyribonucleotides were synthesised by 1st Base
Laboratory, Malaysia.
2.16
Differential Staining for Bacterial
2.16.1 Gram Staining
A bacterial suspension was first heat-fixed on a glass slide. The smear was
then flooded with crystal violet for 1 minute before washing off the excess stain.
Following this, the smear was covered with Gram’s iodine for 90 seconds and
washed. Decolourisation was carried out by adding few drops of 95% ethyl alcohol
for few seconds and the action of alcohol was stopped by rinsing the slide with water.
Lastly, the smear was counterstained with safranin for 30 seconds, washed and airdried before examination under oil immersion.
56
2.16.2 Acid-Fast Staining (Ziehl-Neelsen Method)
The heat-fixed slides were suspended over boiling water and flooded with
carbol fuchsin for 5 minutes. The stain was replenished upon evaporation. Then,
the slides were cooled and decolourised with acid-alcohol. Decolourisation was
stopped by rinsing with distilled water. Next, the smears were counterstained with
methylene blue for 30 seconds and the excess stain was rinsed off with distilled
water. The slides were air-dried and examined under oil immersion. Acid fast
organisms will retain the red colour of carbol fuchsin.
2.16.3 Spore Staining
The heat fixed slide was flooded with malachite green and placed onto warm
hot plate for 2 to 3 minutes. The stain was replenished as needed due to
evaporation. The slide was then cooled before rinsing with distilled water.
Subsequently, smear was counterstained with safranin for 30 seconds and rinsed
again with distilled water. Finally, the slide was blotted dry with Whatman filter
paper and examined under oil immersion. Spore formers will retain the malachite
green colours.
57
2.17
Biochemical Tests
Results and observations of biochemical tests were compared with Bergey’s
Manual of Systematic Bacteriology (Holt et al., 1994) in order to identify the
bacterium.
2.17.1 Oxidase Test
Tetramethyl-ρ-phenylenediaminedihydrochloride (1 %) was dotted on filter
paper. Then, a single colony was streaked on the dot and observed for the colour
changes. An instant formation of blue colour displayed a positive result and
negative result indicated by no colour changes.
2.17.2 Catalase Test
A fresh colony was placed on a microscopic slide. A few drops of 3 % H2O2
was flow slowly on the colony and observed for bubbling reaction. An immediate
bubbling reaction indicated catalase positive result while negative result was
indicated by no bubbling reaction.
58
2.17.3 Citrate Utilisation Test
A single colony from a culture was streaked on Simmon Citrate agar plate
and incubated at 37 °C for 48 hours and observed for change in colour after 2 days.
Growth with an intense blue colour indicated a positive result while negative result
was indicated by no growth and/or no colour changes on the plate.
2.17.4 Gelatine Liquefaction Test
Inoculums were stabbed into nutrient broth with 12 % gelatine to a depth of
1 inch. The tube was then incubated at 25 °C for several days. The medium was
then cooled in fridge for 30 minutes to determine the digestion of gelatine.
Observation of gel formation was done after cooling the medium. Liquefaction of
the medium indicates positive result while negative result was indicated by no
change in the medium.
2.17.5 Lactose Utilisation Test
A single colony was streaked on MacConkey agar plate and incubated
overnight at 37 °C. Growth and colour changes were scrutinised. Pinkish colonies
indicate positive result while no growth or whitish colonies indicate negative result.
59
2.17.6 Motility Test
A single colony was stabbed into the centre of the motility test medium to a
depth of 1 inch and incubated at 37 °C for 18 hours. Motile bacteria readily migrate
from the stab line and cause cloudiness. Non-motile bactera will grow along stab
line only while the surrounding medium remains clear.
2.18
HPLC Analysis of Growth Medium
Samples of growth medium were analysed using HPLC in order to monitor
the disappearance of 3-chloropropionate and 2,2-dichloropropionate. Samples were
filtered through nitrocellulose 0.2 μm filters (Sartorious) to remove bacteria cells
and particles which could damage the HPLC equipment.
Samples were separated using an isocratic elution with a mobile phase
containing potassium sulphate (20 mM): acetonitile (60:40) in deionised water.
Samples were detected with a UV detector equipped with a Supelco C-18 column
(250 × 4.6 mm, particle size of 5μm) using a flow rate of 2 ml/min.
60
2.19
Preparation of Cell Free Extracts
Cell-free extracts were prepared from bacterial cells in mid- to late
exponential phase of growth. Cells from 100 ml culture were harvested by
centrifugation at 10,000 g for 10 minutes at 4°C. The cell pellets were resuspended
in 20 ml of 0.1M Tris-acetate buffer pH 7.6 and centrifuged at 10,000 g for 10
minutes at 4 °C. Finally, the cells were resuspended in 4 ml of 0.1 M Tris-acetate
buffer pH 7.6 and maintained at 0 °C for ultrasonication in a Vibra CellsTM
ultrasonicator (Sonics & Materials Inc) operating at 10 % amplitude for 3 minutes.
Unbroken cells and cell wall material were removed by centrifugation at 20,000g for
15 minutes at 4°C.
2.20 Measurement of Protein Concentration
The concentration of protein was measured using Bradford assay (Bradford,
1976). Bradford reagent was prepared by dissolving Coomassie Blue G-25 (25 mg)
in 12.5 ml of ethanol. Phosphoric acid (25 ml) was added and then the volume made
up to 250 ml with distilled water. The solution was filtered through three layers of 3
mm paper (Whatman). Samples were made up to 100 μl and added to 1000 μl of
Bradford reagent, colour was developed for 5 minutes and then the absorbance at
595 nm was measured against a blank. A standard curve over the range 0-30 mg
bovine serum albumin (BSA) was used (Appendix K).
61
2.21 Assay for Dehalogenase Activity
Dehalogenase activity was determined as total chloride released at 30 °C in
an incubation mixture containing:
1.
0.1 M Tris-acetate buffer (pH 7.6)
9.5 ml
2.
0.1 M halogenated aliphatic acid
0.1 ml
3.
Distilled water and enzyme to a
final volume of
10.0 ml
After 5 minutes equilibration at 30 °C, the reaction was initiated by adding
cell-free extract. Samples (1.0 ml) were removed at appropriate intervals and
assayed for halide ions.
2.22
Assay for Halide Ion
Measurement of free halide released during the dehalogenation reaction was
carried out by an adaptation of the method of Bergman and Sanik (1957). Sample (1
ml) was added into 100 μl of 0.25 M ammonium ferric sulphate in 9 M nitric acid
and mixed thoroughly. Mercuric thiocyanate-saturated ethanol (100 μl) was then
added and the solution was mixed by vortexing. The colour was allowed to develop
for 10 minutes and measured at A460nm in a Jenway 6300 series spectrophotometer.
Halide concentration was determined by comparison of the absorbance of the test
sample against a standard curve of known concentration of halide.
62
2.23
Standard Curve for Chloride Ions
A standard curve for chloride ion was constructed using sodium chloride
within the range of 0-0.5 μmol. Standard was known concentration of sodium
chloride in 100 mM Tris-acetate buffer pH 7.6 with colour developed as described in
section 2.24. The standard curve is shown in Appendix L.
2.24
Technique for Protein Analysis
SDS polyacrylamide gel electrophoresis (SDS-PAGE) was carried out based
on protocol of Laemmli (1970). SDS-PAGE mini-gels were prepared using the
mini-Protean II kit (Biorad). The composition formulation for mini-gel is described
in Table 2.5.
Glass plates and spacers were assembled in the casting system and the
separating gel was poured, covered with a layer of water saturated with butanol (use
the top layer), and allowed to set for one hour. The overlay solution was then
removed and the stacking gel was poured. A comb was inserted without creating air
bubbles, to form the sample wells and the gel was allowed to set for 30 minutes.
63
Table 2.5: Preparation of acrylamide gel for SDS-PAGE
____________________________________________________________________
Resolving Gel
Stacking Gel
12 % acrylamide
4 % acrylamide
____________________________________________________________________
Distilled water
4.35 ml
1.5 M Tris.Cl pH 8.8
2.50 ml
0.5 M Tris.Cl pH 6.8
3.15 ml
-
-
1.26 ml
10 % SDS
100 μl
50 μl
40 % Acrylamide/Bisacrylamide
3.00 ml
0.50 ml
10 % (NH4)2S2O8
50 μl
25 μl
Tetramethyl-ethylenediamine
10 μl
10 μl
(TEMED)
___________________________________________________________________
The mini-gel was placed in an electrophoresis tank and the electrodes were
covered with 1x Tris-glycine running buffer solution (5x stock: 1.5 % [w/v] Tris
base, 7.2 % [w/v] glycine and 0.5 % [w/v] SDS).
Samples were prepared by heating an appropriate amount of protein for 3
minutes at 100 °C with 0.3 volume of sample buffer (50 mM Tris-HCL, pH 6.8, 10
% [w/v] glycerol, 2 % [w/v] SDS, 5 % [v/v] 2-β-mercaptoethanol, 0.05 % [w/v]
bromophenol blue).
Gels were generally run for 2 hours at a constant voltage of 150 V. The
protein bands were stained with Coomassie blue R250 solution (comprising 0.5%
[w/v] Coomasie blue R250 in 45% [v/v] methanol; 10% acetic acid) for 2-3 hours.
Gels were then destained in a solution of 7.5% [v/v] acetic acid; 5% [v/v] methanol
for 3-16 hours. Gels were calibrated using Fermentas Protein Molecular Weight
Marker (Figure 2.4)
64
2.25
Computer Analysis
Sequence analysis for 16S rRNA gene was carried out using GeneRunner
(Hastings Software Inc., Hastings on Hudson, NY). International databases were
searched using the BLASTn program (Altschul et al., 1997). Gene characterisation
of putative dehrP gene was carried out using AnnHyb 4.0. Nucleotide and deduced
protein analysis was carried out using BLASTn and BLASTx
(www.ncbi.nlm.nih.gov/blast). Prediction of protein physio-chemical properties was
carried out using ProtParam Tool (http://kr.expasy.org/cgi-bin/protparam).
Hydrophobic character was analysed using TMHMM maintained by Centre for
Biological Analysis (www.cbs.dtu.dk/services/) and finally the putative conserve
domain was searched using CD server (www.ncbi.nlm.nih.gov). Summary of the
programs used in this course of study was listed in Table 2.6.
Table 2.6: Computing utilities used in this study
Programs
Utility
AnnHyb
Restriction mapping,
Protein Composite Analysis
GeneRunner
Primer design and analysis
BLAST
Multiple sequence alignment for protein
function comparison
TMHMM
Hydrophobic character prediction
PortParam
Protein physio-chemical properties prediction
CD server
Putative conserve domain analysis
65
CHAPTER III
ISOLATION AND CHARACTERISATION OF
3-CHLOROPROPIONATE DEGRADING BACTERIUM
3.1
Introduction
The 3-chloropropionic acid is classified as chlorinated monocarboxylic acids
or β-chloro substituted haloalkanoates. This compound can be considered as a
possible chemical inclusion in certain herbicides and is carcinogenic and genotoxic
to the animal and human (Alexander 1981). The chemical structure was shown in
Figure 3.1.
Many soil microorganisms were capable of utilising halogen-substituted
organic acids as their sole sources of carbon and energy (Jensen 1963; Bollag and
Alexander 1970; Hardman and Slater 1981a; Schwarze et al., 1997; Olaniran et al.,
2001). Halogen-substituted organic acids were poorly degraded. Very few
literatures have reported the degradation of β-chloro substituted haloalkanoates such
as 3-chloropropionate. This study was hence considered important since
degradation of 3-chloropropionate was poorly understood compared to well studied
α-chloro substituted haloalkanoates such as 2,2-dichloropropionate and 2chloropropionate. In addition, further interest was generated by this subject, when it
became apparent that α-chloroalkanoate degrading microorganisms were unable to
dechlorinate the β-substituded haloalkanoates, which differed only in chloride
substitution.
66
O
OH
C
H
C
Cl
C
α
ß
H
H
H
Figure 3.1: Structure of 3-chloropropionic acid
Hydrolytic dehalogenation represents key position in the degradation of
haloaliphatic compounds such as 3-chloropropionate. Until now, only few
microorganisms were reported as capable of dehalogenating 3-chloropropionate and
utilising it as their sole carbon source. Among those reported were Micrococcus
denitrificans (Bollag and Alexander, 1971), Clostridium kluyveri (Hashimoto and
Simon, 1975), Corynebacterium sp. (Yokota et al., 1986) and a variety of soil
microorganism including P.pickettii strain SH1 (Hughes, 1988). However,
dehalogenases from these organisms were not fully characterised and the pathway
was not elucidated. For that particular reason, it was interesting to study a novel
dehalogenase enzyme which can degrade 3-chloropropionate.
In the current study, a batch culture method was used for the enrichment and
selection of microorganism which can utilise 3-chloropropionate as the sole source
of carbon and energy. The 3-chloropropionate utilising bacterium was then
identified using 16S rRNA gene sequencing. The ability of the bacterium in
degrading 3-chloropropionate was further confirmed by HPLC analysis and enzyme
assay using crude cell free extract.
67
3.2
Results
3.2.1 Isolation of 3-Chloropropionate Degrading Bacteria
A mixed culture from UTM plantation soil sample was streaked onto 20 mM
3-chloropropionate minimal medium. Several morphologically different colonies
were observed. Colonies formed were repeatedly streaked on the same type of
medium. Only one of them grew well in 3-chloropropionate liquid minimal medium
and picked for further analysis. This bacterium was designated as bacterium A.
3.2.2 Identification of Bacterium A by 16S rRNA Gene Sequencing
3.2.2.1 DNA Analysia of Bacterium A
Genomic DNA from bacterium A was prepared using Wizard genomic DNA
kit (Promega) as in Figure 3.2. PCR reaction was carried out using the prepared
genomic DNA as a template for 16S rRNA gene amplification. The fragment
amplified was approximately 1.6 kb as in Figure 3.3. The PCR condition generated a
single fragment.
68
Lane
1
2
kb
Figure 3.2 : Agarose gel electrophoresis of undigested genomic DNA
Lane 1: Promega 1 kb DNA ladder
Lane 2: Genomic DNA (0.25 μg/μl) prepared from bacterium A
69
Lane
1
Figure 3.3:
2
3
4
5
6
kb
The PCR amplified 16S rRNA gene fragment
Lane 1: Negative control without DNA template
Lane 2: Positive control using E.coli genomic DNA as template
Lane 3: The amplified 16S rRNA DNA fragment
Lane 4: Negative control without FD1 primer
Lane 5: Negative control without rP1 primer
Lane 6: Fermentas MassRuler™ DNA Ladder, High Range
70
3.2.2.2 Sequencing of the 16S rRNA Gene
The 16S rRNA gene fragment was sequenced using primers FD1 and rP1.
Figure 3.4 showed the partial nucleotide sequence of 16S rRNA gene from the
isolated bacterium. The sequence comprised of 1437 nucleotides lacking the very
proximal 5’ and terminal 3’ regions corresponding to the universal primers used.
This sequence was submitted to the GenBank with accession number of AM 231909
(Appendix A).
The 16S rRNA gene sequence was compared to the sequences in the
GenBank database comprises of NCBI (U.S. National Center for Biotechnology
Information), EMBL (the European Molecular Biology Laboratory) and DDBJ (the
DNA Data Bank of Japan) using BLASTn. The result revealed that the 3chloropropionate degrading bacterium has a 99% identity match (e-value = 0) with
Rhodococcus sp. (Figure 3.5) and it was further designated as Rhodococcus sp.
strain HN2006A. Moreno (1996) suggested that a prokaryote’s 16S rRNA
sequence with less than 97% identity to any other sequence should be considered as
a new species. However, this was not the case in the current experiment.
71
1 atgcaagtcg aacgatgaag cccagcttgc tgggtggatt agtggcgaac gggtgagtaa
61 cacgtgggtg atctgccctg cactctggga taagcctggg aaactgggtc taataccgga
121 tatgacctcg ggatgcatgt ccaggggtgg aaagtttttc ggtgcaggat gagcccgcgg
181 cctatcagct tgttggtggg gtaatggcct accaaggcga cgacgggtag ccggcctgag
241 agggcgaccg gccacactgg gactgagaca cggcccagac tcctacggga ggcagcagtg
301 gggaatattg cacaatgggc gcaagcctga tgcagcgacg ccgcgtgagg gatgacggcc
361 ttcgggttgt aaacctcttt cacccatgac gaagcgcaag tgacggtagt gggagaagaa
421 gcaccggcca actacgtgcc agcagccgcg gtaatacgta gggtgcgagc gttgtccgga
481 attactgggc gtaaagagct cgtaggcggt ttgtcgcgtc gtctgtgaaa tcccgcagct
541 caactgcggg cttgcaggcg atacgggcag actcgagtac tgcaggggag actggaattc
601 ctggtgtagc ggtgaaatgc gcagatatca ggaggaacac cggtggcgaa ggcgggtctc
661 tgggcagtaa ctgacgctga ggagcgaaag cgtgggtagc gaacaggatt agataccctg
721 gtagtccacg ccgtaaacgg tgggcgctag gtgtgggttt ccttccacgg gatccgtgcc
781 gtagccaacg cattaagcgc cccgcctggg gagtacggcc gcaaggctaa aactcaaagg
841 aattgacggg ggcccgcaca agcggcggag catgtggatt aattcgatgc aacgcgaaga
901 accttacctg ggtttgacat gtaccggacg actgcagaga tgtggttccc cttgtggccg
961 gtagacaggt ggtgcatggc tgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg
1021 caacgagcgc aacccttgtc ctgtgttgcc agcacgtgat ggtggggact cgcaggagac
1081 tgccggggtc aactcggagg aaggtgggga cgacgtcaag tcatcatgcc ccttatgtcc
1141 agggcttcac acatgctaca atggtcggta cagagggctg cgataccgtg aggtggagcg
1201 aatcccttaa agccggtctc agttcggatc ggggtctgca actcgacccc gtgaagtcgg
1261 agtcgctagt aatcgcagat cagcaacgct gcggtgaata cgttcccggg ccttgtacac
1321 accgcccgtc acgtcatgaa agtcggtaac acccgaagcc ggtggcctaa ccccttgtgg
1381 gagggagccg tcgaaggtgg gatcggcgat tgggacgaag tcgtaacaag gtagccg
Figure 3.4:
Bacterium A 16S rRNA partial sequence lacking the very
proximal 5’ and terminal 3’ regions (AM 231909)
72
Bacterium A
ATGCAAGTCGAACGATGAAGCCCAGCTTGCTGGGTGGATTAGTGGCGAACGGGTGAGTAA
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
ATGCAAGTCGAACGATGAAGCCCAGCTTGCTGGGTGGATTAGTGGCGAACGGGTGAGTAA
60
CACGTGGGTGATCTGCCCTGCACTCTGGGATAAGCCTGGGAAACTGGGTCTAATACCGGA
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CACGTGGGTGATCTGCCCTGCACTCTGGGATAAGCCTGGGAAACTGGGTCTAATACCGGA
120
TATGACCTCGGGATGCATGTCCAGGGGTGGAAAGTTTTTCGGTGCAGGATGAGCCCGCGG
|||||||||||||||||||||| |||||||||||||||||||||||||||||||||||||
TATGACCTCGGGATGCATGTCCTGGGGTGGAAAGTTTTTCGGTGCAGGATGAGCCCGCGG
180
CCTATCAGCTTGTTGGTGGGGTAATGGCCTACCAAGGCGACGACGGGTAGCCGGCCTGAG
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CCTATCAGCTTGTTGGTGGGGTAATGGCCTACCAAGGCGACGACGGGTAGCCGGCCTGAG
240
AGGGCGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTG
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
AGGGCGACCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTG
300
GGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCC
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
GGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGAGGGATGACGGCC
360
TTCGGGTTGTAAACCTCTTTCACCCATGACGAAGCGCAAGTGACGGTAGTGGGAGAAGAA
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
TTCGGGTTGTAAACCTCTTTCACCCATGACGAAGCGCAAGTGACGGTAGTGGGAGAAGAA
420
GCACCGGCCAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTGTCCGGA
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
GCACCGGCCAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTGTCCGGA
480
ATTACTGGGCGTAAAGAGCTCGTAGGCGGTTTGTCGCGTCGTCTGTGAAATCCCGCAGCT
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
ATTACTGGGCGTAAAGAGCTCGTAGGCGGTTTGTCGCGTCGTCTGTGAAATCCCGCAGCT
540
CAACTGCGGGCTTGCAGGCGATACGGGCAGACTCGAGTACTGCAGGGGAGACTGGAATTC
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CAACTGCGGGCTTGCAGGCGATACGGGCAGACTCGAGTACTGCAGGGGAGACTGGAATTC
600
CTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGGTCTC
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CTGGTGTAGCGGTGAAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGGTCTC
660
TGGGCAGTAACTGACGCTGAGGAGCGAAAGCGTGGGTAGCGAACAGGATTAGATACCCTG
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
TGGGCAGTAACTGACGCTGAGGAGCGAAAGCGTGGGTAGCGAACAGGATTAGATACCCTG
720
GTAGTCCACGCCGTAAACGGTGGGCGCTAGGTGTGGGTTTCCTTCCACGGGATCCGTGCC
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
GTAGTCCACGCCGTAAACGGTGGGCGCTAGGTGTGGGTTTCCTTCCACGGGATCCGTGCC
780
GTAGCCAACGCATTAAGCGCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGG
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
GTAGCCAACGCATTAAGCGCCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGG
840
AATTGACGGGGGCCCGCACAAGCGGCGGAGCATGTGGATTAATTCGATGCAACGCGAAGA
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
AATTGACGGGGGCCCGCACAAGCGGCGGAGCATGTGGATTAATTCGATGCAACGCGAAGA
900
ACCTTACCTGGGTTTGACATGTACCGGACGACTGCAGAGATGTGGTTCCCCTTGTGGCCG
||||||||||||||||||||||||||||||||||||||||||||||| ||||||||||||
ACCTTACCTGGGTTTGACATGTACCGGACGACTGCAGAGATGTGGTTTCCCTTGTGGCCG
960
1020
Rhodoccus
988
GTAGACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCG
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
GTAGACAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCG
Bacterium A
1021 CAACGAGCGCAACCCTTGTCCTGTGTTGCCAGCACGTGATGGTGGGGACTCGCAGGAGAC
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1048 CAACGAGCGCAACCCTTGTCCTGTGTTGCCAGCACGTGATGGTGGGGACTCGCAGGAGAC
1080
Rhodoccus
Bacterium A
Rhodoccus
Bacterium A
Rhodoccus
Bacterium A
Rhodoccus
Bacterium A
Rhodoccus
Bacterium A
Rhodoccus
Bacterium A
Rhodoccus
Bacterium A
Rhodoccus
Bacterium A
Rhodoccus
Bacterium A
Rhodoccus
Bacterium A
Rhodoccus
Bacterium A
Rhodoccus
Bacterium A
Rhodoccus
Bacterium A
Rhodoccus
Bacterium A
Rhodoccus
Bacterium A
Rhodoccus
Bacterium A
Rhodoccus
1
28
61
88
121
148
181
208
241
268
301
328
361
388
421
448
481
508
54
568
601
628
661
688
721
748
781
808
841
868
901
928
961
87
147
207
267
327
387
447
507
567
627
687
747
807
867
927
987
1047
1107
73
Bacterium A
Rhodoccus
Bacterium A
Rhodoccus
Bacterium A
Rhodoccus
Bacterium A
Rhodoccus
Bacterium A
Rhodoccus
Bacterium A
Rhodoccus
1081 TGCCGGGGTCAACTCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGCCCCTTATGTCC
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1108 TGCCGGGGTCAACTCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGCCCCTTATGTCC
1140
1141 AGGGCTTCACACATGCTACAATGGTCGGTACAGAGGGCTGCGATACCGTGAGGTGGAGCG
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1168 AGGGCTTCACACATGCTACAATGGTCGGTACAGAGGGCTGCGATACCGTGAGGTGGAGCG
1200
1201 AATCCCTTAAAGCCGGTCTCAGTTCGGATCGGGGTCTGCAACTCGACCCCGTGAAGTCGG
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1228 AATCCCTTAAAGCCGGTCTCAGTTCGGATCGGGGTCTGCAACTCGACCCCGTGAAGTCGG
1260
1261 AGTCGCTAGTAATCGCAGATCAGCAACGCTGCGGTGAATACGTTCCCGGGCCTTGTACAC
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1288 AGTCGCTAGTAATCGCAGATCAGCAACGCTGCGGTGAATACGTTCCCGGGCCTTGTACAC
1320
1321 ACCGCCCGTCACGTCATGAAAGTCGGTAACACCCGAAGCCGGTGGCCTAACCCCTTGTGG
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1348 ACCGCCCGTCACGTCATGAAAGTCGGTAACACCCGAAGCCGGTGGCCTAACCCCTTGTGG
1380
1381 GAGGGAGCCGTCGAAGGTGGGATCGGCGATTGGGACGAAGTCGTAACAAGGTAGCCG
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1408 GAGGGAGCCGTCGAAGGTGGGATCGGCGATTGGGACGAAGTCGTAACAAGGTAGCCG
1167
1227
1287
1347
1407
1437
1464
Figure 3.5: Sequence comparison of bacterium A 16S ribosomal RNA gene
(Percent identity = 99 %; e-value = 0)
Table 3.1: Top 5 entries in the database that shows highest identity to bacterium
A
Bacteria
Sequence Identity
Rhodococcus sp. MCEPFL2
99%
Rhodococcus pyridinivorans
99%
Rhodococcus sp. MOP100
99%
Rhodococcus sp. T104
99%
Rhodococcus rhodochrous
99%
74
3.2.3 Bacteria Morphology, Staining and Biochemical Characterisation
Identification using 16S rRNA gene sequence was supported by the
morphological and biochemical analysis. Colonies of Bacterium A are observed as
rough surface, a smooth margin and a raised elevation (Table 3.1). It also formed
milky-orange colonies on nutrient agar and 3-chloropropionate-containing medium.
Bacterium A was gram-positive rod in chains. The cells were acid-fast and no
spores were demonstrated by malachite green staining. Bacterium A also
demonstrated the ability to use lactose, gelatin liquefaction, producing catalase and
grew on citrate. However, the isolates could not produce oxidase and was nonmotile (Table 3.2).
The biochemical test results were compared to Bergey’s Manual of
Systematic Bacteriology (Holt et al., 1994). The results supported the 16S rRNA
analysis as Rhodococcus sp.
75
Table 3.2: Colony characteristic on 3-chloropropionate minimal medium
Characteristics
Observation
Size of colony
Small (10-15 mm)
Pigmentation
Milky-orange pigment
Form (Shape of the colony)
Smooth Circular
Margin (Outer edge of colony)
Entire (Sharply defined, even)
Elevation
Raised (Slightly elevated)
Table 3.3: Morphological and biochemical characteristics of Rhodococcus sp.
Biochemical test and staining
Result
Gram Stain
Gram Positive (Dark Blue)
Acid Fast
Positive (Blue)
Spore Stain
No Spore
Grow Behavior
Aerobic
Oxidase
Negative
Catalase
Positive
Citrate
Positive
Gelatin Liquefaction
Positive
Lactose Utilisation
Positive
Motility
Negative
76
3.2.4 Growth of Rhodococcus sp. HN2006A in 3-Cloropropionate Minimal
Medium
Rhodococcus sp. HN2006A was inoculated into 100 ml PJC minimal liquid
medium containing 5 mM, 10 mM, 20 mM and 40 mM 3-chloropropionate as the
sole source of carbon, respectively. The flasks were incubated at 30°C in a rotary
incubator at 180 rpm. The maximum growth was achieved in 20mM 3chloropropionate minimal medium. Figure 3.6 showed the growth patterns of
bacteria in different substrate concentration. No growth was observed at 40 mM 3chloropropionate suggesting that the substrate was toxic to the cell at higher
concentration. The cell doubling time was calculated by applying data acquired
from triplicate reading (Appendix B) into the semi log graph as summarised in Table
3.4.
77
Growth curves on 5, 10, 20 and 40 mM 3-chloropropionate
Figure 3.6 :
for Rhodococcus sp. HN2006A
Table 3.4: Summary of growth properties of Rhodococcus sp. HN2006A in
different 3- chloropropionate concentration
3-chloropropionate
concentration (mM)
Maximum Medium
Turbidity (A680nm)
Mean doubling time
(H)
5
0.518±0.002
14.35±0.19
10
0.914±0.013
12.12±0.28
20
1.388±0.002
11.72±0.26
40
NG*
NG*
*NG: no growth
78
3.2.5 Growth of Rhodococcus sp. HN2006A in Other Halogenated Compounds
The ability of Rhodococcus sp. HN2006A to utilise a variety of halogenated
carbon source was studied. The interest in investigation of Rhodococcus sp.
HN2006A dehalogenase’s affinity towards other halogenated compound became
apparent when many dehalogenase isolated had broad substrate specificity. For
instance, 2-halosubstituent degrading organism such as Rhizobium sp. was able to
degrade D,L-2-cloropropionate, 2,2-dichloropropionate (2,2-DCP),
monochloroacetate , dichloroacetate, and trichloroacetate but unable to dechlorinate
carbon no. 3 of 3-haloalkanoate such as 3-chloropropionate which differed only in
chlorine substitution position.
Figure 3.7 showed the growth curve of Rhodococcus sp. HN2006A when
grown on different halogenated substrate. 3-bromopropionate was the only substrate
utilised by the isolate as sole sources of carbon and energy (Table 3.4). This
experiment suggested that Rhodococcus sp. HN2006A dehalogenase enzyme system
could not act on other substrates except for 3-chloropropionate and 3bromopropionate. Another reason could be the inability of the bacteria to uptake 2chloropropionate, 2-bromopropionate, 2,2-dichloropropionate and 2,3dichloropropionate into the cell.
79
Table 3.5: Growth properties of Rhodococcus sp. HN2006A in different
substrate
Growth Substrate
Maximum Medium
(20mM)
Turbidity (A680nm)
3-chloropropionate
1.388±0.002
2-chloropropionate
NG
NG
2,2-dichloropropionate
NG
NG
2,3-dichlopropionate
NG
NG
3-bromopropionate
0.590±0.002
2-bromopropionate
NG
Mean Doubling time (H)
11.72±0.26
22.27±0.21
NG
*NG : No Growth
Figure 3.7:
Growth curve on 20 mM of various halogenated compound
for Rhodococcus sp. HN2006A
80
3.2.6
HPLC Analysis of Growth Medium
This experiment was carried out to determine the ability of the organism to
utilise 3-chloropropionate using HPLC method. At the end of the experiment, the
amount of 3-chloropropionate left in the growth medium will be measured.
3.2.6.1
3-chloropropionate Calibration Curve
A standard curve was constructed from a series of known concentration of 3chloropropionate within the range of 0 to 20 mM. Table 3.5 shows data used to
construct the calibration curve for 3-chloropropionate whereas Figure 3.8 shows the
calibration curve constructed.
3.2.6.2 Detection of 3-chloropropionate in Growth Medium
Rhodococcus sp. HN2006A was grown in 20 mM 3-chloropropionate
minimal medium. Sample of the growth medium was then analysed using HPLC at
daily intervals to monitor the presence of 3-chloropropionate. The chromatogram
shows the area of the peak corresponding to 3-chloropropionate decreased from 20
mM of 3-chloropropionate at day 0 to 0 mM at day 2. The results were summarised
in Table 3.6.
From the results obtained, it was concluded that 3-chloropropionate was fully
utilised by Rhodococcus sp. HN2006A since there was no traces of 3chloropropionate in the growth medium after 48 hours whereas control of 3chloropropionate minimal medium without bacteria did not show any substrate
decrement. Nevertheless, depletion of 3-chloropropionate coupled with bacteria
81
growth suggesting that 3-chloropropionate was carbon and energy source for
Rhodococcus sp. HN2006A. Figure 3.9 shows the peak of 3-chloropropionate in the
chromatogram from day 0 to day 2.
Table 3.6:
No
Data used to construct the 3-chloropropionate calibration curve
Peak Name
1
3-chloropropionate
1
12.198
Amount
(mM)
5
2
3-chloropropionate
2
22.129
10
3
3-chloropropionate
3
46.048
20
Figure 3.8:
Level
Mean Area
Calibration curve for 3-chloropropionate
82
Comparison between the 3-chloropropionate consumed and turbidity
Table 3.7:
of Rhodococcus growth medium
Day
Amount of
Medium Turbidity
3-chloropropionate left
(A680nm)
0
100 %
0.172
1
87 %
0.446
2
0%
1.363
83
(i)
(ii)
(iii)
Figure 3.9: HPLC elution profile of medium from cells utilising 3-chloropropionate
as source of carbon. Medium was analysed at i) day 0, (ii) day 1 and (iii) day 2
84
3.2.7 Dehalogenase Activity in Cell Free Extracts of 3-chloropropionate
Grown Bacteria
The disappearance of 3-chloropropionate in the growth medium suggested
that the substrate was used as the sole source of carbon. Enzyme assay therefore
was carried out to determine the presence of dehalogenase enzyme in the cell. Cell
free extracts were prepared from bacteria grown on 3-chloropropionate as sole
carbon source and were assayed for dehalogenase activity using 1 mM 3chloropropionate as substrate. Enzyme localisation had been done and dehalogenase
enzyme produced was found to be intracellular enzyme since resuspended cell debris
did not possess any activity towards the substrate. By measuring the rate of chloride
ion released from the substrate, the average specific activity of dehalogenase on
these extracts was found to be 0.013 μmol Cl-/min/mg protein. Details for
calculation of enzyme activity was described in Appendix E.
85
3.3
Discussion
Towner and Cockayne (1993) described identification of variations in
nucleotide sequence would allow the most precise identification and typing of
microorganism. In current investigation, 16S rRNA of 3-chloropropionate
degrading bacteria shared more than 99% to Rhodococcus sp. Therefore, we can
possibly confirmed that the 3-chloropropionate degrading bacteria isolated belongs
to Rhodococcus sp. This result was supported by biochemical test and staining
properties. Rhodococcus sp. are aerobic, Gram positive actinomycetes of high G+C
content and capable of morphological differentiation in response to their
environment (e.g., cocci or filaments). These widely occurring organisms are of
considerable environmental and biotechnological importance due to their broad
metabolic diversity and array of unique enzymatic capabilities.
Rhodococcus sp. are of interest to the pharmaceutical, environmental,
chemical and energy sectors. Specific applications include the desulphurisation of
fossil fuels (Whyte et al., 1998) and the industrial production of acrylamide.
Rhodococcus are well suited for bioremediation due to their capacity for long term
survival in soil, their exceptional ability to degrade hydrophobic and halogenated
pollutants (Maeda et al., 1995; Seto et al., 1995; Fournier et al., 2002; Haroune et al.,
2002) even in the presence of more readily assimilable carbon sources, and their
ability to accumulate high levels of heavy metals. For example, Rhodococcus sp.
was well known in degradation of herbicide thiocarbamate (de Schrijver et al., 1997)
and herbicide S-Ethyl dipropylthiocarbamate (Shao and Behki, 1995).
The ability of Rhodococcus sp. in biodegradation and bioremediation had
been studied at molecular level as well. Their genes and plasmids responsible for
biodegradation was widely studied (Bosma et al., 1995; Kosono et al., 1997; Whyte
et al., 2002). All these showed that Rhodococcus sp. could had great potential in
biodegradation and bioremediation of various xenobiotic including halogenated
compound and were parallel to our result by which Rhodococcus sp. HN 2006A is a
3-chloropropionate degrading bacteria.
86
Growth experiment (Figure 3.7) showed that Rhodococcus sp. HN2006A
was able to grow in 3-chloropropionate minimal media supplied with 5 mM to 20
mM, respectively. However, growth in 40 mM 3-chloropropionate minimal media
was inhibited. This suggested that the concentration of 40 mM 3-chloropropionate
was toxic and could not be tolerated by the bacteria. Growth of the bacterium in the
medium was coupled with the utilisation of 3-chloropropionate as sole carbon source.
This was confirmed by measurement of 3-chloropropionate concentration in the
growth medium using HPLC. As shown in section 3.2.6, the degradation and
utilisation of 3-chloropropionate was initiated from the beginning of the growth.
Table 3.7 clearly showed that when bacteria number (turbidity; A680nm) increased in
the medium, 3-chloropropionate concentration drops gradually. As suggested by
Hughes (1988), growth of microorganism using 3-chloropropionate required cofactor addition such as yeast extract in order to increase growth rates and to achieve
higher 3-chloropropionate catabolism efficiency. However, it’s not in this case since
all 3-chloropropionate was fully utilised by Rhodococcus sp. HN2006A.
Although accurate and precise detection and quantification of 3chloropropionate had been developed as demonstrated by Hymer and Cheever (2004)
using gas chromatography (GC), this project was the first to develop a simple and
effective method to measure the levels of 3-chloropropionate using HPLC. There
were several other ways to follow the utilisation of halogenated compounds by
bacteria. A popular method was to determine the release of halides from
halogenated compounds using a spectrophotometric method modified by Bergman
and Sanik (1957) to estimate the chlorine in naptha. That particular method had
been widely adapted but involves toxic reagents and was more suitable to detect
chloride concentration lower than 1mM. Other methods include colorimetry
titration of halides to detect monochloroacetate as described by Slater et al. (1985)
and microplate fluorimetric assay to determine the biodegradation of 2chloropropionate (Marchesi, 2003). Besides, using bioluminescence method, a
whole-cell bacterial sensor to monitor the amount of 2-chloropropionate higher than
100 mg/l had been developed by Tauber et al. (2001).
87
The ability of Rhodococcus sp. HN2006A to degrade other halogenated
substrates was tested. The Rhodococcus sp. HN2006A was not able to utilise other
than 3-chloropropionate and 3-bromopropionate suggested the specificity of
dehalogenases. For example, dehL and dehD was shown to be specific for 2chloropropionate and do not act on 2,2-dichloropropionate nor 3-chloropropionate
(Cairns et al., 1996) whereas enzyme prepared from Micrococcus denitrificans by
Bollag and Alexander (1970) was shown to be specific to 3-chloropropionate and do
not act on 2-chloropropionate nor 2,2-dichloropropionate. The results from current
findings provide further evidence for the importance of the position of the halogen
substituent in governing the susceptibility of chlorinated aliphatic acids to microbial
attack.
Apart from that the uptake system may play a role in transport of a substrate
into the cell. Rhodococcus sp. HN2006A may lack of a haloacid transport system
for certain halogenated compound to transport the substrate into the cell.
Haloalkanoic acids are negatively charged at physiological pH values, and it is likely
that these substrates need to be transported through the membrane by a carrier
protein. Therefore, dehalogenase associated permease has been proposed to mediate
the uptake of haloacid into the cell. For example, a putative monochloropropionic
acid permease from Rhizobium sp. NHG3 was found to transport D,L2chloropropionate and 2,2-dichloropropionate into the cell (Higgins et al., 2005).
Besides, Xanthobacter autotrophicus GJ10 which able to grow on dichloroethane
and haloalkanoic acids possess a dhlC gene which encodes a protein with significant
similarity to the family of Na+-dependent symport proteins. The similarity to Na+dependent transport proteins suggested that dhlC encodes for a protein that has an
uptake function. Southern blot analysis had shown that a region homologous to
dhlC does not present in X. autotrophicus XD, a strain which could not grew with
haloalkanoic acids (van der Ploeg & Jenssen, 1995).
Bollag and Alexander (1970) had reported a M. denitrificans strain which
was able to degrade 3-chloropropionate with a specific activity of 0.062 μmol Cl/min/mg protein in cell free extracts. In the current study, specific activities of
dehalogenase present in Rhodococcus sp. HN2006A crude cell free extract was only
0.013 μmol Cl-/min/mg protein ( 5 times lower than the M. denitrificans ). If
88
Rhodococcus sp. HN2006A was a slow grower compared to M. denitrificans, then it
was not surprised that specific activity of dehalogenase from Rhodococcus sp.
HN2006A had much lower enzyme specific activity. However, growth rate of
Micrococcus in 3-chloropropionate miminal medium has not been discussed by
Bollag and Alexander (1970).
Nevertheless, dehalogenase from crude extracts of P. pickettii strain SH1
was assayed for its activity by Hughes (1988). A cell doubling time of 8 hours and
dehalogenase activity of 0.05 μmol Cl-/min/mg protein was reported. Lower
dehalogenase activity from Rhodococcus sp. HN2006A in the current study agreed
by the slow growth in 20 mM 3-chloropropionate minimal medium compared to P.
pickettii strain SH1.
89
3.4
Conclusion
A bacterial isolated from soil were found to have good potential in degrading
3-chloropropionate. The organisms were identified as Rhodococcus sp. HN2006A
with 99% identity based on 16S rRNA sequence. It grew well in 5mM, 10mM and
20mM of 3-chloropropionate but growth was inhibited at 40mM of 3chloropropionate minimal medium. The ability of Rhodococcus in degrading 3chloropropionate was confirmed with HPLC analysis. The bacterium also grew in
3-bromopropionate.
Production of dehalogenase enzyme by Rhodococcus sp.
HN2006A was confirmed by enzyme assay. Crude cell free extract of Rhodococcus
showed week activity toward 3-chloropropionate with specific activity of 0.013
μmol Cl-/min/mg protein agreed by the slow growth in 20 mM 3-chloropropionate
minimal medium.
90
CHAPTER IV
ISOLATION AND CHARACTERISATION OF
2,2-DICHLOROPROPIONATE DEGRADING BACTERIA
4.1
Introduction
Halogenated compounds are extensively used as herbicides, insecticides,
fungicides, insulators and lubricants (Chapelle, 1993). Dalapon or 2,2dichloropropionic acid is an example of herbicide and plant growth regulator that
used to control specific annual and perennial grasses like Quackgrass, Bermuda
grass, Johnson grass as well as rushes. It is selective, meaning that it kills only
certain plants, while sparing non-target types of vegetation (Ashton and Crafts,
1973).
Generally, 2,2-dichloropropionic acid does not readily bind or adsorb to soil
particles. Even in muck soil, as little as 20% of applied 2,2-dichloropropionic acid
may be adsorbed. Since it is not adsorb to soil particles, 2,2-dichloropropionic acid
had a high degree of mobility in all soil types and leaching occurs. However, 2,2dichloropropionic acid movement in soil may prevented by rapid breakdown of the
herbicide into naturally-occurring compound by soil microorganisms.
Biodegradation is the main route of 2,2-dichloropropionic acid disappearance from
soil.
91
Figure 4.1: The molecular structure of 2,2-dichloropropionic acid (Dalapon)
Some soil microorganisms utilised halogenated aliphatic compound as sole
sources of carbon and energy and liberate the halogen atoms in the form of ions (Clor Br-). This reaction is called dehalogenation and was catalysed by the
dehalogenase enzyme (Jensen, 1960). 2,2-dichloropropionic acid is readily removed
from the soil by variety of microorganisms by dehalogenation, for example:
Pseudomonas, Agrobacterium, Nocardia, Alcaligens, Arthrobacter, Bacillus sp.
(Jensen, 1957a; Foy, 1975) and Rhizobium sp. (Leigh et al., 1986).
Since microbial dehalogenation of 2,2-dichloropropionic acid is an important
step in 2,2-dichloropropionic acid detoxification (Schwarze et al., 1997), it was thus
interesting to isolate a microorganism that is capable of degrading the halogenated
aliphatic compounds in the environment. In the current study, a microorganism
capable of degrading 2,2-dichloropropionate was isolated and characterised. The
bacterium was identified based on 16S rRNA gene analysis. It was hoped that such
studies would give a greater understanding of the dehalogenase enzyme system and
2,2-dichloropropionate-degrading microorganisms.
92
4.2
Results
4.2.1 Isolation of 2,2-dichloropopionate Degrading Bacteria
A mixed culture from UTM plantation was streaked onto 20 mM 2,2dichloropropionate minimal medium. Three morphologically different colonies
were observed. Colonies formed were repeatedly streaked on the same type of
medium. Only one of them grew well in 2,2-dichloropropionate liquid medium and
picked for further analysis. This bacterium was designated as bacterium B.
4.2.2 Identification of Bacterium B by 16S rRNA Gene Analysis
PCR amplification of the 16S rRNA gene from genomic DNA of bacterium
B revealed a single fragment of approximately 1.6 kb. The 16S rRNA gene
fragment was sequenced using FD1 and rP1 primers. Figure 4.3 showed the partial
nucleotide sequence of 16S rRNA gene from the isolated bacterium. The sequence
comprises of 1307 nucleotides lacking the very proximal 5’ and terminal 3’ regions
corresponding to the universal primers used. This sequence was submitted to the
GenBank with accession number of AM 231910 (Appendix F).
The 16S rRNA gene sequence was compared to the sequences in the
GenBank database. The result revealed that the 2,2-dichloropropionate degrading
bacteria has a 99% identity match (e-value = 0) with Methylobacterium sp. (Figure
4.4). Thus, it was clear that the bacterium B belongs to Methylobacterium sp. and it
was further designated as Methylobacterium sp. strain HN2006B. It shared 99 %
identity to a series of bacteria listed in the database (Table 4.1).
93
Lane
1
2
3
4
5
6
kb
Figure 4.2:
Agarose gel electrophoresis of undigested genomic DNA and 16S
rRNA gene fragment
Lane 1: Genomic DNA (0.25 μg/μl) prepared from bacterium B
Lane 2: The amplified 16S rRNA DNA fragment (1.6 kb)
Lane 3: Negative control without DNA template
Lane 4: Negative control without FD1 primer
Lane 5: Negative control without rP1 primer
Lane 6: 1 KB DNA ladder (Invitrogen)
94
1 tgagtaacgc gtgtgaacgt gccttccggt tcggaataac cctgggaaac tagggctaat
61 accggatacg cccttatggg gaaaggttta ctgccggaag atcggcccgc gtctgattag
121 ctagttggtg gggtaacggc ctaccaaggc gacgatcagt agctggtctg agaggatgat
181 cagccacact gggactgaga cacggcccag actcctacgg gaggcagcag tggggaatat
241 tggacaatgg gcgcaagcct gatccagcca tgccgcgtga gtgatgaagg ccttagggtt
301 gtaaagctct tttatccggg acgataatga cggtaccgga ggaataagcc ccggctaact
361 tcgtgccagc agccgcggta atacgaaggg ggctagcgtt gctcggaatc actgggcgta
421 aagggcgcgt aggcggcgtt ttaagtcggg ggtgaaagcc tgtggctcaa ccacagaatg
481 gccttcgata ctgggacgct tgagtatggt agaggttggt ggaactgcga gtgtagaggt
541 gaaattcgta gatattcgca agaacaccgg tggcgaaggc ggccaactgg accattactg
601 acgctgaggc gcgaaagcgt ggggagcaaa caggattaga taccctggta gtccacgccg
661 taaacgatga atgccagctg ttggggtgct tgcaccgcag tagcgcagct aacgctttga
721 gcattccgcc tggggagtac ggtcgcaaga ttaaaactca aaggaattga cgggggcccg
781 cacaagcggt ggagcatgtg gtttaattcg aagcaacgcg cagaacctta ccatcctttg
841 acatggcgtg ttacccagag agatttgggg tccacttcgg tggcgcgcac acaggtgctg
901 catggctgtc gtcagctcgt gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc
961 cacgtcctta gttgccatca ttcagttggg cactctaggg agactgccgg tgataagccg
1021 cgaggaaggt gtggatgacg tcaagtcctc atggccctta cgggatgggc tacacacgtg
1081 ctacaatggc ggtgacagtg ggacgcgaag gagcgatctg gagcaaatcc ccaaaagccg
1141 tctcagttcg gattgcactc tgcaactcga gtgcatgaag gcggaatcgc tagtaatcgt
1201 ggatcagcat gccacggtga atacgttccc gggccttgta cacaccgccc gtcacaccat
1261 gggagttggt cttacccgac ggcgctgcgc caaccgcaag gaggcag
Figure 4.3:
Bacterium B 16S rRNA partial sequence lacking the very proximal 5’
and terminal 3’ regions (AM 231910)
95
Bacterium B
1
Methylobacterium 69
Bacterium B
61
Methylobacterium
129
Bacterium B
121
Methylobacterium
189
Bacterium B
181
Methylobacterium
249
Bacterium B
241
Methylobacterium
309
Bacterium B
301
Methylobacterium
369
Bacterium B
361
Methylobacterium
429
Bacterium B
421
Methylobacterium
489
Bacterium B
481
Methylobacterium
549
Bacterium B
541
Methylobacterium
609
Bacterium B
601
Methylobacterium
669
Bacterium B
661
Methylobacterium
729
Bacterium B
721
Methylobacterium
789
Bacterium B
781
Methylobacterium
849
Bacterium B
841
Methylobacterium
909
Bacterium B
901
Methylobacterium 969
Bacterium B
961
Methylobacterium 1029
Bacterium B
1021
Methylobacterium 1089
Bacterium B
1081
Methylobacterium 1149
TGAGTAACNCGTGTGAACGTGCCTTCCGGTTCGGAATAACCCTGGGAAACTAGGGCTAAT
|||||||| |||| ||||||||||||||||||||||||||||||||||||||||||||||
TGAGTAACGCGTGGGAACGTGCCTTCCGGTTCGGAATAACCCTGGGAAACTAGGGCTAAT
60
ACCGGATACGCCCTTATGGGGAAAGGTTTACTGCCGGAAGATCGGCCCGCGTCTGATTAG
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
ACCGGATACGCCCTTATGGGGAAAGGTTTACTGCCGGAAGATCGGCCCGCGTCTGATTAG
120
CTAGTTGGTGGGGTAACGGCCTACCAAGGCGACGATCAGTAGCTGGTCTGAGAGGATGAT
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CTAGTTGGTGGGGTAACGGCCTACCAAGGCGACGATCAGTAGCTGGTCTGAGAGGATGAT
180
CAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATAT
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATAT
240
TGGACAATGGGCGCAAGCCTGATCCAGCCATGCCGCGTGAGTGATGAAGGCCTTAGGGTT
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
TGGACAATGGGCGCAAGCCTGATCCAGCCATGCCGCGTGAGTGATGAAGGCCTTAGGGTT
300
GTAAAGCTCTTTTATCCGGGACGATAATGACGGTACCGGAGGAATAAGCCCCGGCTAACT
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
GTAAAGCTCTTTTATCCGGGACGATAATGACGGTACCGGAGGAATAAGCCCCGGCTAACT
360
TCGTGCCAGCAGCCGCGGTAATACGAAGGGGGCTAGCGTTGCTCGGAATCACTGGGCGTA
|||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CCGTGCCAGCAGCCGCGGTAATACGAAGGGGGCTAGCGTTGCTCGGAATCACTGGGCGTA
420
AAGGGCGCGTAGGCGGCGTTTTAAGTCGGGGGTGAAAGCCTGTGGCTCAACCACAGAATG
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
AAGGGCGCGTAGGCGGCGTTTTAAGTCGGGGGTGAAAGCCTGTGGCTCAACCACAGAATG
480
GCCTTCGATACTGGGACGCTTGAGTATGGTAGAGGTTGGTGGAACTGCGAGTGTAGAGGT
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
GCCTTCGATACTGGGACGCTTGAGTATGGTAGAGGTTGGTGGAACTGCGAGTGTAGAGGT
540
GAAATTCGTAGATATTCGCAAGAACACCGGTGGCGAAGGCGGCCAACTGGACCATTACTG
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
GAAATTCGTAGATATTCGCAAGAACACCGGTGGCGAAGGCGGCCAACTGGACCATTACTG
600
ACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCG
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
ACGCTGAGGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCG
660
TAAACGATGAATGCCAGCTGTTGGGGTGCTTGCACCGCAGTAGCGCAGCTAACGCTTTGA
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
TAAACGATGAATGCCAGCTGTTGGGGTGCTTGCACCGCAGTAGCGCAGCTAACGCTTTGA
720
GCATTCCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGGCCCG
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
GCATTCCGCCTGGGGAGTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGGCCCG
780
CACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGCAGAACCTTACCATCCTTTG
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGCAGAACCTTACCATCCTTTG
840
ACATGGCGTGTTACCCAGAGAGATTTGGGGTCCACTTCGGTGGCGCGCACACAGGTGCTG
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
ACATGGCGTGTTACCCAGAGAGATTTGGGGTCCACTTCGGTGGCGCGCACACAGGTGCTG
900
CATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACC
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACC
960
CACGTCCTTAGTTGCCATCATTCAGTTGGGCACTCTAGGGAGACTGCCGGTGATAAGCCG
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CACGTCCTTAGTTGCCATCATTCAGTTGGGCACTCTAGGGAGACTGCCGGTGATAAGCCG
1020
CGAGGAAGGTGTGGATGACGTCAAGTCCTCATGGCCCTTACGGGATGGGCTACACACGTG
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CGAGGAAGGTGTGGATGACGTCAAGTCCTCATGGCCCTTACGGGATGGGCTACACACGTG
1080
CTACAATGGCGGTGACAGTGGGACGCGAAGGAGCGATCTGGAGCAAATCCCCAAAAGCCG
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CTACAATGGCGGTGACAGTGGGACGCGAAGGAGCGATCTGGAGCAAATCCCCAAAAGCCG
1140
128
188
248
308
368
428
488
548
608
668
728
788
848
908
968
1028
1088
1148
1208
96
Bacterium B
1141
Methylobacterium 1209
Bacterium B
1201
Methylobacterium 1269
Bacterium B
1261
Methylobacterium 1329
Figure 4.4:
TCTCAGTTCGGATTGCACTCTGCAACTCGAGTGCATGAAGGCGGAATCGCTAGTAATCGT
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
TCTCAGTTCGGATTGCACTCTGCAACTCGAGTGCATGAAGGCGGAATCGCTAGTAATCGT
1200
GGATCAGCATGCCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCAT
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
GGATCAGCATGCCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCAT
1260
GGGAGTTGGTCTTACCCGACGGCGCTGCGCCAACCGCAAGGAGGCAG
|||||||||||||||||||||||||||||||||||||||||||||||
GGGAGTTGGTCTTACCCGACGGCGCTGCGCCAACCGCAAGGAGGCAG
1268
1328
1307
1375
Sequence comparison for bacterium B 16S rRNA gene (percentage
identity = 99 %; e-value = 0)
Table 4.1:
Top 5 entries in the database that showed highest identity to
bacterium B
Bacteria
Sequence Identity
Methylobacterium sp. F05
99%
Methylobacterium sp. F15
99%
Methylobacterium fujisawaense
99%
Methylobacterium sp. F73
99%
Methylobacterium sp. A1
99%
97
4.2.3 Bacteria Morphology, Staining and Biochemical Characterisation
The identification of bacterium B using 16S rRNA gene sequence was
supported by the bacterial staining and biochemical analysis. Bacterium B colony
was observed as a rough surface, a smooth margin and a raised elevation. It formed
pink colonies on nutrient agar and 2,2-dichloropropionate minimal medium (Table
4.2). It was a gram-negative rod bacterium. The cells were acid-fast and no spores
were demonstrated by malachite green staining. Bacterium B also demonstrated the
ability in utilising lactose, gelatin liquefaction, producing catalase, oxidase, motile
and grew on citrate (Table 4.3).
The biochemical test results were compared to Bergey’s Manual of
Systematic Bacteriology (Holt et al., 1994) and agreed well with the finding of 16S
rRNA analysis.
98
Table 4.2: Colony characteristic on 2,2-dichloropropionate minimal medium
Characteristics
Observation
Size of colony
Small
Pigmentation
pink
Form (Shape of the colony)
Smooth Circular
Margin (Outer edge of colony)
Entire (Sharply defined, even)
Elevation
Raised (Slightly elevated)
Table 4.3: Morphological and biochemical characteristics of Methylobacterium sp.
Biochemical test and staining
Result
Gram Stain
Gram negative (Pink)
Acid Fast
Positive (Blue)
Spore Stain
No Spore
Grow Behavior
Aerobic
Oxidase
Positive
Catalase
Positive
Citrate
Positive
Gelatin Liquefaction
Positive
Lactose Utilisation
Positive
Motility
Positive
99
4.2.4 Growth of Methylobacterium sp. HN2006B in 2,2-dichloropropionate
Minimal Medium
Methylobacterium sp. HN2006B was inoculated in 100 ml PJC minimal
broth medium containing 5 mM, 10 mM, 20 mM and 40 mM 2,2-dichloropropionate
as the sole source of carbon, respectively. The flask was incubated at 30°C in a
rotary incubator at 180 rpm. The maximum growth was achieved in 20mM 2,2dichloropropionate minimal medium. Figure 4.5 showed the growth patterns of
bacteria in different substrate concentration. Methylobacterium sp. HN2006B grew
readily on 5 to 20 mM 2,2-dichloropropionate. No growth was observed when
growth at 40 mM suggested that the substrate was toxic to the cell at higher
concentration. The cell doubling time was calculated by applying data acquired
from triplicate reading (Appendix G) into the semi log graph as summarised in Table
4.4.
100
Time (Hour)
Figure 4.5: Growth curves on 5, 10, 20 and 40 mM 2,2-dichloropropionate for
Methylobacterium sp. HN2006B.
101
Table 4.4: Growth properties of bacteria in different 2,2-dichloropropionate
concentration
Substrate
Maximum Medium
Concentration (mM)
Turbidity (A680nm)
Mean doubling time (H)
5
0.280±0.001
25.41±1.04
10
0.502±0.004
19.50±0.44
20
1.053±0.002
20.32±0.79
40
NG*
NG*
*NG: no growth
102
4.2.5 Growth of Methylobacterium sp. HN2006B in Other Halogenated
Compounds
Table 4.5 showed growth at various types of halogenated compound. D,L-2chloropropionatate was the only other halogenated compound, apart from 2,2dichloropropionate that supported growth of the isolate when supplied as the sole
carbon and energy source. However, both of these compounds failed to support
growth when present in minimal liquid media at concentration of 40mM. The
compounds were toxic to the cell at higher concentration.
Figure 4.6 showed the growth curve of Methylobacterium sp. HN2006B
when grown on 2,2-dichloropropionate and D,L-2-chloropropionate, with doubling
time of 20 and 26 hours, respectively. This experiment suggested that
Methylobacterium sp. HN2006B could only act on chloride attached to carbon
number 2. Other reason might be a nutrient uptake system was not available for
other substrate than 2,2-dichloropropionate and D,L-2-chloropropionate.
103
Table 4.5: Growth properties of Methylobacterium sp. HN2006B in different
substrates
Substrate (20mM)
Maximum Medium
Mean doubling time (H)
Turbidity (A680nm)
3-chloropropionate
NG
NG
D,L-2-chloropropionate
1.090±0.002
26.09±0.25
2,2-dichloropropionate
1.053±0.002
20.32±0.79
2,3-dichloropropionate
NG
NG
3-bromopropionate
NG
NG
NG : No Growth
104
Figure 4.6: Growth curve of Methylobacterium sp. HN2006B when grown on
different halogenated substrate (Appendix H)
105
4.2.6
HPLC Analysis of Growth Medium
This experiment was carried out to determine the ability of the organism to
utilise 2,2-dichloropropionate using HPLC method. At the end of the experiment,
the amount of 2,2-dichloropropionate left in the growth medium will be measured.
4.2.6.1
2,2-Dichloropropionate Calibration Curve
A standard curve was constructed from a series of known concentration of
2,2-dichloropropionate within the range of 0 to 20 mM. Table 4.6 showed the data
used to construct the calibration curve for 2,2-dichloropropionate whereas Figure 4.7
showed the calibration curve constructed.
106
4.2.6.2
Detection of 2,2-dichloropropionate in growth medium
Methylobacterium sp. HN2006B was grown in 20 mM 2,2-
dichloropropionate minimal medium. Growth medium was then analysed using
HPLC at daily intervals to monitor the presence of 2,2-dichloropropionate. The
chromatogram showed the area of the peak corresponding to 2,2-dichloropropionate
decreased from 20 mM at day 0 to 0 mM at day 4. The results were summarised in
Table 4.7.
It was concluded that 2,2-dichloropropionate was fully utilised by
Methylobacterium sp. HN2006B since traces of 2,2-dichloropropionate was not
observed in the growth medium at the 4th days of incubation (Figure 4.8) whereas
control of 2,2-dichloropropionate minimal medium without bacteria did not show
any substrate decrement. Nevertheless, depletion of 2,2-dichloropropionate was
coupled with bacteria growth suggested that 2,2-dichloropropionate was carbon and
energy source for Methylobacterium sp.
107
Table 4.6: Data used to construct the 2,2-dichloropropionate calibration curve
No
Peak Name
Level
Mean Area
Amount
(mM)
1
2,2-dichloropropionic acid
1
56.236
5
2
2,2-dichloropropionic acid
2
130.323
10
3
2,2-dichloropropionic acid
3
274.321
20
Figure 4.7: Calibration curve for 2,2-dichloropropionate
Table 4.7:
Comparison between the 2,2-dichloropropionate consumed and
turbidity of Methylobacterium growth medium
Days
Bacteria Growth
(A680nm)
Amount of 2,2dichloropropionate left
0
0.102
100 %
1
0.199
86 %
2
0.428
72 %
3
0.712
35%
4
1.302
0%
108
(i)
(ii)
(iii)
Figure 4.8: HPLC elution profile of medium from cells utilising 2,2-
dichloropropionate as source of carbon. Medium was analysed at i) day 0, ii) day 2
and (iii) day 4
109
4.2.7 Dehalogenase Activity in Cell Free Extracts of 2,2-Dichloropropionate
Grown Bacteria
The disappearance of 2,2-dichloropropionate in the growth medium
suggested that the substrate was used as the sole source of carbon. Current
experiment will be carried out to determine the presence of dehalogenase enzyme in
the bacteria system.
Enzyme localisation had been carried out and dehalogenase enzyme
produced was found to be intracellular enzyme since resuspended cell debris did not
possess any activity towards the substrate. Cell free extracts were prepared from
bacteria grown on 2,2-dichloropropionate as sole carbon source and were assayed
for dehalogenase activity using 1 mM 2,2-dichloropropionate as substrate. By
measuring the rate of chloride ion released from the substrate, the average specific
activity of dehalogenase on these extracts was found to be 0.039 μmol Cl-/min/mg
protein. Details for calculation of enzyme activity was described in Appendix I.
110
4.3
Discussion
In the current investigation, the 16S rRNA sequence of the 2,2dichloropropionate degrading bacteria shared more than 99% homology to
Methylobacterium sp. Thus the 2,2-dichloropropionate degrading bacteria isolated
belongs to Methylobacterium sp. This was further supported by staining properties
and biochemical analysis.
The 2,2-dichloropropionate degrading bacteria belongs to genus
Methylobacterium was also supported by its ability to produce a pink pigment.
Certain Methylobacterium species are known to be pink-pigmented bacteria that
produce so called pink slime. For example, a Methylobacterium sp. which is known
to be responsible for pink slime produced a (10→3)-galactan polysaccharide (EPS)
was described by Verhoef et al. (2003).
Methylobacterium is a facultative Methylobacteriumlotroph, meaning that it
has the ability to grow by reducing carbon compounds with one or more carbon
atoms but no carbon-carbon bonds (Madigan et al., 2003). It grows on
methylobacteriumlamine, methanol, and C2, C3, and C4 compounds, including the
methanol emitted by the stomata of plants. They are non-motile rod-shaped and are
obligate aerobic; they are also called PPFMs - pink-pigmented facultative
Methylobacteriumlotrophs.
Methane is the end product of anaerobic degradation of organic matter;
therefore, methane-oxidising bacteria can be found in such habitats as in wetland
rice fields (Eller and Frenzel, 2001). This bacterium can be found mostly in soils,
on leaves, and in other parts of plants (Lidstrom and Christoserdova, 2002; Aken et
al., 2004), or even dust, air, freshwater and aquatic sediments (Gallego et al., 2005).
One species, Methylobacterium podarium, is thought to be part of the natural human
foot microflora. Methylobacterium have even been found living inside the human
mouth. Samples have been isolated from the tongue, supra- and subgingival plaques
that have shown growth of Methylobacterium, especially M. thiocyanatum.
111
Methylobacterium was well known for their ability in degrading
chloromethane or dichloromethane as the sole carbon and energy source (Vannelli et
al., 1998, 1999; Studer, 2001; McDonald et al., 2002). However, no
Methylobacterium that able to degrade 2,2-dichloropropionate was reported so far.
Therefore, this novel strain of Methylobacterium sp. that is able to degrade 2,2dichloropropionate deserved more interest.
There are four basic criteria which must be fulfilled in order for a given
halogenated compound to be utilised by an organism as sole source of carbon and
energy. Firstly, the organism must either possess or syhthesise dehalogenase in
response to the halogenated compound which is capable of removing the substituent
halogen(s) from the compound. Secondly, the dehalogenation product should be
non-toxic and easily converted to an intermediate of the organism’s central
metabolic pathway. Thirdly, the halogenated compound should be able to enter cell
either passively or by active transport in order to reach the site of dehalogenase
activity, and finally, the halogenated compound should be non-toxic to the organism
at normal intracellular concentrations (Hughes, 1988). These criteria were satisfied
when Methylobacterium sp. HN2006B grew on 2,2-dichloropropionate and D,L-2chloropropionate as sole source of carbon and energy. The isolate grew more
rapidly on 2,2-dichloropropionate compared to D,L-2-chloropopionate, possibly
because the organism was originally isolated using 2,2-dichloropropionate. Both
halogenated compound failed to support growth at concentration exceeding 40mM;
presumably the intracellular concentration of halo-aliphatic acid had reached a toxic
level.
Pseudomonas putida PP3 isolated from a microbial community by Senior et
al. (1976) utilised 2,2-dichloropropionate as sole carbon source, the specific growth
rates of the organism on 2,2-dichloropropionate at concentration of 0.5 g carbon L-1
in continuous culture was 8.33 hours. However, mean generation time for P. putida
PP3 growing on 2,2-dichloropropionate in closed culture system was not mentioned.
Allison (1981) examined the growth of Rhizobium sp. on 2,2-dichloropropionate in
closed cultures system with the doubling time of 12 hours in liquid medium. In
current study, the doubling time of Methylobacterium sp. HN2006B in the same
substrate was around 20 hours, or 2 times slower compared to Rhizobium sp. The
112
difference maybe due to different affinity towards 2,2-dichloropropionate between
the two organisms.
Allison (1981) described that differential plots of culture extinction at 680
nm against medium chloride ion concentration were linear during exponential
growth of Rhizobium sp. in 2,2-dichloropropionate minimal medium, indicating that
the rate of 2,2-dichloropopionate dehalogenation was proportional to the growth rate
of the Rhizobium sp. Since 2,2-dichloropropionate was the sole carbon source in the
experiment, the results also indicated that the dehalogenation product was being
utilised by the organism for metabolism and growth.
In order to further confirm the relationship between the 2,2dichloropropionate and bacterial growth, HPLC was employed in current study. As
showed in section 4.2.6, the degradation and utilisation of 2,2-dichloropropionate
was initiated from the beginning of the growth. Table 4.7 clearly illustrates that
when the bacteria number increased in the medium, 2,2-dichloropropionate
concentration drops gradually. HPLC results indicating that growth of the bacterium
in the medium was proportional to the utilisation of 2,2-dichloropropionate in the
medium.
Dehalogenase activity was detected in cell-free extracts of Methylobacterium
sp. HN2006B grown on 2,2-dichloropropionate by measuring the rate of halide ion
released from the substrate. Specific activity was expressed as μmol Cl-/min/mg
protein. The specific activity of dehalogenase present in Methylobacterium sp.
HN2006B cell extract was 0.039 μmol Cl-/min/mg protein. The average specific
activity of dehalogenase in cell free extract of Rhizobium sp. described by Allison
(1981) was found to be 0.243 μmol Cl-/min/mg protein. In current study, specific
activities of dehalogenase present in Methylobacterium crude cell free extract was 5
times lower compared to Rhizobium sp. Since Methylobacterium sp. HN2006B was
a slow grower (2 times slower as described earlier) compared to Rhizobium sp, it
was not surprised that specific activity of dehalogease from Methylobacterium sp.
HN2006B had much lower enzyme specific activity.
113
Nevertheless, in a recent study by Schwarze et al. (1997), two
Agrobacterium tumefaciencs species, A. tumefaciens RS4 and A. tumefaciens RS5
were found to grow on 2,2-dichloropropionate with a doubling time of 8.5 and 4.7
hours, respectively. Their dehalogenase specific activity in crude extract were 0.247
μmol Cl-/min/mg protein and 0.647 μmol Cl-/min/mg protein, respectively. These
further indicated that the growth rate was correlated with dehalogenase specific
activity. Slower growth rate in Methylobacterium sp. HN2006B in the current study
could be caused by lower dehalogenase activity.
114
4.4
Conclusion
A bacterial isolated from soil were found to have a good potential in
degrading 2,2-dichloropropionate. It was identified based on 16S rRNA analysis as
Methylobacterium sp. HN2006B with 99% identity. The Methylobacterium
HN2006B grew well in 5mM, 10mM and 20mM of 2,2-dichloropropionate minimal
medium but growth was inhibited in 40mM of 2,2-dichloropropionate. The ability
of Methylobacterium sp. in degrading 2,2-dichloropropionate was confirmed with
HPLC analysis. The bacterium also grew in D,L-2-chloropropionate. Production of
dehalogenase enzyme by Methylobacterium sp. HN2006B was confirmed by
enzyme assay. Crude cell free extract of Methylobacterium sp. HN2006B showed
week activity toward 2,2-dichloropropionate with specific activity of 0.039 μmol Cl/min/mg protein agreed by the slow growth in 20 mM 2,2-dichloropropionate
minimal medium.
CHAPTER V
ANALYSIS AND CLONING OF RHIZOBIUM SP. dehrP GENE
5.1
Introduction
Dehalogenase producing microorganisms are normally selected for their
ability to utilise halogenated alkanoic acids or haloalkanes as carbon and energy
sources. However, in order for this event to take place, the halogenated compounds
must first be transported into the cell because all dehalogenases that have been
identified to date are intracellular enzymes, of which may exist as free enzymes in
the cell or periplasmic membrane bound (Tsang et al., 2001).
Dehalogenase associated permease has been proposed to mediate the uptake
of haloacid into the cell. It is apparent that most substances do not passively enter
the cell, and thus transport processes are critical to cellular function. Van der Ploeg
and Janssen (1995) suggested that haloakanoic acids are negatively charged at
physiological pH values, and therefore it is likely that these substrates need to be
transported through the membrane by a carrier protein.
116
Rhizobium sp. was found to produce three dehalogenases, collectively known
as DehD, DehL and DehE. DehD and DehL are stereospecific for D-2chloropropinate and L-2-chloropropionate respectively, whereas DehE can act on
both D,L-2-chloropropionate and 2,2-dichloropropionate. Escherichia coli K-12
strain NM522 transformed with plasmid pSC1 (dehD+, dehL+; dehrP+?) was
endowed with the novel ability to grow at the expense of D,L-2-chloropropionate.
However, Escherichia coli transformed with pJS 771 (dehE+) was not able to grow
on D,L-2-chloropropionate and 2,2-dichloropropionate. pJS 771 insert contain only
dehE gene without further upstream nor downstream fragment whereas pSC1
contained a truncated open reading frame upstream of dehD gene (Figure 5.1).
Therefore, it was hypothesised that the pSC1 plasmid has the fifth open reading
frame associated with permease that enable the E.coli to uptake the haloacid into the
cell.
Nevertheless, the 3832bp of nucleotide sequence upstream of dehD in
pSC1 insert had been sequenced (Figure 5.2) and was aligned to all known sequence
in GeneBank using BLASTn tool (Choy 2004). A distinct 98% identity was
achieved at position +1766-3832 of the nucleotide sequence with the dehP gene of
Agrobacterium sp. NHG3 (Higgins et al., 2005). Since dehP of Agrobacterium sp.
NHG3 is a gene that encodes for the putative mono chloro propionic acid permease,
this strongly suggested that a putative haloacid permease gene may be located within
upstream region of dehD in pSC1.
In current investigation, the sequence of putative haloacid permease gene in
pSC1 was further analysed and the putative gene was cloned into a plasmid for
further study.
117
(i)
(ii)
Figure 5.1: Plasmid used in this course of study
(i) pSC1 showing dehD and dehL. The putative haloacid permease dehrP gene was
hypothesise to locate at upstream of dehD (Cairns et al., 1996)
(ii) pJS 771 showing dehE insert without upstream nor downstream fragment that
possibly contain an extra gene such as permease (Stringfellow et al., 1997)
MCS: multi cloning site
118
EcoRI site
1 gaattccaga agatgggttc cgtgggagcc aagacagcag tgcgtttcgg gaagccccgc
61 cgcgaacgcg aagtgacatc gtgcaagcgg cagtggagca gtgcggatca gtccgtcgtg
121 cagcgcttca cttgggcata agcgaatcca ccgttaagcg aaaattgcgc agcagccgct
181 gaccatcact gtgtgacctg tgagctgggt tcacgcagct cacagtccat catgactgca
241 taggccatat ttgacccttg caggtcaaaa ttgacctgac ctgatgacat cttttatggc
301 gtcatcgtgg taacctattg aagcatatat atattttaga ttggcatgga tcttgcgctg
361 tctcccttgt taattgggag atccatatgt cagaaatgtt acttaccgac gtcctcgtca
421 tcggcgaggg ctgcgcgggc caaacggcgg cgctcgccgt cgccgaagag ggctgcgatg
481 ttatcctgct tggcgacggc cgtccgccga gcaccgcgat ctcgactggc ttcctgacct
541 ttgcggcgca cgaggggttt ggtcgcgatc aactccatca agcgatgtcc gaagtcaccg
601 gcaaaggcct gtgtgatcgc actctcttga ggcgtttggt tgacgaagcc cctcaggaaa
661 tggctgcagc gatcaaggct tacaacatcc cgttcgacaa ggttgaacgg ggcttgcggg
721 cacggcgcgc gcttgggaag agtgggcgcg aacttctctc aggttacgag gctgattaca
781 gcgagcacgg tcaaatcgaa gacatgaccg ggctcatgat ggagttctcc tcaacgcacg
841 gcacggcgct ctatgcgcaa ctgcgaaagg cagtgaaggc gaatctcaaa attcggcggt
901 gccgtggcag cgcgctggtt ctcgaaccga acacaatccg tcgtcggtgc tcttgtcgat
961 ggtcaaccca cgacgattgt tgcccgcacc attatcatag ctacgggcgg gctccagggc
1021 ctctacgagt tcacggatac gcctgaaacg ctcacaggcg acggacacgg tttagcattc
1081 gaggctggcg ccgctctcgt tgatatggaa tttgtccaat tctatccgct cgcggtgcgc
1141 gaagcggcga ttcctcccat tttcctttac ccagactttc cgaaactctc gacgctcgtg
1201 aatgatcgcg gtgaaaacgt ccttgcaaag catctggggg aggactctca atacctggca
1261 gatcttcata attgggatca tctttcggcg gttatccagt ccgagatcat cgaaggccag
1321 cgcgtctacg ttgactttcg gcagaccaag gccgccgaat tgggcggcag actctttgac
1381 cggtacgttc ctgtcaaaat tcataccgaa cttccgcgag cggccggtgg aggtcgcacc
1441 gtccgcacat ttcacaatcg gcgggcttag agtcgacgag aacggacgaa cgaaccttcc
1501 gcacatttat gccgttggcg aagttgcggg cggagtgcat ggcgcaaatc ggcacggcgg
1561 cacggcgctg gtcgaagcga tcacctttgg ccgcatcgcc ggccgtcatg cggctcacag
1621 cctcaacagc gaggcggtac gtaggaacat atcttcgctg ccgccggagc ggcggtctgg
1681 cccccctcct cgacttgccg gcctgatggc cgacttgcgc cacgcaaacc aaatggcgct
1741 cgggccgatc cgcgatgggc ggcgccttca ggatgccgga ctacgcttgg ctgcgttgcg
1801 tgaggaagtg cggagctcag gctgggagag ctacgctcaa atgcaagagg tcttgcgcct
1861 cgaccgcggc atcgttcttt ccgactgcct tcgccaagcg atgctgcgca gaaccgaaac
1921 acgcggcacc catgcgcgat cggatcatcc ggaggcccgc gacgcctggc tgaaaaagca
1981 agtcgttcgc atcgcaggcg acgtgctgca ttttgaggat gcgcctcttt agcagctgtg
dehrP?
2041 tcgcctacca aaaatcaaca atatccgagg gaacacccaa tgactacgac tctagtcgcc
2101 cgtacttcat cagccggtcg catgacacgc gaggagcgca aagtgatctt cgcctcctcg
2161 ctcggtactg tcttcgaatg gtacgatttc tttctatatg gctcactcgc cgctatcatc
2221 ggcgcgacct ttttcaagga ctttccgcca gccacacaag ccatattcgc gctccttgct
2281 ttcgcggctg gctcgcttgt tcggactttc ggcgcactca tttttggccg tctcggcgat
2341 atgattgggc gcaaatatac cttcctcgta accatcctga tcatgggtct gtcgacgttc
2401 gtggtcggcc ttctaccggg ttcggacacc attggacttg cggcccctac gatcctgatt
2461 ctcctccgcc tgcttcaggg cctcgcgctc ggcggtgagt acgggggcgc cgctgtttac
2521 gaggccgagc atgctccgcc cggacgccgc ggcttttata caagttggat tcaaacaact
2581 gcgacgggtg gcctcttcct atcgctcctt gtcattctcg gaactcgctc attgctgggc
2641 gaggaatcct tcacgtcttg gggctggcgc gtgccgttct tgttgtccgt ggtactgctc
2701 ggcatctcgg tatggatccg catgcagctc aacgagtcgc ccgtgttcca gcgcatgaag
2761 gcagagggca aggcgtccaa ggctcccttg agggaggcat ttgcgcactg gcctaacgca
2821 aggctcgctc tggttgccct tttcggcatg gtcgccggcc aagccgtggt ctggtacacg
2881 ggccaattct atgtgctgtt cttcctgcag agcattctca aggtagacgg cttcacgacg
2941 acgctactta tctgttggtc tttgctgctg ggatctggct tcttcgtttt ctttggctgg
3001 ttatccgatc gcatcggacg caagccgatt atgatcgccg gctgcctcct tgctgttgtg
3061 acttactttc cgatcttcga ggcaatcacc gaacgagcga atcccacttt agccaaggcg
3121 atttcggagg tgaaggtcac ccgtggtctc ccgacccggt cgaatgcggg aatctattca
3181 atccggtcgg aacccgcgtg ttcacatcgt cttgtgacgt tgcccgagcc tatcttgctc
3241 agagttcggt gcgatatgat cgtcaggcag gtgctgcagg agagccaact cgcgtgctcg
3301 tcaatggcac cgaggtaaag ttcgatacgg cgcagcttaa ggaatcccag actcgcatga
3361 cggcagcgct gcaggcagcg ggttacccca aacccggcca atctgccgcg gtgcatatga
3421 cgcatgcctt tgatttcggc gaacctcaag tcttaccgct cattggcctg ctgttcatcc
3481 tggcgattta tgtcacgatg gtctacggcc ctatggcggc agctctcgtc gaactcttcc
3541 ctgctcgcat ccgctattcc gggttgtctc ttccctacca catcggaaat ggttggtttg
3601 gcgggctcct cccagcggca gcttttgcaa tggtggccca aacgggcgac atctatttcg
3661 ggctctggta cccggattgt cattgctgca gttcaccgtt gtggtcggcc tgatatggct
3721 tcccgaaacc aaagatcgtg acatacacgc agtggactag cggataggca accggcggac
dehD
3781 ctgccacagg gcgggctcac tatttcaatt aagaggaata atgatcaata tg
Figure 5.2: The full nucleotide sequence of the upstream of dehD gene
showing the presence of putative haloalkanoic acid permease (dehrP) gene (in blue
font). EcoRI site located at multiple cloning site of pUC 19 was in red (gaattcc)
whereas start codon for dehD gene was in green (atg) (Choy, 2004)
119
5.2
Results
5.2.1 Analysis of Putative dehrP Gene
5.2.1.1 Protein Translation and Sequence Comparison
From the complete nucleotide sequence of putative dehrP gene given in
Figure 5.2, the dehrP gene was then converted into a 412 amino acid sequence
(Figure 5.3). The reading frame of the Rhizobium sp. dehrP gene consisted of a
1239 bp nucleotides sequence, which encoded a 412 amino acids protein with a
subunit molecular weight of 45 kDa and a theoretical pI of 9.78 (Table 5.1). The
results were obtained from analysis using ProtParam (Gasteiger et al., 2005).
The deduced amino acid sequence was then subjected for comparison with
protein sequence in the database. Only two protein sequences with haloacid
permease ability were deposited in the database, which is a) putative
monochloropropionic acid permease DehP from Agrobacterium sp. NHG3 (Higgins
et al., 2005) and b) haloacid-specific transporter Deh4P from Burkholderia cepacia
MBA4 (Chung et al., 2003). Figure 5.4 showed that DehrP protein sequence was
found with significant identity (86%) with Agrobacterium sp. NHG3 putative mono
chloro propionic acid permease (DehP) whereas Figure 5.5 showed that comparison
between DehrP and Deh4P only score 62% sequence identity. Table 5.2 showed top
6 sequences that showed significant alignments.
120
M T T T L V A R T S S A G R M T R E E R
1 ATGACTACGACTCTAGTCGCCCGTACTTCATCAGCCGGTCGCATGACACGCGAGGAGCGC
61
K V I F A S S L G T V F E W Y D F F L Y
AAAGTGATCTTCGCCTCCTCGCTCGGTACTGTCTTCGAATGGTACGATTTCTTTCTATAT
121
G S L A A I I G A T F F K D F P P A T Q
GGCTCACTCGCCGCTATCATCGGCGCGACCTTTTTCAAGGACTTTCCGCCAGCCACACAA
181
A I F A L L A F A A G S L V R T F G A L
GCCATATTCGCGCTCCTTGCTTTCGCGGCTGGCTCGCTTGTTCGGACTTTCGGCGCACTC
241
I F G R L G D M I G R K Y T F L V T I L
ATTTTTGGCCGTCTCGGCGATATGATTGGGCGCAAATATACCTTCCTCGTAACCATCCTG
301
I M G L S T F V V G L L P G S D T I G L
ATCATGGGTCTGTCGACGTTCGTGGTCGGCCTTCTACCGGGTTCGGACACCATTGGACTT
361
A A P T I L I L L R L L Q G L A L G G E
GCGGCCCCTACGATCCTGATTCTCCTCCGCCTGCTTCAGGGCCTCGCGCTCGGCGGTGAG
421
Y G G A A V Y E A E H A P P G R R G F Y
TACGGGGGCGCCGCTGTTTACGAGGCCGAGCATGCTCCGCCCGGACGCCGCGGCTTTTAT
481
T S W I Q T T A T G G L F L S L L V I L
ACAAGTTGGATTCAAACAACTGCGACGGGTGGCCTCTTCCTATCGCTCCTTGTCATTCTC
541
G T R S L L G E E S F T S W G W R V P F
GGAACTCGCTCATTGCTGGGCGAGGAATCCTTCACGTCTTGGGGCTGGCGCGTGCCGTTC
601
L L S V V L L G I S V W I R M Q L N E S
TTGTTGTCCGTGGTACTGCTCGGCATCTCGGTATGGATCCGCATGCAGCTCAACGAGTCG
661
P V F Q R M K A E G K A S K A P L R E A
CCCGTGTTCCAGCGCATGAAGGCAGAGGGCAAGGCGTCCAAGGCTCCCTTGAGGGAGGCA
721
F A H W P N A R L A L V A L F G M V A G
TTTGCGCACTGGCCTAACGCAAGGCTCGCTCTGGTTGCCCTTTTCGGCATGGTCGCCGGC
781
Q A V V W Y T G Q F Y V L F F L Q S I L
CAAGCCGTGGTCTGGTACACGGGCCAATTCTATGTGCTGTTCTTCCTGCAGAGCATTCTC
841
K V D G F T T T L L I C W S L L L G S G
AAGGTAGACGGCTTCACGACGACGCTACTTATCTGTTGGTCTTTGCTGCTGGGATCTGGC
901
F F V F F G W L S D R I G R K P I M I A
TTCTTCGTTTTCTTTGGCTGGTTATCCGATCGCATCGGACGCAAGCCGATTATGATCGCC
961
G C L L A V V T Y F P I F E A I T E R A
GGCTGCCTCCTTGCTGTTGTGACTTACTTTCCGATCTTCGAGGCAATCACCGAACGAGCG
1021
N P T L A K A I S E V K V T R G L P T R
AATCCCACTTTAGCCAAGGCGATTTCGGAGGTGAAGGTCACCCGTGGTCTCCCGACCCGG
1081
S N A G I Y S I R S E P A C S H R L V T
TCGAATGCGGGAATCTATTCAATCCGGTCGGAACCCGCGTGTTCACATCGTCTTGTGACG
1141
L P E P I L L R V R C D M I V R Q V L Q
TTGCCCGAGCCTATCTTGCTCAGAGTTCGGTGCGATATGATCGTCAGGCAGGTGCTGCAG
1201
E S Q L A C S S M A P R *
GAGAGCCAACTCGCGTGCTCGTCAATGGCACCGAGGTAA
Figure 5.3:
The deduced amino acid sequence of dehrP
121
Table 5.1: Amino acid sequence composition of DehrP
Amino
acid
Ala (A)
Number
Percentage
41
10.0 %
Arg (R)
27
6.6 %
Asn (N)
4
1.0 %
Asp (D)
7
1.7 %
Cys (C)
5
1.2 %
Gln (Q)
11
2.7 %
Glu (E)
17
4.1 %
Gly (G)
38
9.2 %
His (H)
3
0.7 %
Ilw (I)
27
6.6 %
Leu (L)
57
13.8 %
Lys (K)
10
2.4 %
Met (M)
10
2.4 %
Phe (F)
30
7.3 %
Pro (P)
18
4.4 %
Ser (S)
29
7.0 %
Thr (T)
30
7.3 %
Trp (W)
9
2.2 %
Tyr (Y)
10
2.4 %
Val (V)
29
7.0 %
Number of amino acids
:
412
Molecular weight
Theoretical pI
:
:
45 kDa
9.78
122
123
124
Table 5.2: Summary of entries obtained from BLAST search with highest identity to
DehrP
Sequence
Sources
Identity
References
Putative mono chloro
Agrobacterium sp.
86 %
Higgins et al., 2005
propionoic acid
NHG3
permease
General substrate
Rhodopseudomonas 63 %
transporter:Major
palustris BisB5
Copeland et al., 2005
facilitator superfamily
MFS_1
Possible MFS
Rhodopseudomonas 63 %
transporter
palustris CGA009
Haloacid-specific
Burkholderia
transporter
cepacia MBA4
Integral inner membrane
Bradyrhizobium
metabolite transport
japonicum USDA
protein
110
Putative transport
Sinorhizobium
transmembrane protein
meliloti
Larimer et al., 2004
62 %
Chung et al., 2003
61 %
Kaneko et al., 2002
61 %
Capela et al., 2001
125
5.2.1.2 Analysis of Putative Conserved Domain
A domain is a compact, locally folded region of tertiary structure. Multiple
domains are especially common in the larger globular proteins, whereas small
proteins tend to be single folded domain. Domains of a protein are identifiable by
their scaffold sequence signatures (the motifs in the protein amino-acid texts that
remain recognizable despite extensive divergent evolution). Knowing the domain
architecture underlying the putative DehrP is very important because it may give
hints about its potential biochemical or cellular function.
NCBI conserved domain search (CD server) results suggested that a sugar
transporter (sugar_tr) domain was found within 22 to 239 amino acid residues of
DehrP proteins (Figure 5.6). Although only 211 of 448 conserved domains residues
were matched, the findings nevertheless agreeing well with the hypothesis that
DehrP protein is a transporter protein.
dehrP:
Sugar:
22
1
VIFASSLGTVFEWYDFFLYGSLAAIIGATFF-----KDFPPATQAIFALLAFAAGSLVRT
VALVAALGGFLFGYDTGVIGGFATLIDFLFFFGGLTSSGACASSTVLSGLVVSIFFVGRF
76
60
dehrP:
Sugar:
77
61
FGALIFGRLGDMIGRKYTFLVTILIMGLSTFVVGLLPGSDTIGLAAPTILILLRLLQGLA
IGSLFAGKLGDRFGRKKSLLIALVLFVIGSLLSGAAPGA-------FYLLIVGRVLVGLG
136
113
dehrP:
Sugar:
137
114
LGGEYGGAAVYEAEHAPPGRRGFYTSWIQTTATGGLFLSLLVILGTRSLLGEESFTSWGW
VGGASVLVPMYISEIAPKALRGALGSLYQLGITIGILVAAIIGLGLN-----KTSGSWGW
196
168
dehrP:
Sugar:
197
169
RVPFLLSVVLLGISVWIRMQLNESPVFQRMKAEGKASKAPLRE
RIPLGLQLVPAILLLIGLFFLPESPRWLVLKGRLEEARAVLAK
239
211
Figure 5.6: Results of the conserved domain search. A sugar transporter was
found within DehrP protein with very significant socre (e-value = 6e-16) when
analysed using CD server (Marchler and Bryant, 2004)
126
5.2.1.3 Hydrophatic Character: Analysis of Transmembrane Segments
Hydrophobic regions in a protein could represent membrane spanning
segments in proteins that anchor themselves into a membrane. DehrP protein
sequence was subjected to primary structure analysis using TMHMM to find
transmembrane helices (TM helix) in that particular protein (Sonnhammer et al.,
1998).
Figure 5.7 showed the predicted transmembrane region in DehrP. It
suggested that most regions of DehrP protein were labels as TM helix
(transmembrane helix, showed in red colour). This coincided with the prediction
that DehrP is a transmembrane transport protein that facilitated the transport of
halogenated compound into bacteria cell.
Figure 5.7: Plot of probabilities of TM helix (transmembrane segment) found in
DehrP
127
5.2.1.4 Further Analysis of Putative dehrP Gene and Protein Sequence
The dehP and deh4P code for 558 and 552 amino acids respectively, while
the putative dehrP only codes for 412 amino acids (Figure 5.4 and Figure 5.5).
There are about 140 amino acids residues “missing” from the C-terminal. The Cterminal of both DehP and Deh4P are likely to have some important roles since
several conserved motifs are found within the C-terminal region from multiple
alignment. For example “FPARIRY” and “SLPYHIGNGWFGG” which shown in
red font in Figure 5.8. Besides, alignment of the putative DehrP amino acid
sequence with DehP showed almost perfect homology for the first 355 amino acids
but the match abruptly breaks down after amino acid number 356 (Figure 5.4).
In addition, bacterial genes are often arranged in operon and transcripted as a
long polycistronic mRNAs. Since there are about 400 nucleotides between the stop
codon of the putative dehrP and the start codon of dehD (Figure 5.2), these give rise
to a strong suspicion that a frame-shift mutation has occurred in dehrP, which could
have changed the translation of the amino acids sequence, and introduced a
premature stop codon, thus truncating off the last 140 amino acids.
Therefore, the putative dehrP gene sequence was reexamined carefully and
realigned to DehP and Deh4P gene in order to reveal any deletions or insertions
within the dehrP gene. As shown in Figure 5.9, two nucleotides insertion of C
residue were found at nucleotide position 1063 and 1073. These caused a shift in
the reading frame during translation and result in a complete change in the amino
acid sequence in the C terminal direction from the point of mutation which caused
the match between DehrP and DehP breaks down after amino acid number 356
(Figure 5.4).
When the mismatches in the DNA sequences were compensate virtually, the
full frame DehrP shows high identity to DehP (94%) and Deh4P (63%) indicating
that the putative dehrP sequence does have the capacity to code for a full length 543
amino acids protein (Figure 5.10).
128
DehrP
DehP
Deh4P
MTTTLVARTSSAGRMTREERKVIFASSLGTVFEWYDFFLYGSLAAIIGATFFKDFPPATQ 60
MTTTLVARTSSAGRMTREERKVIFASSLGTVFEWYDFFLYGSLAAIIGATFFKDFPPATQ 60
-MATIEGRAAPAP-ITSEERRVIFASSLGTVFEWYDFYLAGSLAIYISRTFFSGVNPAAG 58
: :*: .*::.* :* ***:****************:* **** *. ***... **:
DehrP
DehP
Deh4P
AIFALLAFAAGSLVRTFGALIFGRLGDMIGRKYTFLVTILIMGLSTFVVGLLPGSDTIGL 120
AIFALLAFAAGSLVRTFGALIFGRLGDMIGRKYTFLVTILIMGLSTFVVGLLPGSDTIGL 120
FVFTLLGFAAGFAVRPFGAIVFGRLGDMIGRKYTFLATILLMGLSTFVVGLLPGYGTIGM 118
:*:**.**** **.***::***************.***:************* .***:
DehrP
DehP
Deh4P
AAPTILILLRLLQGLALGGEYGGAAVYEAEHAPPGRRGFYTSWIQTTATGGLFLSLLVIL 180
AAPTILILLRLLQGLALGGEYGGAAVYVAEHSPPGRRGFYTSWIQTTATGGLFLSLLVIL 180
TAPVVFIAMRMLQGLALGGEYGGAATYVAEHAPSNKRGAWTAWIQTTATLGLFISLLVIL 178
:**.::* :*:**************.* ***:*..:** :*:******* ***:******
DehrP
DehP
Deh4P
GTRSLLGEESFTSWGWRVPFLLSVVLLGISVWIRMQLNESPVFQRMKAEGKASKAPLREA 240
GTRSLLGEESFTSWGWRVPFLLSVVLLGISVWIRMQLNESPVFQRMKAKGKASKAPLREA 240
SVRSLLNEDTFAAWGWRVPFLVSIVLLAVSVWIRMQLHESPVFERIKAEGKTSKAPLSEA 238
..****.*::*::********:*:***.:********:*****:*:**:**:***** **
DehrP
DehP
Deh4P
FAHWPNARLALVALFGMVAGQAVVWYTGQFYVLFFLQSILKVDGFTTTLLICWSLLLGSG 300
FAHWPNARLALVALFGMVAGQAVVWYTGQFYVLFFLQSILKVDGFTTTLLICWSLLLGSG 300
FGQWKNLKIVLLALFGLTAGQAVVWYTGQFYTLFFLTQTLKVDGTSANMLVAVALLIGTP 298
*.:* * ::.*:****:.*************.**** . ***** ::.:*:. :**:*:
DehrP
DehP
Deh4P
FFVFFGWLSDRIGRKPIMIAGCLLAVVTYFPIFEAITERANPTLAKAISEVKVTR----- 355
FFVFFGWLSDRIGRKPIMIAGCLLAXVTYFPXXEAITERANPTLAKAIXEVKVTVVSDPV 360
FFLFFGSLSDKIGRKPIIMAGCLIAALTYFPLFKALAHYTNPKLEAATLQAPITMIADPS 358
**:*** ***:******::****:* :**** :*::. :**.* * :. :*
DehrP
DehP
Deh4P
---------------------------------------GLPTR---------------S 361
ECGNLFNPVGTRVFTSSCDVARAYLAQNSVRYDRQAGAAGEPTRV-----LVNGTEVKFD 415
ECSFQFNPVGTAKFTSSCDVAKGALSKAGLNYENITAPAGTVAQIRIGDKVVDAYDGKAA 418
* ::
DehrP
DehP
Deh4P
NAGIYSIRSEPACSHRLVTLPEP-----ILLRVRCDMIVRQVLQES----------QLAC 406
TAQLKESQTRVTAALQAAGYPKPGQSAAVHMTHAFDFGKPQVLPLIGLLFILAIYVTMVY 475
DAKARGAEFEQTLSKSLETAGYPAKADPALINWPMSILILTILVLY---------VTMVY 469
*
. . : :
*
:
.:
:*
:.
DehrP
DehP
Deh4P
SSMAPR------------------------------------------------------ 412
GPMAAALVELFPARIRYSGLSLPYHIGNGWFGGLLPAAAFAMVAQTGDIYFGLWYPIVIA 535
GPLAAMLVEMFPARIRYTSMSLPYHIGNGWFGGFLPATAFAIIAARGNIYSGLWYPIVIA 529
..:*.
DehrP
DehP
Deh4P
----------------------AVTVVVGLIWLPETKDRDIHALD 558
SVAFVIGTLFVKETKGSSTYDAD 552
Figure 5.8: Multiple Alignment of DehrP with (i) the putative monochloropropionic
acid permease (DehP) from Rhizobium sp. NHG3 by Higgins et al., 2005 and (ii) the
putative haloacid permease (Deh4) from Burkholderia cepacia by Chung et al.,
2003.
Note: Identity (*)
Strongly similar (:)
Weakly similar (.)
129
mutated
original
ATGACTACGACTCTAGTCGCCCGTACTTCATCAGCCGGTCGCATGACACGCGAGGAGCGC 60
ATGACTACGACTCTAGTCGCCCGTACTTCATCAGCCGGTCGCATGACACGCGAGGAGCGC 60
************************************************************
mutated
original
AAAGTGATCTTCGCCTCCTCGCTCGGTACTGTCTTCGAATGGTACGATTTCTTTCTATAT 120
AAAGTGATCTTCGCCTCCTCGCTCGGTACTGTCTTCGAATGGTACGATTTCTTTCTATAT 120
************************************************************
mutated
original
GGCTCACTCGCCGCTATCATCGGCGCGACCTTTTTCAAGGACTTTCCGCCAGCCACACAA 180
GGCTCACTCGCCGCTATCATCGGCGCGACCTTTTTCAAGGACTTTCCGCCAGCCACACAA 180
************************************************************
mutated
original
GCCATATTCGCGCTCCTTGCTTTCGCGGCTGGCTCGCTTGTTCGGACTTTCGGCGCACTC 240
GCCATATTCGCGCTCCTTGCTTTCGCGGCTGGCTCGCTTGTTCGGACTTTCGGCGCACTC 240
************************************************************
mutated
original
ATTTTTGGCCGTCTCGGCGATATGATTGGGCGCAAATATACCTTCCTCGTAACCATCCTG 300
ATTTTTGGCCGTCTCGGCGATATGATTGGGCGCAAATATACCTTCCTCGTAACCATCCTG 300
************************************************************
mutated
original
ATCATGGGTCTGTCGACGTTCGTGGTCGGCCTTCTACCGGGTTCGGACACCATTGGACTT 360
ATCATGGGTCTGTCGACGTTCGTGGTCGGCCTTCTACCGGGTTCGGACACCATTGGACTT 360
************************************************************
mutated
original
GCGGCCCCTACGATCCTGATTCTCCTCCGCCTGCTTCAGGGCCTCGCGCTCGGCGGTGAG 420
GCGGCCCCTACGATCCTGATTCTCCTCCGCCTGCTTCAGGGCCTCGCGCTCGGCGGTGAG 420
************************************************************
mutated
original
TACGGGGGCGCCGCTGTTTACGAGGCCGAGCATGCTCCGCCCGGACGCCGCGGCTTTTAT 480
TACGGGGGCGCCGCTGTTTACGAGGCCGAGCATGCTCCGCCCGGACGCCGCGGCTTTTAT 480
************************************************************
mutated
original
ACAAGTTGGATTCAAACAACTGCGACGGGTGGCCTCTTCCTATCGCTCCTTGTCATTCTC 540
ACAAGTTGGATTCAAACAACTGCGACGGGTGGCCTCTTCCTATCGCTCCTTGTCATTCTC 540
************************************************************
mutated
original
GGAACTCGCTCATTGCTGGGCGAGGAATCCTTCACGTCTTGGGGCTGGCGCGTGCCGTTC 600
GGAACTCGCTCATTGCTGGGCGAGGAATCCTTCACGTCTTGGGGCTGGCGCGTGCCGTTC 600
************************************************************
mutated
original
TTGTTGTCCGTGGTACTGCTCGGCATCTCGGTATGGATCCGCATGCAGCTCAACGAGTCG 660
TTGTTGTCCGTGGTACTGCTCGGCATCTCGGTATGGATCCGCATGCAGCTCAACGAGTCG 660
************************************************************
mutated
original
CCCGTGTTCCAGCGCATGAAGGCAGAGGGCAAGGCGTCCAAGGCTCCCTTGAGGGAGGCA 720
CCCGTGTTCCAGCGCATGAAGGCAGAGGGCAAGGCGTCCAAGGCTCCCTTGAGGGAGGCA 720
************************************************************
mutated
original
TTTGCGCACTGGCCTAACGCAAGGCTCGCTCTGGTTGCCCTTTTCGGCATGGTCGCCGGC 780
TTTGCGCACTGGCCTAACGCAAGGCTCGCTCTGGTTGCCCTTTTCGGCATGGTCGCCGGC 780
************************************************************
mutated
original
CAAGCCGTGGTCTGGTACACGGGCCAATTCTATGTGCTGTTCTTCCTGCAGAGCATTCTC 840
CAAGCCGTGGTCTGGTACACGGGCCAATTCTATGTGCTGTTCTTCCTGCAGAGCATTCTC 840
************************************************************
mutated
original
AAGGTAGACGGCTTCACGACGACGCTACTTATCTGTTGGTCTTTGCTGCTGGGATCTGGC 900
AAGGTAGACGGCTTCACGACGACGCTACTTATCTGTTGGTCTTTGCTGCTGGGATCTGGC 900
************************************************************
mutated
original
TTCTTCGTTTTCTTTGGCTGGTTATCCGATCGCATCGGACGCAAGCCGATTATGATCGCC 960
TTCTTCGTTTTCTTTGGCTGGTTATCCGATCGCATCGGACGCAAGCCGATTATGATCGCC 960
************************************************************
mutated
original
GGCTGCCTCCTTGCTGTTGTGACTTACTTTCCGATCTTCGAGGCAATCACCGAACGAGCG 1020
GGCTGCCTCCTTGCTGTTGTGACTTACTTTCCGATCTTCGAGGCAATCACCGAACGAGCG 1020
************************************************************
mutated
original
AATCCCACTTTAGCCAAGGCGATTTCGGAGGTGAAGGTCACCCGTGGTCTCCCGACCCGG 1080
AATCCCACTTTAGCCAAGGCGATTTCGGAGGTGAAGGTCACC-GTGGTCTCC-GACCCGG 1078
****************************************** ********* *******
130
mutated
original
TCGAATGCGGGAATCTATTCAATCCGGTCGGAACCCGCGTGTTCACATCGTCTTGTGACG 1140
TCGAATGCGGGAATCTATTCAATCCGGTCGGAACCCGCGTGTTCACATCGTCTTGTGACG 1138
************************************************************
mutated
original
TTGCCCGAGCCTATCTTGCTCAGAGTTCGGTGCGATATGATCGTCAGGCAGGTGCTGCAG 1200
TTGCCCGAGCCTATCTTGCTCAGAGTTCGGTGCGATATGATCGTCAGGCAGGTGCTGCAG 1198
************************************************************
mutated
original
GAGAGCCAACTCGCGTGCTCGTCAATGGCACCGAGGTAA--------------------- 1239
GAGAGCCAACTCGCGTGCTCGTCAATGGCACCGAGGTAAAGTTCGATACGGCGCAGCTTA 1258
***************************************
mutated
original
-----------------------------------------------------------AGGAATCCCAGACTCGCATGACGGCAGCGCTGCAGGCAGCGGGTTACCCCAAACCCGGCC 1318
mutated
original
-----------------------------------------------------------AATCTGCCGCGGTGCATATGACGCATGCCTTTGATTTCGGCGAACCTCAAGTCTTACCGC 1378
mutated
original
-----------------------------------------------------------TCATTGGCCTGCTGTTCATCCTGGCGATTTATGTCACGATGGTCTACGGCCCTATGGCGG 1438
mutated
original
-----------------------------------------------------------CAGCTCTCGTCGAACTCTTCCCTGCTCGCATCCGCTATTCCGGGTTGTCTCTTCCCTACC 1498
mutated
original
-----------------------------------------------------------ACATCGGAAATGGTTGGTTTGGCGGGCTCCTCCCAGCGGCAGCTTTTGCAATGGTGGCCC 1558
mutated
original
-----------------------------------------------------------AAACGGGCGACATCTATTTCGGGCTCTGGTACCCGGATTGTCATTGCTGCAGTTCACCGT 1618
mutated
original
-------------TGTGGTCGGCCTGA 1632
Figure 5.9: DNA alignment of putative dehrP gene sequence revealed two cytosine
residue insertions at nucleotide position 1063 and 1073 respectively
Note: “Mutated” refer to the cloned putative dehrP gene in the current study.
“Original” refer to dehP sequence reported by Higgins et al. (2005).
131
DehrP
DehP
Deh4P
MTTTLVARTSSAGRMTREERKVIFASSLGTVFEWYDFFLYGSLAAIIGATFFKDFPPATQ 60
MTTTLVARTSSAGRMTREERKVIFASSLGTVFEWYDFFLYGSLAAIIGATFFKDFPPATQ 60
-MATIEGRAAPAP-ITSEERRVIFASSLGTVFEWYDFYLAGSLAIYISRTFFSGVNPAAG 58
: :*: .*::.* :* ***:****************:* **** *. ***... **:
DehrP
DehP
Deh4P
AIFALLAFAAGSLVRTFGALIFGRLGDMIGRKYTFLVTILIMGLSTFVVGLLPGSDTIGL 120
AIFALLAFAAGSLVRTFGALIFGRLGDMIGRKYTFLVTILIMGLSTFVVGLLPGSDTIGL 120
FVFTLLGFAAGFAVRPFGAIVFGRLGDMIGRKYTFLATILLMGLSTFVVGLLPGYGTIGM 118
:*:**.**** **.***::***************.***:************* .***:
DehrP
DehP
Deh4P
AAPTILILLRLLQGLALGGEYGGAAVYEAEHAPPGRRGFYTSWIQTTATGGLFLSLLVIL 180
AAPTILILLRLLQGLALGGEYGGAAVYVAEHSPPGRRGFYTSWIQTTATGGLFLSLLVIL 180
TAPVVFIAMRMLQGLALGGEYGGAATYVAEHAPSNKRGAWTAWIQTTATLGLFISLLVIL 178
:**.::* :*:**************.* ***:*..:** :*:******* ***:******
DehrP
DehP
Deh4P
GTRSLLGEESFTSWGWRVPFLLSVVLLGISVWIRMQLNESPVFQRMKAEGKASKAPLREA 240
GTRSLLGEESFTSWGWRVPFLLSVVLLGISVWIRMQLNESPVFQRMKAKGKASKAPLREA 240
SVRSLLNEDTFAAWGWRVPFLVSIVLLAVSVWIRMQLHESPVFERIKAEGKTSKAPLSEA 238
..****.*::*::********:*:***.:********:*****:*:**:**:***** **
DehrP
DehP
Deh4P
FAHWPNARLALVALFGMVAGQAVVWYTGQFYVLFFLQSILKVDGFTTTLLICWSLLLGSG 300
FAHWPNARLALVALFGMVAGQAVVWYTGQFYVLFFLQSILKVDGFTTTLLICWSLLLGSG 300
FGQWKNLKIVLLALFGLTAGQAVVWYTGQFYTLFFLTQTLKVDGTSANMLVAVALLIGTP 298
*.:* * ::.*:****:.*************.**** . ***** ::.:*:. :**:*:
DehrP
DehP
Deh4P
FFVFFGWLSDRIGRKPIMIAGCLLAVVTYFPIFEAITERANPTLAKAISEVKVTVVSDPV 360
FFVFFGWLSDRIGRKPIMIAGCLLAXVTYFPXXEAITERANPTLAKAIXEVKVTVVSDPV 360
FFLFFGSLSDKIGRKPIIMAGCLIAALTYFPLFKALAHYTNPKLEAATLQAPITMIADPS 358
**:*** ***:******::****:* :**** :*::. :**.* * :. :*:::**
DehrP
DehP
Deh4P
ECGNLFNPVGTRVFTSSCDVARAYLAQSSVRYDRQAGAAGEPTRVLVNGTEVKFDTAQLK 420
ECGNLFNPVGTRVFTSSCDVARAYLAQNSVRYDRQAGAAGEPTRVLVNGTEVKFDTAQLK 420
ECSFQFNPVGTAKFTSSCDVAKGALSKAGLNYENITAPAGTVAQIRIGDKVVDAYDGKAA 418
**. ****** ********:. *:: .:.*:. :..** ::: :... *.
.:
DehrP
DehP
Deh4P
ESQTR-------MTAALQAAGYPKPGQSAAVHMTHAFDFGEPQVLPLIGLLFILAIYVTM 473
ESQTR-------VTAALQAAGYPKPGQSAAVHMTHAFDFGKPQVLPLIGLLFILAIYVTM 473
DAKARGAEFEQTLSKSLETAGYPAKADPALINWPMS-----------ILILTILVLYVTM 467
::::*
:: :*::**** .:.* :: . :: :
:: :* :* **.:****
DehrP
DehP
Deh4P
VYGPMAAALVELFPARIRYSGLSLPYHIGNGWFGGLLPAAAFAMVAQTGDIYFGLWYP-- 531
VYGPMAAALVELFPARIRYSGLSLPYHIGNGWFGGLLPAAAFAMVAQTGDIYFGLWYPIV 533
VYGPLAAMLVEMFPARIRYTSMSLPYHIGNGWFGGFLPATAFAIIAARGNIYSGLWYPIV 527
****:** ***:*******:.:*************:***:***::* *:** *****
DehrP
DehP
Deh4P
--DCHCCSSPLWSA----------- 543
IAAVTVVVGLIWLPETKDRDIHALD 558
IASVAFVIGTLFVKETKGSSTYDAD 552
. ::
Figure 5.10: Multiple alignment of full size DehrP after compensating the frame
shift mutation with DehP (Higgins et al., 2005) and Deh4P (Chung et al., 2003).
The C terminal of DehrP shows high identity to DehP (94%) and Deh4P (63%)
indicating that the putative dehrP sequence does have the capacity to code for a full
length 543 amino acids protein.
Note: Identity (*)
Strongly similar (:)
Weakly similar (.)
132
5.2.2 Cloning of the Putative dehrP Gene
As discussed in 5.2.1.4, two cytosine residue insertions at nucleotide
position 1063 and 1073 respectively were found in putative dehrP gene. Thus, it is
interesting to study whether the truncated DehrP is fully functional or not. Cloning
of dehrP gene was therefore carried out.
5.2.2.1 Restriction Map Analysis of Haloacid Permease (dehrP) Gene
Internal restriction sites on the haloacid permease (dehrP) gene were
predicted using AnnHyb computer program (Figure 5.11). It was important to
investigate the presence of internal restriction sites in order to avoid from using
restriction enzymes that may cut the putative dehrP gene internally during cloning.
Table 5.3 showed a list of restriction enzymes that do not cut in the putative dehrP
gene. An NdeI and an EcoRI site were selected to be incorporated into the PCR
primers for cloning.
Thus, a pair of PCR primers, fnde1 (5’- ggaacaccatatgactacgactctag) and
reco1 (5’- gggaattcaaatcaaaggcatgcgtcatat) were designed for introducing NdeI and
EcoRI sites for construction of dehrP gene expression plasmid. The introduced
NdeI and EcoRI restriction sites were underlined.
133
dehrP
ATGACTACGACTCTAGTCGCCCGTACTTCATCAGCCGGTCGCATGACACGCGAGGAGCGC
1 ---------+---------+---------+---------+---------+---------+ 60
TACTGATGCTGAGATCAGCGGGCATGAAGTAGTCGGCCAGCGTACTGTGCGCTCCTCGCG
HinfI
Csp6I
TspDTI BsiEI NlaIII
MnlI
PleI
RsaI
BsrFI FatI MwoI
MwoI
BfaI
Hin4I
CviJI
HpyF10VI HinP1I
MlyI
HpaII
BstUI HpyF10VI
PshAI
HhaI
61
121
181
241
301
361
AAAGTGATCTTCGCCTCCTCGCTCGGTACTGTCTTCGAATGGTACGATTTCTTTCTATAT
---------+---------+---------+---------+---------+---------+ 120
TTTCACTAGAAGCGGAGGAGCGAGCCATGACAGAAGCTTACCATGCTAAAGAAAGATATA
MboI
MboII
BseRI Csp6I BbsI
Csp6I
BseRI
MnlI HpyCH4III
RsaI
DpnI
MnlI
BstBI
BstKTI
RsaI
TaqI
MboII
GGCTCACTCGCCGCTATCATCGGCGCGACCTTTTTCAAGGACTTTCCGCCAGCCACACAA
---------+---------+---------+---------+---------+---------+ 180
CCGAGTGAGCGGCGATAGTAGCCGCGCTGGAAAAAGTTCCTGAAAGGCGGTCGGTGTGTT
CviJI
AciI
HinP1I
AciI CviJI MwoI
Fnu4HI
BstUI
Cac8I
HpyF10VI
TauI
HhaI
EciI
GCCATATTCGCGCTCCTTGCTTTCGCGGCTGGCTCGCTTGTTCGGACTTTCGGCGCACTC
---------+---------+---------+---------+---------+---------+ 240
CGGTATAAGCGCGAGGAACGAAAGCGCCGACCGAGCGAACAAGCCTGAAAGCCGCGTGAG
CviJI MwoI HhaI HpyF10VI Fnu4HI Cac8I
Hpy188I HinP1I
HpyF10VI
AciI Cac8I
HhaI
BstUI MwoI
BstUI CviJI
HinP1I
MwoI
HpyF10VI
CviJI
TauI
ATTTTTGGCCGTCTCGGCGATATGATTGGGCGCAAATATACCTTCCTCGTAACCATCCTG
---------+---------+---------+---------+---------+---------+ 300
TAAAAACCGGCAGAGCCGCTATACTAACCCGCGTTTATATGGAAGGAGCATTGGTAGGAC
EaeI
BglI
HinP1I
MaeIII MnlI
CviJI MwoI
HhaI
FokI BclI
HaeIII HpyF10VI
Hpy188III
BceAI BsmAI
BstF5I
BsmBI
MboI
ATCATGGGTCTGTCGACGTTCGTGGTCGGCCTTCTACCGGGTTCGGACACCATTGGACTT
---------+---------+---------+---------+---------+---------+ 360
TAGTACCCAGACAGCTGCAAGCACCAGCCGGAAGATGGCCCAAGCCTGTGGTAACCTGAA
XcmI NlaIII AccI Hpy99I
CviJI BslI
Hpy188I
BccI
Hin4I HpyCH4IV
HaeIII StyD4I
DpnI
Hin4I
TaiI
HpaII
BstKTI
SalI
Hin4I
NciI
FatI
TaqI
Hin4I
ScrFI
BslI
HincII
Hpy8I
GCGGCCCCTACGATCCTGATTCTCCTCCGCCTGCTTCAGGGCCTCGCGCTCGGCGGTGAG
---------+---------+---------+---------+---------+---------+ 420
CGCCGGGGATGCTAGGACTAAGAGGAGGCGGACGAAGTCCCGGAGCGCGAGCCGCCACTC
AciI
MboI
HinfI
AciI
EcoNI CviJI HhaI AciI
Fnu4HI
DpnI TfiI
BseRI MnlI Eco57I BstUI MnlI
Sau96I
BstKTI
Cac8I
Eco57MI
CviJI
AlwI
EciI BslI HaeIII
HaeIII
Hpy188III
EcoO109I
TauI
Sau96I HinP1I
NlaIV
134
421
481
541
601
661
721
TACGGGGGCGCCGCTGTTTACGAGGCCGAGCATGCTCCGCCCGGACGCCGCGGCTTTTAT
---------+---------+---------+---------+---------+---------+ 480
ATGCCCCCGCGGCGACAAATGCTCCGGCTCGTACGAGGCGGGCCTGCGGCGCCGAAAATA
Csp6I BanI MspA1I
CviJI MwoI MwoI EciI
AciI CviJI
RsaI HphI TauI Hpy8I HaeIII Cac8I StyD4I
BsaJI
KasI
MnlI FatI
AciI
BsaHI Fnu4HI
BsaHI
HpyF10VI HpaII BtgI HgaI
HinP1I
NlaIII NciI
Fnu4HI
NarI
NspI
ScrFI
AciI
NlaIV
SphI
BstUI
SfoI
HpyF10VI
MspA1I
HhaI
TauI
AciI
SacII
BbeI
TauI
Fnu4HI
HaeII
ACAAGTTGGATTCAAACAACTGCGACGGGTGGCCTCTTCCTATCGCTCCTTGTCATTCTC
---------+---------+---------+---------+---------+---------+ 540
TGTTCAACCTAAGTTTGTTGACGCTGCCCACCGGAGAAGGATAGCGAGGAACAGTAAGAG
HinfI
Hpy99I
MboII
TfiI
CviJI
EarI
MmeI
HaeIII
MnlI
GGAACTCGCTCATTGCTGGGCGAGGAATCCTTCACGTCTTGGGGCTGGCGCGTGCCGTTC
---------+---------+---------+---------+---------+---------+ 600
CCTTGAGCGAGTAACGACCCGCTCCTTAGGAAGTGCAGAACCCCGACCGCGCACGGCAAG
Hpy188I
BseYI
HinfI
HpyCH4IV CviJI BstUI BceAI
BsrDI
MnlI
BmgBI
Cac8I Cac8I
TfiI
TaiI
HinP1I
HhaI
TTGTTGTCCGTGGTACTGCTCGGCATCTCGGTATGGATCCGCATGCAGCTCAACGAGTCG
---------+---------+---------+---------+---------+---------+ 660
AACAACAGGCACCATGACGAGCCGTAGAGCCATACCTAGGCGTACGTCGAGTTGCTCAGC
BsaJI RsaI
SfaNI BstKTI HpyCH4V
HinfI
BtgI Csp6I
BamHI FatI Fnu4HI
BbvI
TspGWI
BstYI
AlwI AluI
MboI
Cac8I
DpnI
NlaIII
NlaIV
NspI
AciI SphI
AlwI TseI
CviJI
CCCGTGTTCCAGCGCATGAAGGCAGAGGGCAAGGCGTCCAAGGCTCCCTTGAGGGAGGCA
---------+---------+---------+---------+---------+---------+ 720
GGGCACAAGGTCGCGTACTTCCGTCTCCCGTTCCGCAGGTTCCGAGGGAACTCCCTCCGT
PleI
HinP1I
MnlI HgaI BsaJI NlaIV FalI
MnlI
MlyI
FatI
TspDTI StyI CviJI
MnlI
HhaI
BsaHI MwoI
SmlI
NlaIII
HpyF10VI
TTTGCGCACTGGCCTAACGCAAGGCTCGCTCTGGTTGCCCTTTTCGGCATGGTCGCCGGC
---------+---------+---------+---------+---------+---------+ 780
AAACGCGTGACCGGATTGCGTTCCGAGCGAGACCAACGGGAAAAGCCGTACCAGCGGCCG
FalI BpuEI
CviJI
MwoI
FatI
BsrFI
HinP1I CviJI
MwoI
HpyF10VI
NlaIII CviJI
FspI
HaeIII
Cac8I
NgoMIV
HhaI
BsrI
HpyF10VI
HpaII
TspRI
Cac8I
EaeI
NaeI
HaeIII
135
781
841
901
961
1021
1081
CAAGCCGTGGTCTGGTACACGGGCCAATTCTATGTGCTGTTCTTCCTGCAGAGCATTCTC
---------+---------+---------+---------+---------+---------+ 840
GTTCGGCACCAGACCATGTGCCCGGTTAAGATACACGACAAGAAGGACGTCTCGTAAGAG
MwoI BceAI
Csp6I Sau96I
MboII
BsmI
HpyF10VI BslI RsaI
CviJI
SfcI
SmlI
CviJI
Hpy8I HaeIII
HpyCH4V
BsaJI
Tsp509I
PstI
BtgI
BslI
PflMI
AAGGTAGACGGCTTCACGACGACGCTACTTATCTGTTGGTCTTTGCTGCTGGGATCTGGC
---------+---------+---------+---------+---------+---------+ 900
TTCCATCTGCCGAAGTGCTGCTGCGATGAATAGACAACCAGAAACGACGACCCTAGACCG
BpuEI
CviJI
Hpy99I
HgaI
BbvI
BstYI CviJI
AccI
Hpy188III
TseI
MboI
Hpy8I
BceAI
Fnu4HI DpnI
Hpy99I
BseYI BstKTI
TTCTTCGTTTTCTTTGGCTGGTTATCCGATCGCATCGGACGCAAGCCGATTATGATCGCC
---------+---------+---------+---------+---------+---------+ 960
AAGAAGCAAAAGAAACCGACCAATAGGCTAGCGTAGCCTGCGTTCGGCTAATACTAGCGG
AlwI MboII
CviJI
Hpy188I
Hpy188I CviJI BsaBI BsrFI
MboI
MwoI Cac8I
MboI NgoMIV
DpnI
HpyF10VI
DpnI
BsiEI
SfaNI HgaI
BstKTI
BstKTI
HpaII
PvuI
GGCTGCCTCCTTGCTGTTGTGACTTACTTTCCGATCTTCGAGGCAATCACCGAACGAGCG
---------+---------+---------+---------+---------+---------+ 1020
CCGACGGAGGAACGACAACACTGAATGAAAGGCTAGAAGCTCCGTTAGTGGCTTGCTCGC
Cac8I
MnlI
Hpy188I
MnlI
HphI
NaeI
MaeIII
MboI TaqI
BbvI
Tsp45I
DpnI MboII
CviJI
BstKTI
TseI
Fnu4HI
AATCCCACTTTAGCCAAGGCGATTTCGGAGGTGAAGGTCACCCGTGGTCTCCCGACCCGG
---------+---------+---------+---------+---------+---------+ 1080
TTAGGGTGAAATCGGTTCCGCTAAAGCCTCCACTTCCAGTGGGCACCAGAGGGCTGGGCC
HinfI
CviJI
Hpy188I BstEII
Hpy188III
TfiI
BsaJI
MnlI MaeIII
BsaI HpaII
StyI
Tsp45I
BsmAI
BsaJI
StyD4I
BtgI
NciI
HphI
ScrFI
HphI
Tth111I
TCGAATGCGGGAATCTATTCAATCCGGTCGGAACCCGCGTGTTCACATCGTCTTGTGACG
---------+---------+---------+---------+---------+---------+ 1140
AGCTTACGCCCTTAGATAAGTTAGGCCAGCCTTGGGCGCACAAGTGTAGCAGAACACTGC
BsiEI AciI
XmnI
BsaWI Hpy188I
FauI
MaeIII
TaqI
BsmI
HpaII
NlaIV
Hpy8I
Tsp45I
FauI
BsiEI AciI
HpyCH4IV
HinfI
MmeI
TfiI
BstUI
136
1141
1201
TTGCCCGAGCCTATCTTGCTCAGAGTTCGGTGCGATATGATCGTCAGGCAGGTGCTGCAG
---------+---------+---------+---------+---------+---------+ 1200
AACGGGCTCGGATAGAACGAGTCTCAAGCCACGCTATACTAGCAGTCCGTCCACGACGTC
TaiI
CviJI MwoI
Hpy188I
BspCNI DpnI
BspMI Fnu4HI
AvaI
HpyF10VI
BseMII BstKTI
AarI HpyF10VI
DdeI
MboI
AlwNI
BbvI PstI
BstAPI
MwoI
TseI
SfcI
HpyCH4V
GAGAGCCAACTCGCGTGCTCGTCAATGGCACCGAGGTAA
---------+---------+---------+--------- 1260
CTCTCGGTTGAGCGCACGAGCAGTTACCGTGGCTCCATT
CviJI
BstUI BsiHKAI BanI
MnlI
Cac8I
NlaIV
Bsp1286I
BsaJI
Figure 5.11:
The predicted restriction enzyme map of putative haloacid permease
(dehrP) gene fragment using computer programme-AnnHyb
137
Table 5.3 : Endonucleases that would not restrict haloacid permease (dehrP) gene
AatII
AgeI
ApaLI
AvrII
BcgI
BmrI
BsaXI
BspHI
BstXI
DraIII
FseI
MluI
NheI
PfoI
PsiI
RsrII
SexAI
SrfI
TatI
Acc65I
AhdI
ApoI
BaeI
BciVI
BmtI
BsaXI
BsrBI
BstZ17I
DrdI
FspAI
MscI
NotI
PmeI
PspGI
SacI
SfiI
SspI
XbaI
AclI
AleI
AscI
BaeI
BfrBI
BplI
BsgI
BsrGI
Bsu36I
EagI
HindIII
MseI
NruI
PmlI
PspOMI
SanDI
SgrAI
StuI
XhoI
AfeI
AloI
AseI
BanII
BglII
BpmI
BsiWI
BssHII
BtsI
EcoICRI
HpaI
MslI
NsiI
PpiI
PsrI
SapI
SmaI
SwaI
XmaI
AflII
AloI
AsiSI
BbvCI
BlpI
Bpu10I
BsmFI
BssSI
ClaI
EcoRI
KpnI
NcoI
PacI
PpiI
PsrI
SbfI
SnaBI
TaqII
ZraI
AflIII
ApaI
AvaII
BcgI
Bme1580I
BsaAI
BspEI
BstNI
DraI
EcoRV
MfeI
NdeI
PciI
PpuMI
PvuII
ScaI
SpeI
TaqII
138
5.2.2.2 Analysis of pSC1 Plasmid
This experiment was carried out for checking the plasmid pSC1. pSC1
plasmid was prepared from overnight culture of E.coli strain Novablue grown in
LB/amp medium at 37 °C. The plasmid was then subjected to EcoRI endonuclease.
The EcoRI fragments were analysed by gel electrophoresis. Two sharp bands were
observed indicating that the 9.2 kb plasmid was completely digested. The top band
was a 6.5 kb DNA insert whereas the lower band was a 2.7 kb vector (Figure 5.12).
This analysis confirmed the plasmid constructed by Cairns et al. (1996). pSC1 was
then used for further investigation.
139
Lane
1
2
3
kb
Figure 5.12 : Agarose gel electrophoresis of pSC1
Lane 1:
1kb DNA Ladder
Lane 2:
Undigested pSC1 (control)
Lane 3:
pSC1 digested with EcoRI
140
5.2.2.3 PCR Analysis of Haloacid Permease (dehrP) Gene
The synthesised primers (fnde1 and reco1) were subsequently used for
amplification of the putative dehrP gene using pSC1 as DNA template. Lane 2 of
Figure 5.14 showed that the putative dehrP gene was successfully amplified from
pSC1, yielding a fragment of approximately 1.3 kb in size. The PCR conditions
gave a single fragment, while the control containing no added template DNA or
without PCR primers yielded no amplification product. The 1.3 kb PCR product
then subjected for further analysis.
The PCR product was re-sequenced to confirm that there is no mutation
during PCR. The full sequence was obtained using custom-synthesised
oligonucleotides primers designed from the sequence already obtained (see Figure
5.13 for sequencing strategy). Complete sequence of dehrP gene was obtained and
compared to initial sequence reported by Choy (2004). Alignment between these
two sequences showed 100% identity suggested that the gene did not mutate during
the cloning process (data not shown). The sequence of dehrP gene was then
submitted to Gene Bank with an accession number of AM 260971 (Appendix J). To
express the dehrP gene, it was then cloned into an appropriate plasmid vector.
Figure 5.13: Sequencing strategy of dehrP gene. Primers used for sequencing of
dehrP gene include fnde1, fnde2 and reco1.
141
kb
Lane
Figure 5.14:
1
2
3
4
5
Isolation of the haloacid permease (dehrP) gene from pSC1 via
PCR amplification
Lane 1: 1KbDNA ladder (Promega)
Lane 2: The isolated putative haloacid permease (dehrP) gene.
Lane 3: Negative control omitting DNA template
Lane 4: Negative control omitting fnde1 primer
Lane 5: Negative control omitting reco1 primer
142
5.2.2.4 Engineering of Plasmid pet 43.1a
The PCR product was digested with EcoRI and NdeI before incorporated into
the vector pET 43.1a which was initially digested using the same restriction
enzymes. In Figure 5.16, lane 4 shows pET 43.1a was double digested with NdeI
and EcoRI. Two bands were produced, which were 5.6 kb and 1.6 kb respectively.
The 5.6 kb band was subsequently excised from the gel and used for cloning
whereas the 1.6 kb band which contains the Nus.Tag was discarded. Figure 5.15
showed the 1.6 kb fragment digested with EcoRI and NdeI was discarded, whereas
the 5.6 kb fragment was used as a vector.
Figure 5.15: Plasmid map of pET 43.1a. The 1.6 kb fragment between EcoRI
(654bp) and NdeI (2317) was digested and discarded while the 5.6 kb fragment was
retained to ligate with putative dehrP insert (1.3 kb)
143
kb
Lane
Figure 5.16
1
:
2
3
4
Restriction enzyme digest of pET 43.1a
Lane 1:
1 KB DNA ladder (promega)
Lane 2:
pET 43.1a digested with EcoRI
Lane 3:
pET 43.1a digested with NdeI
Lane 4:
pET 43.1a double digested with EcoRI and NdeI
144
5.2.2.5 Ligation of Haloacid Permease (dehrP) Gene into pET 43.1a
The PCR amplified dehrP gene was then ligated into pET 43.1a plasmid.
The newly constructed plasmid was designated as pHJ (Figure 5.17). The dehrP
gene was controlled by T7 promoter. pHJ was initially transformed into E. coli
strain NovaBlue (Novagen).
In order to confirm whether the newly constructed plasmid carry dehrP, the
pHJ plasmid DNA was prepared from E. coli strain NovaBlue for restriction enzyme
digest analysis (Figure 5.18). The size of pHJ was about 7 kb in total as showed in
lane 3 and 4 after digestion by NdeI or EcoRI, respectively. As a positive control,
Lane 1 showed the haloacid permease (dehrP) gene amplified by PCR. Lane 5
showed that when pHJ was double digested by NdeI and EcoRI, two bands were
produced, which was 5.5 kb and 1.3 kb, respectively. The 1.3 kb band suggested
that the dehrP DNA insert had been successfully cloned into plasmid pET 43.1a.
145
Figure 5.17: The newly constructed plasmid (designated as pHJ) after ligating the
dehrP gene with pET 43.1 plasmid vector. The 1.3 kb fragment of dehrP gene was
showed in green
146
Lane
1
2
3
4
5
6
kb
kb
Figure 5.18: Restriction digestion of pHJ using EcoRI and NdeI
Lane 1:
Reamplified permease gene
Lane 2:
1 KB DNA ladder (Promega)
Lane 3:
pHJ digested with EcoRI
Lane 4:
pHJ digested with NdeI
Lane 5:
pHJ double digested with EcoRI and NdeI
Lane 6:
1 KB DNA ladder (Promega)
147
5.2.2.6 Nucleotide Sequencing of pHJ to Determine the Important Sites
In order to check the various important sites, primer petupstream which
located in plasmid region was used to confirm the sequence of promoter, operator
and ribosomal binding site were intact in the plasmid. As showed in Figure 5.19, the
initiation codon is ATG (methionine, shown in purple). The shine-dalgarno
ribosomal binding site (RBS) upstream of the initiation codon is shown in green.
The lac operator of the plasmid is shown in blue and the T7 promoter is shown in
red.
The newly constructed plasmid (pHJ) can be further characterised by
transforming it into E.coli. Various parameters for expression of DehrP in E.coli
can be optimised. Active DehrP protein will be needed to further characterise this
haloacid transport protein.
148
1 gaaattaata cgactcacta taggggaatt gtgagcggat aacaattccc ctctagaaat
dehrP
61 aattttgttt aactttaaga aggagatata catatgacta cgactctagt cgcccgtact
121 tcatcagccg gtcgcatgac acgcgaggag cgcaaagtga tcttcgcctc ctcgctcggt
181 actgtcttcg aatggtacga tttctttcta tatggctcac tcgccgctat catcggcgcg
241 acctttttca aggactttcc gccagccaca caagccatat tcgcgctcct tgctttcgcg
301 gctggctcgc ttgttcggac tttcggcgca ctcatttttg gccgtctcgg cgatatgatt
361 gggcgcaaat ataccttcct cgtaaccatc ctgatcatgg gtctgtcgac gttcgtggtc
421 ggccttctac cgggttcgga caccattgga cttgcggccc ctacgatcct gattctcctc
481 cgcctgcttc agggcctcgc gctcggcggt gagtacgggg gcgccgctgt ttacgaggcc
541 gagcatgctc cgcccggacg ccgcggcttt tatacaagtt ggattcaaac aactgcgacg
601 ggtggcctct tcctatcgct ccttgtcatt ctcggaactc gctcattgct gggcgaggaa
661 tccttcacgt cttggggctg gcgcgtgccg ttcttgttgt ccgtggtact gctcggcatc
721 tcggtatgga tccgcatgca gctcaacgag tcgcccgtgt tccagcgcat gaaggcagag
781 ggcaaggcgt ccaaggctcc cttgagggag gcatttgcgc actggcctaa cgcaaggctc
841 gctctggttg cccttttcgg catggtcgcc ggccaagccg tggtctggta cacgggccaa
901 ttctatgtgc tgttcttcct gcagagcatt ctcaaggtag acggcttcac gacgacgcta
961 cttatctgtt ggtctttgct gctgggatct ggcttcttcg ttttctttgg ctggttatcc
1021 gatcgcatcg gacgcaagcc gattatgatc gccggctgcc tccttgctgt tgtgacttac
1081 tttccgatct tcgaggcaat caccgaacga gcgaatccca ctttagccaa ggcgatttcg
1141 gaggtgaagg tcacccgtgg tctcccgacc cggtcgaatg cgggaatcta ttcaatccgg
1201 tcggaacccg cgtgttcaca tcgtcttgtg acgttgcccg agcctatctt gctcagagtt
1261 cggtgcgata tgatcgtcag gcaggtgctg caggagagcc aactcgcgtg ctcgtcaatg
1321 gcaccgaggt aa
Figure 5.19: Sequence of haloacid permease (dehrP) gene insert within pHJ
together with some upstream sequence including the T7 promoter, lac operator, and
ribosomal binding site. dehrP gene insert was deposited in Gene Bank with
accession number AM 260971
atg
:
Start Codon
gaagga
:
ribosomal binding site
aattgtgagcggataacaattc
:
lac operator
ttaatacgactcactata
:
T7 promoter
taa
:
Stop Codon
149
5.3
Discussion
Halogenated compounds are extremely important and diverse class of
environmental chemicals. Microbiological research on the degradation of
halogenated compounds has mainly focused on the physiological processes
responsible for their mineralisation and on the enzyme involved in cleavage of the
carbon-halogen bond. The transport system involved in degradation of halogenated
compounds was always neglected. Therefore, more efforts should be channeled to
investigate the transport system involved in dehalogenase system.
In current study, the putative DehrP protein has significant similarities to
various membrane transport proteins in the database strongly suggested that dehrP
encodes a protein that has an uptake function. High sequence identity of DehrP with
putative monochloropropionic acid permease from Agrobacterium sp. NHG3
(Higgins et al., 2005) indicated that these two proteins have a similar
transmembrane structure. Since Agrobacterium sp. NHG3 discussed by Higgins et
al. (2005) belongs to a member of the Rhizobium family, it was not surprise that
haloacid permease gene of Agrobacterium sp. NHG3 and Rhizobium sp. shared high
similarity.
This was further supported by the finding that the coding region of DehrP
contained a sugar (and other) transporters domains. The sugar transporters belong to
a superfamily of membrane proteins responsible for the binding and transport of
various carbohydrates, organic alcohols, and acids in a wide range of prokaryotic
and eukaryotic organisms (Mueckler et al., 1985; Fiegler et al., 1999). These
integral membrane proteins are predicted to comprise twelve membrane spanning
domains. It is likely that the transporters have evolved from an ancient protein
present in living organisms before the divergence into prokaryotes and eukaryotes
(Maiden et al., 1987). A sugar transporter domain found within DehrP agreeing
well with the hypothesis that DehrP protein was a haloacid permease.
150
Studies on the uptake system of haloorganic metabolites are still in its
infancy and not much are known about such systems, this putative dehrP gene
deserves more study. The active uptake of halogenated carboxylic acids was first
observed in Pseudomonas putida PP3 by Slater et al. (1985). Many subsequent
studies on halogen degraders were growth-rate based rather than genetical and
almost all efforts were primarily channeled on the dehalogenase enzymes. At
present, only two transport proteins which functions are for the uptake of
haloorganic compounds was submitted to the international database: (i) the putative
monochloropropionic acid permease from Agrobacterium sp. NHG3 by Higgins et
al. (2005); and (ii) the putative haloacid permease from Burkholderia cepacia by
Tsang (2001).
Nevertheless, accuracy of protein analysis results in this study has some
limitations because comparisons were limited to alignment between amino acid
sequences deduced from nucleotide sequences only. The putative
monochloropropionic acid permease discussed by Higgins et al. (2005) was only a
hypothesised protein whereas other significant matches such as the “General
substrate transporter: Major facilitator superfamily MFS_1” from
Rhodopseudomonas palustris BisB5 (Copeland et al., 2005) was a protein deduced
from genome sequence project and may not be associated with any haloacid uptake
protein. The structure of haloacid permease is yet to be resoled. From the top 6
significant alignment matched (Table 5.2), only haloacid-specific transporter gene
(deh4P) from Burkholderia cepacia MBA4 described by Tsang (2001) had been
cloned for further analysis (Chung et al., 2003).
The haloacid-specific transporter gene was designated as deh4p (Chung et
al., 2003). deh4p is located downstream of the coding sequence of DehIVa. Deh4p
has a putative molecular weight of 59 kDa, whereas the DehrP in this study was 45
kDa in size. The amino acid sequence of deh4p did not show high significant
homology (62 % only) with dehrP. This is not surprising since Rhizobium sp. and
Burkholderia cepacia MBA4 were different bacteria. However, comparison of the
predicted amino acid sequence with the databases showed that Deh4p has the
151
signatures of sugar transport proteins and is an integral membrane protein. These
two characteristics were similar to DehrP and indicate that both DehrP and Deh4P
may play a vital role to mediate the uptake of haloacid into the cell. deh4p had been
cloned and expressed in E. coli and the characterisation of this transporter protein is
still in process as reported by Chung et al. (2003). Therefore, no further comparison
can be carried out between DehrP and Deh4P.
152
5.4
Conclusion
The putative haloacid permease (dehrP) gene from Rhizobium sp. was
isolated by PCR amplification using fnde1 and reco1 primers. The gene was then
subcloned into pET-43.1a(+). The newly constructed plasmid was designated as
pHJ. The nucleotide sequence of dehrP was determined and characterised. The
fifth open reading frame of 1,239 bp encoding for a 412 amino acids was identified
with a molecular weight of 45 kDa and an isoelectric point of 9.78. The putative
DehrP showed 86% sequence homology with putative mono-chloro propionic acid
permease from Agrobacterium sp. NHG3 (Higgins et al., 2005) and 62% sequence
homology with haloacid-specific transporter from Burkholderia cepacia MBA4
(Chung et al., 2003) in the standard databases. Comparison of the predicted amino
acid sequence with the databases also showed that the putative DehrP had the
signatures of sugar transport proteins and was an integral membrane protein. Frame
shift mutation within putative dehrP gene was found. It was caused by nucleotide
insertion at position 1063 and 1073. This is most likely bona fide mutation since
resequencing has been carried out.
153
CHAPTER VI
Concluding Remarks & Suggestions
6.1
Isolation and Characterisation of Haloalkanoic Acid Degrading Bacteria
The results presented in this thesis offer information about the
biodegradation of β-chloro substituted alkanoates (3-chloropropionate) and α-chloro
substituted alkanoates (2,2-dichloropropionate). There are few reports in the
literature concerning catabolism of these compounds, especially 3-chloropropionate.
In addition, the observation made in this research highlighted the differences
between the degradation of β-chloro substituted alkanoates and α-chloro substituted
alkanoates. Bacterial specialised in degrading β-chloro substituted alkanoates such
as Rhodococcus sp. HN2006A in current study was unable to utilise α-chloro
substituted alkanoates whereas bacterial specialised in degrading α-chloro
substituted alkanoates such as Methylobacterium sp. HN2006B in current study was
unable to utilise β-chloro substituted alkanoates.
This general observation agreeing well with the findings from Bollag and
Alexander (1971), Allison (1981), Hughes (1989) and Schwarze et al. (1997), and
indicated the presence in 3-chloropropionate-utiliser or 2,2-dichlorpropionateutiliser a specific enzymes adapted to catalyse conversion of only β-chloro
substituted alkanoates or α-chloro substituted alkanoates, respectively.
154
Soil enrichment and selection experiments clearly demonstrated that 3chloropropionate and 2,2-dichloropionate are readily metabolised by soil
microorganisms, all of which are capable of utilising this substrate as the sole source
of carbon and energy. It was also established by monitoring substrate utilisation
(HPLC analysis) that substrate disappearance correlated directly with the growth of
these organisms.
Most characterised haloacid utilising bacterial to date was carried out in
temperate country such as North America, Japan and many parts of Europe. This
study was the first to isolate and characterised 2,2-dichloropropionate and 3chloropropionate degrading bacteria in tropical country, Malaysia. Therefore,
further characterisation of these two haloalkanoate acid degrading bacteria and their
enzyme system is of interest. O2 electrode studies and whole cell dehalogenase
assays, in conjunction with chromatographic analysis (GC-MS) can be done to
construct a tentative catabolic pathway for 3-chloropropionate and 2,2dichloropropionate. In addition, the identity of other 3-chloropropionate and 2,2dichloropropionate dehalogenase assay products could be determined by isolation of
further mutant strains, defective in certain catabolic enzymes.
Moreover, the precise mechanism whereby dehalogenase attack halogenated
aliphatic acid is not known. Purification of the enzyme to homogeneity, followed by
detailed kinetic, inhibition and chemical modification studies, would help to
elucidate this. Such studies, together with amino acid analysis and protein
sequencing, would indicate also how closely the two dehalogenase were related
from an evolution point of view.
155
6.2
Analysis and Cloning of Rhizobium sp. dehrP Gene
Dehalogenase associated permease has been proposed to mediate the uptake
of haloacid into the cell. In current study, haloacid permease (dehrP) gene of
Rhizobium sp. in pSC1 was cloned. Further characterisation of this transporter
protein is needed. Expression of active DehrP will be carried out. The DehrP
protein will then subjected to gel-shift experiment to test its ability to bind with
haloalkanoate acid molecule. In addition, DehrP structure can be solved by X-ray
diffraction. Only by knowing these structures the mechanism on how DehrP
proteins work can be discovered. However, membrane transport protein was found
to be very difficult to crystallise. Without crystalline samples, x-ray diffraction
could not be used to solve a protein's molecular structure. Of the 20 000 solved
structures in the Protein Data Bank, only a few handfuls correspond to membrane
proteins. Therefore, structure-solving techniques based on nuclear magnetic
resonance (NMR) that do not require crystalline samples and can measure directly
the position and orientation of a protein's alpha helices can be carried out. The
information obtained will shed light on the mechanism of the dehalogenase enzyme
system and helps engineering of enzyme or bacteria used in biotransformation
procedure for chemical and biotechnological industries.
156
REFERENCES
Aislabie, J. M., Richards, N. K., Boul, H. L. (1997). Microbial degradation of DDT
and its residues—a review. New Zealand Journal of Agricultural Research. 40:
269-282.
Aken, B.V., Peres, C. V., Doty, S. L., Jong, M. Y. and chnoor, J. L. (2004).
Methylobacterium populi sp. nov., a novel aerobic, pink-pigmented,
facultatively methylotrophic, methane-utilising bacterium isolated from poplar
trees (Populus deltoidesxnigra DN34). Int J Syst Evol Microbiol. 54: 1191-119.
Alexander, M. (1981). Biodegradation of Chemicals of Environmental Concern.
Science. 211: 132-138.
Allison, N. (1981). Bacterial Degradation of Halogenated Aliphatic Acids. Trent
Polytechnic: Ph.D. Thesis.
Altschul, S. F., Madden, T. L., Schäffer, A. A., Zhang, J., Zhang, Z., Miller, W. and
Lipman, D. J. (1997). Gapped BLAST and PSI-BLAST: A New Generation of
Protein Database Search Programs. Nucleic Acids Res. 25: 3389-3402.
Araoz, B. and Viale, A.A. (2004). Microbial Dehalogenatiob of Polychlorinated
Biphenyls in Aerobic Conditions. Revista Argentina de Microbiologia. 36: 4751.
Ashton, F. M. and Crafts, A. S. (1973). Mode of Action of Herbicides. New York:
Wiley & Sons.
Atlas, R. M. (1996). Principles of Microbiology. 2nd ed. Wm. C. Brown Publishers.
Baggi, G., Bernasconi, S. and Zangrossi, M. (2005). 3-Chloro-, 2,3- and 3,5Dichlorobenzoate Co-Metabolism in a 2-Chlorobenzoate-Degrading
Consortium: Role of 3,5-Dichlorobenzoate as Antagonist of 2-Chlorobenzoate
Degradation: Metabolism and Co-Metabolism of Chlorobenzoates.
Biodegradation. 16(3): 275-282.
Barnum, S. R. (1998). Biotechnology: An Introduction. Wadsworth Publishing
Company.
157
Barriault, D., Durand, J., Maaroufi, H., Eltis, L. D. and Sylvestre, M. (1998).
Degradation of Polychlorinated Biphenyl Metabolites by NaphthaleneCatabolising Enzymes. Appl Environ Microbiol. 64(12): 4637–4642.
Baumann, M. D., Daugulis, A. J. and Jessop, P. G. (2005). Phosphonium Ionic
Liquids for Degradation of Phenol in a Two-Phase Partitioning Bioreactor.
Appl Microbiol Biotechnol. 67: 131–137.
Beckwith, J.R. and Zipser, D. (1970). The Lactose Operon. Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, New York.
Bergmann, J.G. and Sanik, J. Jr. (1957). Determination of Trace Amounts of
Chlorine in Naphtha. Petroleum Chemistry. 29: 241 – 243.
Birge, E. A. (1992). Modern Microbiology Principle & Application. Wm. C. Brown
Publishers.
Bollag, J. M. and Alexander, M. (1971). Bacterial Dehalogenation of Chlorinated
Aliphatic Acids. Soil Biol. Biochem. 3: 91-96.
Bosma, T., Kruizinga, E., deBruin, E., Poelarends, G. J. and Janssen, D. B. (1999).
Utilisation of Trihologenated Propanes by Agrobacterium radiobacter AD1
through Heterologous Expression of the Haloalkane Dehalogenase from
Rhodococcus sp. Strain m15-3. Applied and environmental Micobioogy.
65(10): 4575-4581.
Bradford, M. (1976). A Rapid and Sensitive Method For The Quantitation of
Microgram Quantities of Protein Utilising, The Principle of Protein-Dye
Binding. Analytical Biochemistry, 72, 248-254.
Brokamp, A., B. Happe, and F. R. J. Schmidt. (1997). Cloning and Nucleotide
Sequence of a D,L-Haloalkanoic Acid Dehalogenase Encoding Gene from
Alcaligenes xylosoxidans ssp. denitrificans ABIV. Biodegradation 7:383-396.
Bumpus, J. A. and Aust, S. D. (1987). Biodegradation of DDT [1,1,1-trichloro-2,2bis(4-chlorophenyl)ethane] by the White Rot Fungus Phanerochaete
chrysosporium. Appl Environ Microbiol. 53(9): 2001–2008.
Capela, D., Barloy-Hubler, F., Gouzy, J., Bothe, G., Ampe, F., Batut, J., Boistard, P.,
Becker, A., Boutry, M., Cadieu, E., Dreano, S., Gloux, S., Godrie, T., Goffeau,
A., Kahn, D., Kiss, E., Lelaure, V., Masuy, D., Pohl, T., Portetelle, D., Puhler,
A., Purnelle, B., Ramsperger, U., Renard, C., Thebault, P., Vandenbol, M.,
158
Weidner, S. and Galibert, F. (2001). Analysis of the Chromosome Sequence of
the Legume Symbiont Sinorhizobium meliloti strain 1021. Proc. Natl. Acad.
Sci. 98 (17): 9877-9882
Cairns, S. S., A. Cornish, and R. A. Cooper. (1996). Cloning, Sequencing And
Expression in Escherichia Coli of Two Rhizobium sp. Genes Encoding
Haloalkanoate Dehalogenases of Opposite Stereospecificity. Eur. J. Biochem.
235:744-749
Chapelle, F. H. (1993). Ground Water Microbiology and Geochemistry. New York:
John Wiley and Sons.
Cheah U.B., Lum K.Y. (1994). Pesticide Residue and Microbial Contamination of
Water Resources of Rice Agrosystem in Muda Area. Proceeding of the
Seminar on Impact of Pesticide on the Rice Agrosystem in the Muda area,
Penang: 1-5.
Choy, T. S. (2004). Sequence Analysis of pSC1. Universiti Teknologi Malaysia:
B.Sc. Thesis.
Chung, W. Y. K., Wong, H. P. S. and Tsang, J. S. H. (2003). Cloning and
Characterisation of a 2-Haloacid Permease Gene from Burkholderia cepacia
MBA4. Abstracts of the 1st FEMS Congress of European Microbiologists. June
29 – July3. Ljubljana, Slovenia: 371.
Cole, J. R., Chai, B., Marsh, T. L., Farris, R. J., Wang, Q., Kulam, S. A., Chandra, S.,
McGarrell, D. M., Schmidt, T. M., Garrity, G. M. and Tiedje, J. M. (2003).
The Ribosomal Database Project (RDP-II): Previewing a New Auto Aligner
That Allows Regular Updates and The New Prokaryotic Taxonomy. Nucleic
Acids Res. 31(1): 442-443.
Commandeur, L.C. and Parsons, J.R.(1990) Degradation of Halogenated Aromatic
Compounds. Biodegradation.1(2-3):207-20.
Copeland, A., Lucas, S., Lapidus, A., Barry, K., Detter, J. C., Glavina, T., Hammon,
N., Israni, S., Pitluck, S. and Richardson, P. (2005). Sequencing of the Draft
Genome andAssembly of Rhodopseudomonas palustris BisB5. US DOE Joint
Genome Institute (JGI-PGF): unpublished
159
Curragh, H., Flynn, O., Larkin, M. J., Stafford, T. M., Hamilton, J. T. G. and Harper,
D. B. (1994). Haloalkane Degradation and Assimilation by Rhodococcus
rhodochrous NCIMB 13064. Microbiology 140:1433-1442.
Daugulis, A. J. and McCracken, C. M. (2003). Microbial Degradation of High and
Low Molecular Weight Polyaromatic Hydrocarbons in a Two-Phase
Partitioning Bioreactor by Two Strains of Sphingomonas sp. The Netherlands:
Kluwer Academic Publishers. 1441–1444.
De Schrijver, A., Nagy, I., Schoofs, G., Proost, P., Vanderleyden, J., van Pee, K. H.
(1997). Thiocarbamate Herbicide-Inducible Nonheme Haloperoxide of
Rhodococcus erythropolis N186/21. Applied and Environmental Microbiology.
63(5): 1911-1916.
Ejlertsson, J., Johansson, E., Karlsson, A., Meyerson, U. and Svensson, B. H. (1996).
Anaerobic Processes for Bioenergy and Environment (Part II). Antonie van
Leeuwenhoek (Historical Archive). 69(1): 67-74.
Eller, G. and Frenzel, P. (2001). Changes in Activity and Community Structure of
Methane-Oxidising Bacteria over the Growth Period of Rice. Applied and
Environmental Microbiology. 67(6): 2395-2403.
Embley, T. M., Stackebrant, E. (1996). The Use of 16S Ribosomal RNA Sequences
in Microbial Ecology. In: Pickup, R.W. and Saunders, J. R. ed. Molecular
Approaches to Environmental Microbiology. Ellis Horwood. 39-55.
Farhana, L., Fulthorpe, R. R., Harbour, C., and New, P. B. (1998). Monoclonal
Antibodies to 2,4-Dichlorophenol Hydroxylase as Probes for the 2,4-DDegradative Phenotype. Can. J. Microbiol. 44: 920-928.
Fetzner, S. (1998). Bacterial Dehalogenation. Applied Microbiology and
Biotechnology. 50:633-657
Fournier, D., Halasz, A., Spain, J., Fiurasek, P. and Hawari, J. (2002). Determination
of Key Metabolites during Biodegradation of Hexahydro-1,3,5-Trinitro-1,3,5Triazine with Rhodococcus sp. Strain DN22. Applied and Environmental
Microbiology. 68(1): 166-172.
Foy, C. L. (1975). The Chlorinated Apliphatic Acids in Herbicides-Chemistry,
Degradation and Mode of Action. In: Kearney, P. C. and Kaufman, D. D. ed.
160
Herbicides: Chemistry, degradation And Mode Of Action Second Edition. New
York: M. Dekker. 339-452.
Gallego, V., García, M. T. and Ventosa, A. (2005). Methylobacterium variabile sp.
Nov., A Methylotrophic Bacterium Isolated from an Aquatic Environment. Int
J Syst Evol Microbiol. 55: 1429-1433.
Galli, R., and T. Leisinger. (1985). Specialised Bacterial Strains for the Removal of
Dichloromethane from Industrial Waste. Conservation and Recycling 8:91-100.
Gasteiger, E., Hoogland, C., Gattiker, A., Duvaud, S., Wilkins, M.R., Appel, R.D.,
Bairoch, A. (2005). Protein Identification and Analysis Tools on the ExPASy
Server. In: Walker, J.M. ed. The Proteomics Protocols Handbook, Totowa, N.
J.: Humana Press. 571-607
Ghosal, D., You, I. S., Chatterjee, D. K. and Chakrabarty, A. M. (1985). Microbial
Degradation of Halogenated Compound. Science. 228: 135-142.
Golovleva, L. A., Aharonson, N., Greenhalgh, R., Sethunathan, N. and Vonk, J. W.
(1990). The Role And Limitations Of Microorganisms In The Conversion Of
Xenobiotics. Pure & Appl. Chem. 62(2): 351-364.
Gribble G.W. (1994) The Natural Production of Chlorinated Compounds. Environ
Sci Technol 28:310A-319A
Gschwend, P., Macfarlane, J. K., Newman, K. A. (1985). Volatile Halogenated
Organic Compounds Released to Seawater from Temperate Marine
Macroalgae. Science. 227: 1033-1035.
Han, S. O. and New, P. B. (1994). Effect of Water Availability on Degradation of
2,4-Dichlorophenoxyacetic Acid (2,4-D) by Soil Microorganisms. Soil Biology
& Biochemistry. 26: 1689-1697.
Hardman, D. J. and Slater, J. H. (1981). Dehalogenases in Soil Bacteria. Journal of
General Microbiology. 123: 117-128.
Hardy, S. P. (2002). Human Microbiology. Taylor & Francis.
Hareland, W. A., R. L. Crawford, P. J. Chapman, and S. Daley. 1975. Metabolic
Function and Properties of 4-Hydroxyphenylacetic Acid 1-Hydroxylase from
Pseudomonas acidovorans. J. Bacteriol. 121:272-285.
Haroune, N., Combourieu, B., Desse, P., Sancelme, M., Reemtsma, T., Kloepfer, A.,
Diab, A., Knapp J. S., Baumberg, S. and Delortu, A. M. (2002). Benzothiazole
161
Degradation by Rhodococcus pyridinovorans Strain PA: Evidence of a
Catechol 1,2-Dioxgenase Activity. Applied and Environmental Microbiology.
68(12): 6114-6120.
Hashimoto, K. and Simon, H. (1975). Reductive Dehydrodehalogenation of βHalogenated Fatty Acids and Stereoselective Hydrogenation of α,βUnsaturated Fatty Acids by Clostridium kluyveri. Angewandte Chemie,
International Edition. 14(2): 106 – 107.
Higgins, T. P., Hope, S. J., Effendi, A. J., Dawson, S. and Dancer, B. N. (2005).
Biochemical and Molecular Characterisation of the 2,3-Dichloro-1-Propanol
Dehalogenase and Stereospecific Haloalkanoic Dehalogenases from a
Versatile Agrobacterium sp. Biodegradation. 16(5): 485-492.
Higson, F. K. (1991). Degradation of Xenobiotics by White Rot Fungi. Rev Environ
Contam Toxicol. 122: 111-52.
Hill, K. E., Marchesi, J. R. and Weightman, A. J. (1999). Investigation of two
Evolutionarily Unrelated Halocarboxylic Acid Dehalogenase Gene Families. J.
Bacteriol. 181: 2535-2547.
Hirahara, Y., Ueno, H. and Nakamuro, K. Comparative Photodegradation Study of
Fenthion and Disulfoton under Irradiation of Different Light Sources in
Liquid- and Solid-Phases. Journal of Health Science. 47(2): 129.
Hirsch, P. & Alexander, M. (1960). Microbial Decomposition of Halogenated
Propionic and Acetic Acids. Canadian Journal of Microbiology. 6, 241-249
Holt, J.G., Krieg, N.R., Sneath, P.H.A., Staley, J.T. and Willianms, S.T. (1994).
Bergey’s Manual of Determinative Bacteriology. 9th edition. Baltimo:
Williams and Wilkins.
Hughes, S. (1988). Microbial Growth on 3-Chloropropionic Acid. University of
Wales: Ph.D. Thesis
Hutzinger, O., Veerkamp, W. (1981). Xenobiotic Chemicals with Pollution Potential.
In: Leisinger, T., Cook, A. M. and Butter, T. and Nuesch, J. ed. Microbial
Degradation of Xenobiotic and Recalcitrant Compounds. London: Academic
Press. 3-45.
162
Hymer, C.B., Cheever, K.L. (2004). Development Of A Gas Chromatograpgoc Test
for the Quantification of the Biomaeker 3-Bromopropionic Acid in Human
Urine. Journal of Chromatography B. 802:361-366
Jacob, F. and Monod, J.L. (1961). Genetic Regulatory Mechanisms in the Synthesis
of Proteins. J. Mol. Biol. 3:318-356
Janssen, D. B., A. Scheper, L. DiJkhuizen, and B. Witholt. (1985).Degradation of
Halogenated Aliphatic Compounds by Xanthobacter autotrophicus GJ10. Appl.
Environ. Microbiol. 49:673-677
Janssen, D. B., Bosma, T. and Poelarends, G. J. (1997). Diversity and Mechanisms
of Bacterial Dehalogenation Mechanisms. In: Janssen, D. B., Soda, K. and
Weaver, R. ed. Mechanisms of Biohalogenation and Dehalogenation.
Amsterdam, The Netherlands: Royal Netherlands Academy of Arts and
Sciences. 119-128.
Janssen, D. B., Oppentocht, J. E. and Poelarends, G. J. (2001). Microbial
Dehalogenation. Curr. Opin. Biotechnol. 12: 254-258.
Janssen, D. B., S. Keuning, and B. Witholt. (1987). Involvement of a Quinoprotein
Alcohol Dehydrogenase and an NAD-Dependent Aldehyde Dehydrogenase in
2-Chloroethanol Metabolism in Xanthobacter autotrophicus GJ10. J. Gen.
Microbiol. 133:85-92.
Janssen, D. B., Van Der Ploeg, J. R. and Pries, F. (1995). Genetics and Biochemistry
of Dehalogenating Enzymes. Annu. Rev. Microbiol. 48: 163-191.
Javorekova, S., Stevlikova, T., Labuda, R. and Ondrisik, P. (2001). Influence Of
Xenobiotics On The Biological Soil Activity. Journal of Central European
192 Agriculture. 2: 3-4.
Jensen, H.L. (1957a). Decomposition of Chloro-Substituted Aliphatic Acids by Soil
Bacteria. Canadian Journal of Microbiology, 3: 151-164
Jensen, H.L. (1957b). Decomposition of Chloro-Organic Acids by Fungi. Nature,
180: 1416
Jensen, H. L. (1960). Decomposition of Chloroacetates and Chloropropionates by
Bacteria. Acta Agriculturae Scandinavica. 10: 83-103.
163
Jensen, H. L. (1963). Carbon Nutrition of Some Microorganisms Decomposing
Halogen-Substituted Aliphatic Acids. Acta Agriculturae Scandinavica. 13:
404-412.
Kaneko, T., Nakamura, Y., Sato, S., Minamisawa, K., Uchiumi, T., Sasamoto, S.,
Watanabe, A., Idesawa, K., Iriguchi, M., Kawashima, K., Kohara, M.,
Matsumoto, M., Shimpo, S., Tsuruoka, H., Wada, T., Yamada, M. and Tabata,
S. (2002). Complete Genomic Sequence of Nitrogen-Fixing Symbiotic
Bacterium Bradyrhizobium japonicum USDA110. DNA Res. 9 (6):189-197
Kayser, M. (2001) Genes and Proteins Associated with Dichloromethane
Metabolism in Mwthrylobacterium Dichlotomethanicum DM4. Swiss Federal
Institute of Technology Zurich: Ph.D. Thesis.
Kiely, T., Donaldson, D., Grube, A. (2004). Pesticides Industry Sales and Usage:
2000 and 2001 Market Estimates. EPA-733-R-04-001. Washington, DC:Office
of Pesticide Programs, U.S. Environmental Protection Agency.
Kim, S. and Picardal, F. (2001). Microbial Growth on Dichlorobiphenyls
Chlorinated on Both Rings as a Sole Carbon and Energy Source. Applied and
Environmental Microbiology. 67(4): 1953–1955.
Kitagawa, W., Takami, S., Miyauchi, K., Masai, E., Kamagata, Y., Tiedje, J. M. and
Fukuda, M. (2002). Novel 2,4-Dichlorophenoxyacetic Acid Degradation
Genes from Oligotrophic Bradyrhizobium sp. strain HW13 Isolated from a
Pristine Environment. J Bacteriol. 184(2): 509-18.
Konstantinou, I.K., Zarkadis, A.K. Albanis, T.A. (2001). Photodegradation of
Selected Herbicides in Various Natural Waters and Soils under Environmental
Conditions. Journal of Environmental Quality. 30:121-130.
Kosono, S., Maeda, M., Fuji, F., Aral, H., Kudo, T. (1997). Three of the Seven bphC
Genes of Rhodococcus erythropolis TA421, Isolated from a Termite
Ecosystem, Are Located on an Indigenous Plasmid Associated with Biphenyl
Degradation. Applied and Environmental Microbiology. 63(8): 3282-3285.
Kurihara, T., Liu, J.Q., Nardi-Dei, V., Koshikawa, H., Esaki, N., Soda., K. (1995).
Comprehensive Site-Directed Mutagenesis of L-2-halo Acid Dehalogenase to
Probe Catalytic Amino-Acid-Residues. J. Biochem. 117:1317–132
164
Laemmlli, U.K. (1970). Cleavage of Structural Proteins During the Assembly of the
Head of Bacteriophage T4. Nature, 227, 680-685
Larimer, F. W., Chain, P., Hauser, L., Lamerdin, J., Malfatti, S., Do, L., Land, M. L.,
Pelletier, D. A., Beatty, J. T., Lang, A. S., Tabita, F. R., Gibson, J. L., Hanson,
T. E., Bobst, C., Torres, J. L., Peres, C., Harrison, F. H., Gibson, J. and
Harwood, C. S. (2004). Complete Genome Sequence of the Metabolically
Versatile Photosynthetic Bacterium Rhodopseudomonas palustris. Nat.
Biotechnol. 22 (1), 55-61
Leasure, J. K. (1964). Metabolism of herbicides: The Halogenated Aliphatic Acids.
Journal of Agricutural and Food Chemistry. 12: 40-43.
Leigh, J. A. (1986). Studies on Bacterial Dehalogenase. Trent Polytechnic,
Nottingham, U.K: Ph.D. Thesis.
Leong, K. H., Mohd, M. A., Abdullah, A. R., Anuradha, S. and Tan, L. L. (2002).
UNU Project on EDC Pollution in the East Asian Coastal Hydrosphere: The
Monitoring of 12 Different Pesticides in the Selangor River, Malaysia from
August 2002 to October 2002. Kuala Lumpur, Malaysia: Alam Sekitar
Malaysia Sdn. Bhd.
Lidstrom, M. E. and Chistoserdova, L. (2002). Plants in the Pink: Cytokinin
Production by Methylobacterium. Journal of Bacteriology. 184(7): 1818.
Liu, J. Q., Kurihara, T., Miyagi, M., Esaki, N. and Soda, K. (1995). Reaction
Mechanism of L-2-Haloacid Dehalogenase of Pseudomonas sp. YL. J. Biol.
Chem. 270: 18309-18312.
Madigan, M. T., Martinko, J. M., Parker, J. (2000). Brock Biology of
Microorganism. 9th ed. Prentice Hall. 360-452.
Maeda, M., Chung, S.Y., Song, E., Kudo, T. (1995) “Multiple Genes Encoding 2,3Dihydroxybiphenyl 1,20dioygenase in Gram-Positive Polychlorinated
Biphenyl-Degrading Bacterium Rhodococcus erythropolis TA421. Isolated
from a Termite Ecosystem” Applied and Environmental Microbiology. 61(2):
549-555
Magee, L.A., Colmer, A.R. (1959). Decomposition of 2,2-dichloropropionic Acid by
Soil Bacteria. Canadian Journal of Microbiology, 5, 255-260
165
Maidak, B. L., Larsen, N., McCaughey, M. J., Overbeek, R., Olsen, G. J., Fogel, K.,
Blandy, J. and Woese, C. R. (1994). The Ribosomal Database Project. Nuclei
Acids Res. 22(17): 3485–3487.
Maiden, M.C., Davis, E.O., Baldwin, S.A., Moore, D.C., Henderson, P.J. (1987).
Mammalian and Bacterial Sugar Transport Proteins are Homologous. Nature.
325: 641-643
Marchesi J.R. (2003). A Microplate Fluorimetric Assay for Measuring
Dehalogenase Activity. Journal of microbiological methods. 55: 325-329
Marchler, B.A., Bryant S.H. (2004). CD-Search: Protein Domain Annotations on the
Fly. Nucleic Acids Res. 32:327-331.
Mathews, C. K., Holde, K. E., Ahern, K. G. (2000). Biochemistry. Addison-Wesley
Publishing Company.
McDonald, I.R., Warner, K.L., McAnulla, C., Woodall, C.A., Oremland, R.S.,
Murrell, J.C. (2002). “A Review of Bacterial Methyl Halide Degradation:
Biochemistry, Genetics and Molecular Ecology.” Environ Microbiol. 4(4):193203.
McGrath, J. and Harfoot, C.G. (1997). Reductive Dehalogenation of Halocarboxylic
Acids by the Phototrophic Genera Rhodospirillum and Rhodopseudomonas.
Applied and environmental microbiology. 63(8):3333–3335
Miller, J. H. (1972). Experiments in Molecular Genetics. Cold Spring Harbor, N.Y:
Cold Spring Harbor Laboratory.
Mueckler, M., Caruso, C., Baldwin, S.A., Panico, M., Blench. I., Morris, H.R.,
Allard, W.J., Lienhard, G.E., Lodish, H.F. (1985). Sequence and Structure of a
Human Glucose Transporter. Science 229: 941-945
Nardi-Dei, V., Kurihara, T., Park, C., Miyagi, M., Tsunasawa, S., Soda, K., Esaki, N.
(1999). DL-2-Haloacid Dehalogenase from Pseudomonas sp. 113 is a New
Class of Dehalogenase Catalysing Hydrolytic Dehalogenation not Involving
Enzyme–Substrate Ester Intermediate. J Biol Chem. 274:20977-20981.
Olaniran, A.O., Babalola, G.O., Okoh, A.I. (2001). “Aerobic Dehalogenation
Potentials of Four Bacterial Species Isolated from Soil and Sewage Sludge”.
Chemosphere. 45(1): 45-50
166
PIC Circular XII, (2000), Appendix I: Synopsis of Notifications of Final Regulatory
Action Received Under the Interim PIC Procedure, Part A: Summary of Each
Notification of Final Regulatory Action That has been Verified to Contain All
the Information Required by Annex I of the Convention, United Nations
Environment Programme.
PIC Circular XIV, (2001), Appendix IV: Listing of All Importing Country
Responses Received from Parties as of November 2001, United Nations
Environment Programme
Poelarends, G.J., Wilkens, M., Larkin, M.J., Van Elsas J.D. and Janssen D.B. (1998)
Degradation of 1,3-Dichloropropene by Pseudomonas cichorii 170 Applied
and Environmental Microbiology. 64(8):2931–2936
Priest, F. and Austin, B. (1993). Modern Bacterial Taxonomy. 2nd ed. Chapman &
Hall.
Quensen III, J. F., Mueller, S. A., Jain, M. K. and Tiedje, J. M. (1998). Reductive
Dechlorination of DDE to DDMU in Marine Sediment Microcosms. Science.
280(5364): 722 – 724.
Reeves, R. H. (1997). Phylogenetic Analysis and Implications for Subsurface
Microbiology. In: Amy, P. S. and Haldeman, D. L. ed. The Microbiology of the
Terrestrial Deep Subsurface. Lewis Publishers. 174-176.
Reineke, W. (1984). Microbial Degradation of Halogenated Aromatic Compounds.
In: Gibson, D.T. and Dekker, M. ed. Microbial Degradation of Organic
Compounds. New York. 310-360.
Ridder, I.S., Rozeboom, H.J., Kalk, K.H., Dijkstra, B.W. (1999). Crystal Structures
of Intermediates in the Dehalogenation of Haloalkanoates by L-2-Haloacid
Dehalogenase. J Biol Chem. 274:30672-30678.
Ritter, L., Solomon, K. R. and Foget, J. (1995). Persistent Organic Pollutants: An
Assessment Report on DDT, Aldrin, Dieldrin, Endrin, Chlordane, Heptachlor,
Hexachlorobenzene, Mirex and Toxaphene. United Nations, Geneva: WHO. 144.
Rozgaj, R. (1994). Microbial Degradation of Xenobiotics in the Environment. Arh
Hig Rada Toksikol. 45(2): 189-98.
167
Schwarze, R., Brokamp, A., Schmidt, F.R.J. (1997). “Isolation and Characterisation
of Dehalogenases from 2,2-Dichloropropionate-Degrading Soil Bacteria”.
Current Microbiology. 34:103-109
Senior, E., Bull, A. T. and Slater, J. H. (1976). Enzyme Evolution in a Microbial
Community Growing on the Herbicide Dalapon. Nature. 263: 476-479.
Seto, M., Masai, E., Ida, M., Hatta, T., Kimbara, K., Fukada, M., Yano, K. (1995).
Multiple Polychlorinated Biphenyl Transformation Systems in the GramPositive Bacterium Rhodococcus sp. Strain RHA1. Applied and Environmental
Microbiology. 61(1): 5041-4513
Shao, Z. Q. and Behki, R. (1995). Cloning of the Genes for Degradation of the
Herbicides EPTC (S-Ethyl Dipropylthiocarbamate) and Atrazine from
Rhodococcus sp. Strain TE1”. Applied and Environmental Microbiology. 61(5):
2061-2065.
Shuler, M. L. and Kargi, F. (2002). Bioprocess Engineering: Basic Concepts.
Second Edition. Upper Saddle River, New Jersey : Prentice-Hall, Inc.
Silver, S, Walderhaug, M. (1992). Gene Regulation of Plasmid and Chromosome
Determined Inorganic Ion Transport in Bacteria. Microbiol Rev. 56(1):195–
228.
Siuda, J. F. and De Bernardis, J. F. (1973). Naturally Occurring Halogenated
Organic Compounds. Lloydin. 36: 107-143.
Skelton, P. (1993). Evolution, a Biological and Palaeontalogical approach. Addison
Wesley Publishing Company.
Slater, J. H., Lovatt, D., Weightman, A. J., Senior, E. and Bull, A. T. (1979). The
Growth of Pseudomonas putida on Chlorinated Aliphatic Acids and its
Dehalogenase Activity. J. Gen. Microbiol. 114: 125-136.
Slater, J. H., Weightman, A. J. and Hall, B.G. (1985). Dehalogenase Genes of
Pseudomonas putida PP3 on Chromosomally Located Transposable Elements.
Mol. Biol. Evol. 2: 557-567.
Slater, J.H. (1994) Microbial Dehalogenation of Haloaliphatic Compound. In:
Biochemistry of Microbial Degradation. Eds: Ratledge, C. Kluwer Academic
Publ. The Netherlands.
168
Slater, J.H., Bull, A.T., Hardmam, D.J. (1995). Microbial Dehalogenation.
Biodegradation. 6(3):181-189
Solomons, T.W.G. (1994). Fundamentals of Organic Chemistry. 4th ed. New York:
John Wiley & Sons, Inc.
Sonnhammer, E.L.L., von Heijne, G., and Krogh, A. (1998). A Hidden Markov
Model for Predicting Transmembrane Helices In Protein Sequences. In:
Glasgow, E.J., Littlejohn, T., Major, F., Lathrop, R., Sankoff, D. and Sensen C.
ed. Conf. on Intelligent Systems for Molecular Biology, CA: AAAI Press. 175182
Spirin, A. S. (1986). Ribosome Structure and Protein Biosynthesis. The Benjamin /
Cummings Publishing Company. 83-183.
Stanbury, P. F. and Whitaker, A. (1984). Principles of Fermentation Technology.
Headington Hill Hall, Oxford : Pergamon Press Ltd.
Staub, D.K. and Kohler, H.P.E. (1989). Microbial Degradation of β-Chlorinated
Four-Carbon Aliphatic Acids. Journal of Bacteriology. 1428-1434
Stringfellow, J.M., Cairns, S.S., Cornish, A., Cooper, R.A.(1997). Haloalkanoate
Dehalogenase II (DehE) of a Rhizobium sp.--Molecular Analysis of the Gene
and Formation of Carbon Monoxide from Trihaloacetate by the Rnzyme. Eur J
Biochem. 250(3):789-93.
Studer, A. (2001). Aerobic Mocrobial Degradation of Chloromethane. Swiss Feferal
Institute of Technology Zurich: Ph.D. Thesis.
Talaro, K.P., Talaro, A. (2002). Foundations in Microbiology, 4th edition. MGraw
Hill
Tauber, M., Rosen, R., Shimshon, B. (2001). Whole-Cell Biodetection of
Halogenated Organic Acids. Talanta 55: 959-964
Thomas, A. W., Slater, J. H. and Weightman, A. J. (1992a). The Dehalogenase Gene
dehI from Pseudomonas putida strain PP3 is Carried on an Unusual Mobile
Genetic Element, designated DEH . J. Bacteriol. 174: 1932-1940.
Thomas, A. W., Topping, A. W., Slater, J. H. and Weightman, A. J. (1992b).
Localisation and Functional Analysis of Structural and Regulatory
Dehalogenase Genes , Arried on DEH, a Mobile Genetic Element from
Pseudomonas putida strain PP3. J. Bacteriol. 174: 1941-1947.
169
Topping, A. W., Thomas, A. W., Slater, J. H. and Weightman, A. J. (1995). The
Nucleotide Sequence of a Transposable Haloalkanoic Acid Dehalogenase
Regulatory Gene (dehRI) from Pseudomonas putida strain PP3 and its
Relationship with
54
-Dependent Activators. Biodegradation. 6: 247-255.
Towner, K. J. and Cockayayne, A. (1993). Molecular Methods for microbial
Identification and Typing. Chapman & Hall.
Tsang, J.S.H. and Pang, B.C.M. (2001). “Mutagenic Analysis of Conserved
Residues in Dehalogenese IVa of Burkholderia cepacia MBA4”. FEMS
Microbiology Letters. 204: 135-140.
Umland, J. B. and Bellama, J. M. (1999). General Chemistry. 3rd ed. U.S.A:
International Thomson Publishing, Inc. 282 – 291.
van der Ploeg, J., van Hall, G., Janssen, D.B. (1991). Characterisation of the
Haloacid Dehalogenase from Xanthobacter autotrophicus GJ10 and
sequencing of the dhlB Gene. J. Bacteriol. 173(24):7925-33.
Van der Ploeg, J., and Jassen D.B. (1995). Sequence Analysis of the Upstream
Region of dhlB, the Gene Encoding Haloalkanoic Acid Dehalogenase of
Xanthobacter autotrophicus GJ10. Biodegradation 6: 257-264
Van Pée, K.H., and Unversucht, S. (2003). “Biological Dehalogenation and
Halogenation Reactions.” Chemosphere. 52:299 – 312.
Vannelli, T., Studer, A., Kertesz, M. and Leisinger, T. (1998) Chloromethane
Metabolism by Methylobacterium sp. Strain CM4. Appl. Environ. Microbiol.
64:1933-6.
Vannelli, T., Messmer, M., Studer, A., Vuilleumier, S. and Leisinger, T. (1999) A
Corrinoid-Dependent Catabolic Pathway for Growth of a Methylobacterium
Strain with Chloromethane. Proc. Natl. Acad. Sci. USA. 96:4615-4620.
Verhoef, R., de Waard, P., Schols, H.A., Siika-aho, M., Voragen, A.G.J. (2003)
“Methylobacterium sp. Isolated from a Finnish Paper Machine Produces
Highly Pyruvated Galactan Exopolysaccharide” Carbohydrate Research 338:
1851/1859
Wade, L. G. Jr. (2003). Organic Chemistry. 5th ed. New Jersey: Pearson Education,
Inc.
170
Weightman, A. J., Slater, J. H. and Bull, A. T. (1979). The Partial Purification of
two Dehalogenases from Pseudomonas putida PP3. FEMS Microbiol. Lett. 6:
231-234.
Weightman A.J., Weightman A.L. and Slater, J.H. (1982). “Stereospecificity of 2Monochloropropionate Dehalogenation by the Two Dehalogenases of
Pseudomonas putida PP3:Evidence of Two Different Dehalogenation
Mechanisms”. J. Gen. Microbiol. 131. 1755-1762.
Weightman, A.J., Slater, J.H. (1987). The Problem of Xenobiotics and Recalcitrance.
In: Lynch, J.M., and Hobbie, J.E. eds, Microorganism in Action: Concepts,
Application in Microbial Ecology.” Blackwell Scientific Publication. 322-347.
Wheeler, D.L., Church, D.M., Federhen, S., Lash, A.E., Madden, T.L., Pontius, J.U.,
Schuler, G.D., Schriml, L.M., Sequeira, E, Tatusova, T.A. and Wagner, L.
(2003). Database Resources of the National Center for Biotechnology. Nucleic
Acids Res. 31(1):28-33.
Whyte, L.G., Hawari, J., Zhou, E., Bourbonnier, L., Inniss, W.E., Greer, C.W. (1998)
“Biodegradation of Variable-Chain-Length Alkanes at Low Temperatures by a
Psychrotrophic Rhodococcus sp”. Applied and Environmental Microbiology.
64(7): 2578-2584
Woese, C. R. (1987). Bacterial Evolution. Microbiological Reviews. 51(2): 221–271.
Worsey, M. J., and Williams, P. A. (1975). Metabolism of Toluene and Xylenes by
Pseudomonas putida (arvilla) mt-2: Evidence for a New Function of the TOL
Plasmid. J. Bacteriol. 124:7-13.
Xu, X.Y., Ren, Y. H., Huang, X., Zhen, P. (2004). Advanced on Microbial
Biodegradation of Chlorinated Aromatics and their Molecular Mechanism.
Journal Of Zhejiang University. 30(6): 684-689.
Yokota, T., Fuse, H., Omori, T , and Minoda, Y. (1986). “Microbial Dehalogenation
of Haloalkanes Mediated by Oxygenases or Halidohydrolase.” Agricul. and
Biol. Chem. 50:453 – 460.
Zabik, M. J., Leavitt, R. A. and Su, G. C. C. (1976). Photochemistry of Bioactive
Compounds: A Review of Pesticide Photochemistry. Annual Review of
Entomology. 21: 61-79.
171
APPENDIX A
Rhodococcus sp. HN2006A partial 16S rRNA gene
Accession#: AM231909
Status:
not confidential
Description: Rhodococcus sp. HN2006A partial 16S rRNA gene
Accession#: AM231910
Status:
not confidential
Description: Methylobacterium sp. HN2006B partial 16S rRNA gene
ID
AM231909
standard; genomic DNA; PRO; 1437 BP.
AC
AM231909;
SV
AM231909.1
DT
24-FEB-2006 (Rel. 86, Created)
DT
24-FEB-2006 (Rel. 86, Last updated, Version 1)
DE
Rhodococcus sp. HN2006A partial 16S rRNA gene
KW
16S ribosomal RNA; 16S rRNA gene.
OS
Rhodococcus sp. HN2006A
OC
Bacteria; Actinobacteria; Actinobacteridae; Actinomycetales;
OC
Corynebacterineae; Nocardiaceae; Rhodococcus.
RN
[1]
RP
1-1437
RA
Ng H.;
RT
;
RL
Submitted (22-FEB-2006) to the EMBL/GenBank/DDBJ databases.
RL
Huyop F.Z., Biology Department, Faculty of Science, UniversitiTeknologi
RL
Malaysia, Universiti Teknologi Malaysia, Skudai, Johor, 81310, MALAYSIA.
RN
[2]
RA
Ng H.;
RT
"Degradation of herbicide by soil microorganism and cloning of a haloacid
RT
permease gene";
RL
Unpublished.
FH
Key
Location/Qualifiers
FH
FT
source
1..1437
FT
/country="Malaysia"
FT
/db_xref="taxon:373518"
FT
/mol_type="genomic DNA"
FT
/organism="Rhodococcus sp. HN2006A"
FT
/strain="HN2006A"
FT
/isolation_source="agriculture soil"
FT
rRNA
<1..>1437
FT
/gene="16S rRNA"
FT
/product="16S ribosomal RNA"
XX
SQ
Sequence 1437 BP; 316 A; 354 C; 494 G; 273 T; 0 other;
atgcaagtcg aacgatgaag cccagcttgc tgggtggatt agtggcgaac gggtgagtaa
60
cacgtgggtg atctgccctg cactctggga taagcctggg aaactgggtc taataccgga
120
tatgacctcg ggatgcatgt ccaggggtgg aaagtttttc ggtgcaggat gagcccgcgg
180
cctatcagct tgttggtggg gtaatggcct accaaggcga cgacgggtag ccggcctgag
240
agggcgaccg gccacactgg gactgagaca cggcccagac tcctacggga ggcagcagtg
300
gggaatattg cacaatgggc gcaagcctga tgcagcgacg ccgcgtgagg gatgacggcc
360
ttcgggttgt aaacctcttt cacccatgac gaagcgcaag tgacggtagt gggagaagaa
420
gcaccggcca actacgtgcc agcagccgcg gtaatacgta gggtgcgagc gttgtccgga
480
attactgggc gtaaagagct cgtaggcggt ttgtcgcgtc gtctgtgaaa tcccgcagct
540
caactgcggg cttgcaggcg atacgggcag actcgagtac tgcaggggag actggaattc
600
ctggtgtagc ggtgaaatgc gcagatatca ggaggaacac cggtggcgaa ggcgggtctc
660
tgggcagtaa ctgacgctga ggagcgaaag cgtgggtagc gaacaggatt agataccctg
720
gtagtccacg ccgtaaacgg tgggcgctag gtgtgggttt ccttccacgg gatccgtgcc
780
gtagccaacg cattaagcgc cccgcctggg gagtacggcc gcaaggctaa aactcaaagg
840
aattgacggg ggcccgcaca agcggcggag catgtggatt aattcgatgc aacgcgaaga
900
accttacctg ggtttgacat gtaccggacg actgcagaga tgtggttccc cttgtggccg
960
gtagacaggt ggtgcatggc tgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg
1020
caacgagcgc aacccttgtc ctgtgttgcc agcacgtgat ggtggggact cgcaggagac
1080
tgccggggtc aactcggagg aaggtgggga cgacgtcaag tcatcatgcc ccttatgtcc
1140
agggcttcac acatgctaca atggtcggta cagagggctg cgataccgtg aggtggagcg
1200
aatcccttaa agccggtctc agttcggatc ggggtctgca actcgacccc gtgaagtcgg
1260
agtcgctagt aatcgcagat cagcaacgct gcggtgaata cgttcccggg ccttgtacac
1320
accgcccgtc acgtcatgaa agtcggtaac acccgaagcc ggtggcctaa ccccttgtgg
1380
gagggagccg tcgaaggtgg gatcggcgat tgggacgaag tcgtaacaag gtagccg
1437
172
APPENDIX B
Growth curve of Rhodococcus sp. HN2006A in various concentration of 3chloropropionate
Hours
0
6
12
18
24
30
36
42
48
54
60
Doubling
Time (h)
Maximum
A680nm
A680nm1
0.171
0.176
0.222
0.297
0.451
0.517
0.515
0.503
0.501
0.497
0.491
5mM
A680nm 2
0.169
0.172
0.223
0.301
0.458
0.521
0.516
0.500
0.500
0.500
0.495
A680nm 3
0.170
0.174
0.222
0.296
0.455
0.517
0.513
0.511
0.505
0.503
0.497
Mean/SD
0.170±0.001
0.174±0.002
0.222±0.001
0.298±0.003
0.455±0.004
0.518±0.002
0.515±0.002
0.505±0.006
0.502±0.003
0.500±0.003
0.494±0.003
14.54
14.16
14.36
14.35±0.19
0.517
0.521
0.517
0.518±0.002
Hours
0
6
12
18
24
30
36
42
48
54
60
Doubling
Time (h)
Maximum
A680nm
A680nm 1
0.173
0.179
0.227
0.296
0.448
0.621
0.854
0.901
0.891
0.795
0.793
10mM
A680nm 2
0.166
0.170
0.219
0.288
0.448
0.631
0.862
0.913
0.907
0.812
0.802
A680nm 3
0.177
0.177
0.230
0.303
0.452
0.635
0.860
0.927
0.911
0.805
0.790
Mean/SD
0.172±0.006
0.175±0.005
0.225±0.006
0.296±0.008
0.449±0.002
0.629±0.007
0.859±0.004
0.914±0.013
0.903±0.011
0.854±0.042
0.795±0.006
12.26
11.80
12.31
12.12±0.28
0.901
0.913
0.927
0.914±0.013
173
Hours
0
6
12
18
24
30
36
42
48
54
60
Doubling
Time (h)
Maximum
A680nm
A680nm 1
0.172
0.177
0.221
0.293
0.446
0.621
0.897
1.216
1.363
1.385
1.372
20mM
A680nm 2
0.171
0.180
0.225
0.297
0.459
0.631
0.888
1.212
1.367
1.389
1.383
A680nm 3
0.171
0.179
0.221
0.289
0.429
0.627
0.903
1.222
1.366
1.389
1.380
Mean/SD
0.171±0.001
0.179±0.002
0.222±0.002
0.293±0.004
0.445±0.015
0.626±0.005
0.896±0.008
1.217±0.005
1.365±0.002
1.388±0.002
1.378±0.006
11.71
11.98
11.46
11.72±0.26
1.385
1.389
1.389
1.388±0.002
Hours
0
6
12
18
24
30
36
42
48
54
60
Doubling
Time (h)
Maximum
A680nm
A680nm 1
0.172
0.176
0.183
0.189
0.193
0.196
0.196
0.189
0.181
0.173
0.171
40mM
A680nm 2
0.172
0.179
0.182
0.188
0.190
0.193
0.197
0.195
0.192
0.193
0.179
A680nm 3
0.175
0.173
0.188
0.189
0.190
0.189
0.194
0.196
0.197
0.193
0.185
Mean/SD
0.173±0.002
0.176±0.003
0.184±0.003
0.189±0.001
0.191±0.002
0.196±0.002
0.196±0.002
0.193±0.004
0.190±0.008
0.186±0.012
0.178±0.007
NG
NG
NG
-
NG
NG
NG
-
174
APPENDIX C
Doubling time calculation
Three growth curves were established and comparison was made. By
applying the data acquired from triplicate reading into the semi log graph, doubling
time for isolated bacteria grown in different situation was calculated based on the
formula as stated below. Table 3.4 summarised the results obtained.
Calculation of doubling time
It was known that:
log N t = log N 0 + n • log 2
(3.1)
N0 = the initial population number
Nt = the population at time t
n = the number of generation in time t
Solving for n, the number of generation, where all logarithms are to the base
of 10,
n=
log N t − log N 0
0.301
(3.2)
The rate of growth during the exponential phase in a batch culture can be
expressed in terms of the mean growth rate constant (k)
k=
n log N t − log N 0
=
t
0.301t
(3.3)
175
The time it takes a population to double in size is doubling time (g). It can
be calculated,
t=g
(3.4)
1
g
(3.5)
k=
Therefore, the doubling time should be:
g=
1
k
(3.6)
The doubling time of Rhodococcus sp. could be determined using the
equation below during logarithmic phase:
From equation 3.3:
g=
0.301
slope
(3.7)
Slope = gradient value for the growth curve during exponential phase. The
data are plotted with the A680nm value expressed in logarithm.
176
As an example:
log A680nm
Doubling Time of the Bacteria Grow in 20mM 3CP
0.20
0.10
0.00
-0.10
-0.20
-0.30
-0.40
-0.50
-0.60
y = 0.0257x - 0.6725
0
5
10
15
20
25
30
35
Time (Hour)
The doubling time of Rhodococcus sp. in 20 mM of 3-chloropropionate should be
0.3010
0.0257
=11.71 hours
g rhodococcus =
177
APPENDIX D
Growth curve of Rhodococcus sp. HN2006A
in 20 mM 3-bromopropionate
Hours
0
6
12
18
24
30
36
42
48
54
60
Doubling
Time (h)
Maximum
A680nm
A680nm 1
0.172
0.173
0.184
0.211
0.253
0.301
0.362
0.444
0.521
0.588
0.576
A680nm 2
0.172
0.179
0.182
0.213
0.255
0.310
0.371
0.451
0.532
0.591
0.579
A680nm 3
0.172
0.173
0.188
0.214
0.256
0.303
0.366
0.458
0.529
0.590
0.588
Mean/SD
0.172±0.000
0.175±0.003
0.185±0.003
0.213±0.002
0.255±0.002
0.305±0.005
0.366±0.005
0.451±0.007
0.527±0.006
0.590±0.002
0.581±0.006
22.52
22.18
22.13
22.27±0.212
0.588
0.591
0.59
0.59±0.002
178
APPENDIX E
Enzyme activity calculation for Rhodococcus sp. HN2006A
Raw data of enzyme assay when using cell free extract from Rhodococcus.
Min
A460
Corrected
Cl- (mM)
A460
0
0.508
0
0
5
0.592
0.084
0.076
10
0.597
0.089
0.081
15
0.641
0.133
0.128
20
0.684
0.176
0.174
From the table above, the enzyme activity was 0.0088 μmol Cl¯/mL/min. Since
protein concentration of the cell free extract was 15.76 mg/ml and 400μl of cell free
extract used in 10ml incubation mixture during enzyme assay, the dehalogenase
specific activity can be calculated as below:
[ y μmol Cl- /ml ] / z minute = X μmol Cl- /ml /min
[ X x 1000 /a ] / b mg /ml protein in a sample = specific activity
Where a: Amount of cell free extract: 400/10 = 40 μl/ml
b: Measurement of protein concentration at Abs595 :
(0.283-0.048) / 0.015 = 15.76 mg/ml
Therefore, dehalogenase Specific Activity towards 3-chloropropionate was
0.013 μmol Cl-/min/mg protein.
179
APPENDIX F
Methylobacterium sp. HN2006B partial 16S rRNA gene
ID
AC
SV
DT
DT
DE
KW
OS
OC
OC
RN
RP
RA
RT
RL
RL
RL
RN
RA
RT
RT
RL
FH
FH
FT
FT
FT
FT
FT
FT
FT
FT
FT
FT
XX
SQ
AM231910
standard; genomic DNA; PRO; 1307 BP.
AM231910;
AM231910.1
24-FEB-2006 (Rel. 86, Created)
24-FEB-2006 (Rel. 86, Last updated, Version 1)
Methylobacterium sp. HN2006B partial 16S rRNA gene, strain HN2006B
16S ribosomal RNA; 16S rRNA gene.
Methylobacterium sp. HN2006B
Bacteria; Proteobacteria; Alphaproteobacteria; Rhizobiales;
Methylobacteriaceae; Methylobacterium.
[1]
1-1307
Ng H.;
;
Submitted (22-FEB-2006) to the EMBL/GenBank/DDBJ databases.
Huyop F.Z., Biology Department, Faculty of Science, Universiti Teknologi
Malaysia, Universiti Teknologi Malaysia, Skudai, Johor, 81310, MALAYSIA.
[2]
Ng H.;
"Degradation of herbicide by soil microorganism and cloning of a haloacid
permease gene";
Unpublished.
Key
Location/Qualifiers
source
rRNA
1..1307
/country="Malaysia"
/db_xref="taxon:373519"
/mol_type="genomic DNA"
/organism="Methylobacterium sp. HN2006B"
/strain="HN2006B"
/isolation_source="agriculture soil"
<1..>1307
/gene="16S rRNA"
/product="16S ribosomal RNA"
Sequence 1307 BP; 307
tgagtaacgc gtgtgaacgt
accggatacg cccttatggg
ctagttggtg gggtaacggc
cagccacact gggactgaga
tggacaatgg gcgcaagcct
gtaaagctct tttatccggg
tcgtgccagc agccgcggta
aagggcgcgt aggcggcgtt
gccttcgata ctgggacgct
gaaattcgta gatattcgca
acgctgaggc gcgaaagcgt
taaacgatga atgccagctg
gcattccgcc tggggagtac
cacaagcggt ggagcatgtg
acatggcgtg ttacccagag
catggctgtc gtcagctcgt
cacgtcctta gttgccatca
cgaggaaggt gtggatgacg
ctacaatggc ggtgacagtg
tctcagttcg gattgcactc
ggatcagcat gccacggtga
gggagttggt cttacccgac
//
A; 317 C; 422 G; 261 T; 0 other;
gccttccggt tcggaataac cctgggaaac
gaaaggttta ctgccggaag atcggcccgc
ctaccaaggc gacgatcagt agctggtctg
cacggcccag actcctacgg gaggcagcag
gatccagcca tgccgcgtga gtgatgaagg
acgataatga cggtaccgga ggaataagcc
atacgaaggg ggctagcgtt gctcggaatc
ttaagtcggg ggtgaaagcc tgtggctcaa
tgagtatggt agaggttggt ggaactgcga
agaacaccgg tggcgaaggc ggccaactgg
ggggagcaaa caggattaga taccctggta
ttggggtgct tgcaccgcag tagcgcagct
ggtcgcaaga ttaaaactca aaggaattga
gtttaattcg aagcaacgcg cagaacctta
agatttgggg tccacttcgg tggcgcgcac
gtcgtgagat gttgggttaa gtcccgcaac
ttcagttggg cactctaggg agactgccgg
tcaagtcctc atggccctta cgggatgggc
ggacgcgaag gagcgatctg gagcaaatcc
tgcaactcga gtgcatgaag gcggaatcgc
atacgttccc gggccttgta cacaccgccc
ggcgctgcgc caaccgcaag gaggcag
tagggctaat
gtctgattag
agaggatgat
tggggaatat
ccttagggtt
ccggctaact
actgggcgta
ccacagaatg
gtgtagaggt
accattactg
gtccacgccg
aacgctttga
cgggggcccg
ccatcctttg
acaggtgctg
gagcgcaacc
tgataagccg
tacacacgtg
ccaaaagccg
tagtaatcgt
gtcacaccat
60
120
180
240
300
360
420
480
540
600
660
720
780
840
900
960
1020
1080
1140
1200
1260
1307
180
APPENDIX G
Methylobacterium sp. HN2006B growth in various concentration of 2,2dichloropropionate.
Hours
0
12
24
36
48
60
72
84
96
108
120
Doubling
time (h)
Maximum
A680nm
A680nm 1
5 mM
A680nm 2
A680nm 3
Mean/SD
0.102
0.131
0.209
0.273
0.276
0.279
0.279
0.278
0.273
0.272
0.269
0.107
0.130
0.202
0.265
0.279
0.276
0.272
0.272
0.271
0.270
0.266
0.105
0.134
0.211
0.266
0.279
0.278
0.281
0.276
0.272
0.269
0.268
0.105±0.003
0.132±0.002
0.207±0.005
0.268±0.004
0.278±0.002
0.278±0.002
0.277±0.005
0.275±0.003
0.272±0.001
0.270±0.002
0.268±0.002
24.27
26.31
25.65
25.41±1.04
0.279
0.279
0.281
0.280±0.001
Hours
0
12
24
36
48
60
72
84
96
108
120
Doubling
time (h)
Maximum
A680nm
A680nm 1
0.098
0.129
0.205
0.321
0.453
0.497
0.499
0.493
0.489
0.487
0.482
10 mM
A680nm 2
A680nm 3
0.099
0.121
0.211
0.318
0.454
0.499
0.501
0.499
0.494
0.488
0.100
0.127
0.209
0.315
0.448
0.497
0.507
0.498
0.492
0.489
0.480
0.486
Mean/SD
0.099±0.001
0.126±0.004
0.208±0.003
0.318±0.003
0.452±0.003
0.498±0.001
0.502±0.004
0.497±0.003
0.492±0.003
0.488±0.001
0.484±0.003
19.67
19.00
19.84
19.50±0.44
0.499
0.501
0.507
0.502±0.004
181
Hours
0
12
24
36
48
60
72
84
96
108
120
Doubling
time (h)
Maximum
A680nm
A680nm 1
0.102
0.127
0.199
0.301
0.428
0.538
0.689
0.981
1.032
1.052
1.051
20 mM
A680nm 2
0.100
0.122
0.198
0.315
0.434
0.544
0.699
0.990
1.031
1.055
1.050
A680nm 3
0.099
0.129
0.208
0.310
0.422
0.541
0.691
0.982
1.039
1.054
1.049
Mean/SD
0.100±0.002
0.126±0.004
0.202±0.006
0.309±0.007
0.428±0.006
0.541±0.003
0.693±0.005
0.984±0.005
1.034±0.004
1.054±0.002
1.050±0.001
20.47
19.47
21.03
20.32±0.79
1.052
1.055
1.054
1.053±0.002
Hours
0
12
24
36
48
60
72
84
96
108
120
Doubling
time (h)
Maximum
A680nm
A680nm 1
0.103
0.105
0.109
0.111
0.119
0.121
0.122
0.121
0.121
0.119
0.118
40 mM
A680nm 2
0.102
0.103
0.105
0.110
0.120
0.120
0.123
0.121
0.122
0.118
0.114
A680nm 3
0.103
0.109
0.104
0.118
0.115
0.122
0.123
0.125
0.119
0.119
0.115
Mean/SD
0.103±0.001
0.106±0.003
0.106±0.003
0.113±0.004
0.118±0.003
0.121±0.001
0.123±0.001
0.122±0.002
0.121±0.002
0.119±0.001
0.116±0.002
NG
NG
NG
-
NG
NG
NG
-
182
APPENDIX H
Methylobacterium sp. HN2006B growth in 20mM 2-chloropropionate
Hours
0
12
24
36
48
60
72
84
96
108
120
Doubling
time (h)
Maximum
A680nm
A680nm 1
0.109
0.127
0.186
0.294
0.407
0.502
0.691
0.999
1.087
1.091
1.091
A680nm 2
0.110
0.122
0.179
0.303
0.410
0.511
0.690
0.989
1.088
1.088
1.086
A680nm 3
0.107
0.123
0.183
0.301
0.402
0.507
0.692
1.000
1.089
1.091
1.089
Mean/SD
0.109±0.002
0.124±0.003
0.183±0.004
0.299±0.005
0.406±0.004
0.507±0.005
0.691±0.001
0.996±0.006
1.088±0.001
1.090±0.002
1.089±0.003
26.32
25.82
26.14
26.09±0.25
1.091
1.088
1.091
1.090±0.002
183
APPENDIX I
Enzyme activity calculation for Methylobacterium sp. HN2006B
Min
A460nm
Corrected A460nm
Cl- (mM)
0
0.198
0
0
5
0.242
0.044
0.046
10
0.297
0.099
0.101
15
0.351
0.153
0.155
20
0.394
0.196
0.199
Since 400 μl of cell free extract used in 10ml incubation mixture during enzyme
assay, the dehalogenase specific activity can be calculated as below:
[ y μmol Cl- /ml ] / z minute = X μmol Cl- /ml /min
[ X x 1000 /a ] / b mg /ml protein in a sample = specific activity
Where a: Amount of cell free extract: 400/10 = 40 μl/ml
b: Measurement of protein concentration at Abs595 :
(0.145-0.0122) / 0.021 = 6.45 mg/ml
Therefore, dehalogenase specific activity towards 2,2-dichloropropionate
was 0.039 μmol Cl-/min/mg protein.
184
APPENDIX J
Putative dehrP gene sequence
ID
AC
SV
DT
DT
DE
KW
OS
OC
OC
RN
RP
RA
RL
RL
RL
RN
RA
RT
RL
FH
FT
FT
FT
FT
FT
FT
FT
FT
FT
FT
FT
FT
FT
FT
FT
FT
FT
FT
FT
SQ
AM260971
standard; genomic DNA; PRO; 1239 BP.
AM260971;
AM260971.1
28-APR-2006 (Rel. 87, Created)
28-APR-2006 (Rel. 87, Last updated, Version 0)
Rhizobium sp. UTM dehrP gene for putative haloacid permease
dehrP gene; haloacid permease.
Rhizobium sp. UTM
Bacteria; Proteobacteria; Alphaproteobacteria; Rhizobiales; Rhizobiaceae;
Rhizobium/Agrobacterium group; Rhizobium.
[1]
1-1239
Ng H.;
Submitted (27-APR-2006) to the EMBL/GenBank/DDBJ databases.
Huyop F.Z., Biology Department, Faculty of Science, UniversitiTeknologi
Malaysia, Universiti Teknologi Malaysia, Skudai, Johor, 81310, MALAYSIA.
[2]
Ng H.; Choy T.; Huyop F.Z
"Sequence analysis and cloning of a haloacid permease gene”
Unpublished.
Key
Location/Qualifiers
source
1..1239
/organism="Rhizobium sp. UTM"
/strain="UTM"
/mol_type="genomic DNA"
/db_xref="taxon:382149"
CDS
1..1239
/transl_table=11
/gene="dehrP"
/product="putative haloacid permease"
/experiment="heterologous expression in Escherichia coli"
/protein_id="CAJ98618.1"
/translation="MTTTLVARTSSAGRMTREERKVIFASSLGTVFEWYDFFLYGSLAA
IIGATFFKDFPPATQAIFALLAFAAGSLVRTFGALIFGRLGDMIGRKYTFLVTILIMGL
STFVVGLLPGSDTIGLAAPTILILLRLLQGLALGGEYGGAAVYEAEHAPPGRRGFYTSW
IQTTATGGLFLSLLVILGTRSLLGEESFTSWGWRVPFLLSVVLLGISVWIRMQLNESPV
FQRMKAEGKASKAPLREAFAHWPNARLALVALFGMVAGQAVVWYTGQFYVLFFLQSILK
VDGFTTTLLICWSLLLGSGFFVFFGWLSDRIGRKPIMIAGCLLAVVTYFPIFEAITERA
NPTLAKAISEVKVTRGLPTRSNAGIYSIRSEPACSHRLVTLPEPILLRVRCDMIVRQVL
QESQLACSSMAPR"
Sequence 1239 BP; 202 A; 369 C; 342 G; 326 T; 0 other;
atgactacga ctctagtcgc ccgtacttca tcagccggtc gcatgacacg cgaggagcgc
60
aaagtgatct tcgcctcctc gctcggtact gtcttcgaat ggtacgattt ctttctatat
120
ggctcactcg ccgctatcat cggcgcgacc tttttcaagg actttccgcc agccacacaa
180
gccatattcg cgctccttgc tttcgcggct ggctcgcttg ttcggacttt cggcgcactc
240
atttttggcc gtctcggcga tatgattggg cgcaaatata ccttcctcgt aaccatcctg
300
atcatgggtc tgtcgacgtt cgtggtcggc cttctaccgg gttcggacac cattggactt
360
gcggccccta cgatcctgat tctcctccgc ctgcttcagg gcctcgcgct cggcggtgag
420
tacgggggcg ccgctgttta cgaggccgag catgctccgc ccggacgccg cggcttttat
480
acaagttgga ttcaaacaac tgcgacgggt ggcctcttcc tatcgctcct tgtcattctc
540
ggaactcgct cattgctggg cgaggaatcc ttcacgtctt ggggctggcg cgtgccgttc
600
ttgttgtccg tggtactgct cggcatctcg gtatggatcc gcatgcagct caacgagtcg
660
cccgtgttcc agcgcatgaa ggcagagggc aaggcgtcca aggctccctt gagggaggca
720
tttgcgcact ggcctaacgc aaggctcgct ctggttgccc ttttcggcat ggtcgccggc
780
caagccgtgg tctggtacac gggccaattc tatgtgctgt tcttcctgca gagcattctc
840
aaggtagacg gcttcacgac gacgctactt atctgttggt ctttgctgct gggatctggc
900
ttcttcgttt tctttggctg gttatccgat cgcatcggac gcaagccgat tatgatcgcc
960
ggctgcctcc ttgctgttgt gacttacttt ccgatcttcg aggcaatcac cgaacgagcg
1020
aatcccactt tagccaaggc gatttcggag gtgaaggtca cccgtggtct cccgacccgg
1080
tcgaatgcgg gaatctattc aatccggtcg gaacccgcgt gttcacatcg tcttgtgacg
1140
ttgcccgagc ctatcttgct cagagttcgg tgcgatatga tcgtcaggca ggtgctgcag
1200
gagagccaac tcgcgtgctc gtcaatggca ccgaggtaa
1239
185
APPENDIX K
Standard curve for Bradford assay
0.50
A595
0.40
0.30
y = 0.0147x + 0.0476
0.20
0.10
0.00
0
5
10
15
20
BSA (mg/ml)
25
30
35
186
APPENDIX L
Standard curve for chloride ion assay
0.50
log A460nm
0.40
y = 0.9866x
0.30
0.20
0.10
0.00
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
Chloride ion concentration
`
0.40
0.45
(μmol)
0.50