Current Research Journal of Biological Sciences 4(5): 570-577, 2012
ISSN: 2041-0778
© Maxwell Scientific Organization, 2012
Submitted: May 24, 2012
Accepted: June 23, 2012
Published: September 20, 2012
Molecular Study of nifH1, nifH2, nifH3, nifU, nifV, VF Genes and Classical Approach
Cared out to Identification of Azotobacter chrococcum from Soil
Adel Kamal Khider
Biology Department, College of Education/Scientific Departments, Salahaddin University, Erbil, Iraq
Abstract: The present study aimed to compare classical approach with molecular based method for identification of
Azotobacter chrococcum from soil samples. A. chrococcum was isolated from soil source in Erbil city, Iraq. They
were cultivated under laboratory conditions using Nitrogen free Azotobacter specific medium. A. chrococcum was
present in all soil samples. result shows that A. chrococcum were rod shape, motility occur through the use of
peritrichous flagella, cysts-forming, positive to oxidase, catalase and tryptophanase test, unable to liquefy gelatin,
with insoluble brown or brown-black pigmentation and darken with age. Utilized starch, sucrose, mannitol and
moloanat, but not rhamnose. molecular method based on detection of nifgenes have been successfully applied to
describe A. chrococcum isolated from soil. The PCR products for nifH1 1102bp, nifH2 246bp, nifH3 128bp, nifU
930bp, nifV 1146bp and VF gene 594bp were detected on gel electrophoresis, while no bands observed for negative
control. The isolated bacteria considered Azotobacte chrococcum belonging to Genus Azotobacter.
Keywords: Azotobacter, Azotobacter chrococcum, nif gene, nitrogen fixation, PCR
target of this work was the development of a method for
screening and preliminary recognition of cultures
belonging to genusAzotobacter and identification of
A. chrococcum through detection of nifH1, nifH2,
nifH3, nif U, nifV and VF genes analysis, in order to
individuate the most effective one. This study goal is
particularly important because the unequivocal
characterization of Azotobacter at the species level is
not so easily achieved through classical phenotypic
methods, mainly due to considerable variations in many
phenotypic traits (Jayarao et al., 1992) and this study
conceder the first report for identification of
Azotobacter depending on molecular approach in
Kurdistan region even in Iraq.
INTRODUCTION
Azotobacter is a free-living nitrogen-fixing
bacterium, which is used as a biofertilizer in the
cultivation of most crops. Biological nitrogen fixation is
an essential step in the nitrogen cycle in the biosphere
and it is a major contributor to the nitrogen available to
agricultural crops.
Nitrogen fixation can be considered as one of the
most interesting microbial activity as it makes the
recycling of nitrogen on earth possible and gives a
fundamental contribution to nitrogen homeostasis in the
biosphere (Aquilantia et al., 2004).
Microorganisms catalyze biological nitrogen
fixation with the enzyme nitrogenase, which has been
highly conserved through evolution (Zehr et al., 2003).
Nitrogenases are composed of two proteins that can be
purified
separately:
dinitrogenase
and
dinitrogenasereductase. Dinitrogenase, also referred to
as the MoFe protein or component 1 is a 220 to 240
kDa tetramer of the nifDand nifKgene products that
contains two pairs of two complex metalloclusters
known as the P cluster and the iron molybdenum
cofactor (FeMo-co). Dinitrogenasereductase, also
referred to as the Fe protein or component 2, is a 60
kDa dimer of the product of the nif H gene, which
contains a single (4Fe-4S) cluster at the subunit
interface and two Mg-ATP binding sites, one at each
subunit (Rubio and Ludden, 2008).
The present study was aimed to compare two
strategy for the isolation of Azotobacter from soil.
depending on classical phenotypic approach, the other
MATERIALS AND METHODS
Isolation of bacteria: Isolation strategy of Azotobacter
from ten soil samples collected from Erbil city during
spring 2011, based on streaking of serial soil dilutions
on plates containing Nitrogen Free Jensen,s Medium
(NFJM) (Difco USA), followed method of Becking
(1981). The isolates were purified by streaking on
(NFJM) agar plates.
The bacterial isolates were characterized, using Nfree Jensen,s medium, Potato dextrose agar (Oxoid
England) and Ashbey,s Medium (ASHM) (Alpha
India). Gram-staining characteristics and cell
morphologies were determined by standard methods
(Gerhard et al., 1981). Motility was observed in wet
mount using phase contrast microscope. Preliminary
physiological characterization such as catalase test,
oxidase, Indol, gelatenase, tryptophananase test and
570
Curr. Res. J. Biol. Sci., 4(5): 570-577, 2012
Table 1: The forwards and the reverse primers used
Primer
Sequence (5'-3')
nifH1-F(cagacacgaagaagccgggc)
nifH1-R(gaccagcagcttgttgttga)
nifH2-F(cgccggcgcagtgtttgcgg)
nifH2-R(cactcgttgcagctgtcggc)
nifH3-F(cgatgactgaagactgaacgag)
nifH3-R(aaggtgcggtcaggagagaa)
nifU-F(atgtgggattattcggaaaaa)
nifU-R(tcagcctccatctgccgtggg)
nifV-F(gatggctagggtgatcatcgacga)
nifV-R(gccattcctcctgccgccagttcg)
FV-F(tacagtagcggaaggttagggt)
FV-R(tcagccgccgaccttgatgccg)
Nucleotide
20
20
20
20
22
20
21
22
24
24
22
22
Table 2: Some cultural, morphological and biochemical characteristics of isolated Azotobacter
Insoluble
Soluble
Sample Cell shape
pigment
pigment
Flagella
Catalase
A A1 Rod
Cinnamon
Peritrichous
+
A2 Rod
Brownblack
Peritrichous
+
A3 Rod
Brownblack
Peritrichous
+
B B1
Coccobacili
Redviolet
Peritrichous
+
B2 Coccobacili
Redviolet
Peritrichous
+
B3 Ovoid
Yellowgeenish
Peritrichous
+
B4 Rod
Brown
Peritrichous
+
C C1
Rod
Brown
Peritrichous
+
C2 Rod
Brown
Peritrichous
+
C3 Ovoidrod
Yellowgrenish
Peritrichous
+
D D1 Coccobacili
Redviolet
Peritrichous
+
D2 Coccobacili
Greenish
Peritrichous
+
D3 Coccobacili
Brown
Peritrichous
+
D4 Rod
Greenish
Peritrichous
+
E E1
Rod
Cinnamon
Peritrichous
+
E2
Rod
Cinnamon
Peritrichous
+
E3
Coccobacili
Redviolet
Peritrichous
+
F F1
Ovoidrod
Yellowgrenish
Peritrichous
+
F2
Ovoidrod
Yellowgrenish
Peritrichous
+
F3
Rod
Peritrichous
+
G G1
Ovoid
Redviolet
Peritrichous
+
G2
Coccobacili
Yellowgrenish
Peritrichous
+
G3
Ocobaili
Brown
Peritrichous
+
G4
Rod
Brown
Peritrichous
+
H H1
Ovoid
Yellowgrenish
Peritrichous
+
H2
Rod
Brownblack
Peritrichous
+
H3
Coccobacili
Yellowgrenish
Peritrichous
+
I I1
Ovoid
Yellowgrenish
Peritrichous
+
I2
Rod
Brown
Peritrichous
+
I3
Rod
Brown
Peritrichous
+
J J1
Rod
Cinnamon
Peritrichous
+
J2
Rod
Brownblack
Peritrichous
+
J3
Rod
Yellowgrenish
Peritrichous
+
Reference
Setubal et al. (2009)
Setubal et al. (2009)
Setubal et al. (2009)
Setubal et al. (2009)
Setubal et al. (2009)
Setubal et al. (2009)
Oxidase
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
Gelatenase
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Treptophanase
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
A-J: Soil samples; A1-J3: Isolated N2-fixing bacteria
carbohydrate assimilation test (glucose, malonate,
starch, sucrose, rhaminose, inositol and mannitol)
according to Atlas et al. (1995) and Forbes et al.
(2002).
Maintanus of the isolates: Long-term storage of the
purified isolates was at 20oC in broth medium with 30%
(w/v) glycerol. Short term storage for further
characterization was on (NFJM) plates at 4oC (Delves
et al., 1996).
DNA extraction: Genomic DNA was extracted and
purified from bacterial cells using the QI Aamp DNA
Mini Kit (Jayashreet et al., 2007). PCR amplification of
nitrogenase genes nifH1, nifH2, nifH3, nifU, nifV and
FV genes for A. chrococcum was performed. Genes
sequence were obtained from NCBI site and were
designed in OPERON diagnostic ltd, Germany. The
primer length and melting temperature were designed
with coordination between forward and reverse primers.
The melting and annealing temperature were calculated
following (Womble, 2000). Primers Amplification was
completed using the protocol and reagents followed of
(Rajeswari and Kasthuri, 2009). The programmed
temperature sequence was 96ºC followed by 55ºC for
one minute and 72ºC for 1 min, the temperature
571 Curr. Res. J. Biol. Sci., 4(5): 570-577, 2012
sequence was run for 30 cycles, the final product
extension was conducted at 72ºC for 6 min followed by
4ºC temperature hold. The primers were used acquired
from Operon Biotechnologies, Germany. The forwards
and the reverse primers are showing in Table 1.
Gel electrophoresis: DNA amplification was checked
by electrophoresis of each PCR product in a 1.5% (w/v)
agarose gel, in TBE buffer for 1 h at 3.2 V/cm. Gels
were stained in ethidium bromide for 15 min and
thereafter washed for 5 min. DNA fragments were
visualised at 312 nm with a UV-transilluminator Image
Master VDS (Amersham Biosciences) (Helmut et al.,
2004).
RESULTS
Totally 10 samples were collected in Erbil city,
during April 2011, the intervals of approximately 7
days. All the samples were showing the presence of
Azotobacter. These samples were processed through the
commonly used procedures such as selective media for
isolation N2-fixing bacteria i.e., ASHM. Gram’s
staining, Phase contrast observation for motility,
Catalase test, carbohydrate hydrolysis test and for
identification of free-living diazotrophic organism
i.eAzotobacter from the above samples and that can be
processed, result shows that Azotobacter sp., are motile,
gram negative, oxidase, tryptophanase, catalase and
starch
hydrolysis
positive
Table
2.
The colony morphology of Azotobacter strains were
varying during the isolation in the selective media
NFJM, they were very clear, large, mucoid, watery due
drops like initially. The mother culture was sub cultured
in the same media, 27 colonies were obtained, the
colony morphology differs slightly i.e., small and
circular, convex in nature. The utilization of different
sugars by individual isolates and the subsequent
production of gas and acid are generally used as a
diagnostic indicator to distinguish Azotobacter at
species level Table 3.
According to the mentioned characteristics,
Azotobacter isolates were identified as A. chrococcum.
A. chrococcum were rod shape, motility occur through
the use of peritrichous flagella, cysts-forming, positive
to oxidase, catalase and tryptophanase test, unable to
liquefy gelatin, with insoluble brown or brown-black
pigmentation and darken with age, utilized manittol,
molonate and not rhaminose A3, B1, B2, C2, C3, D1,
D2, E1, F2, F3, G2, H2, H3, I1, I3 and J2 colonies of
different soil samples Table 2 and 3.
Moreover the molecular method based on detection
of nif genes (nitrogenase genes) have been successfully
applied. The DNA was isolated from Azotobacter spp.
cultures. Their banding pattern of DNA on agarose gel
Table 3: Utilization of Different carbon sources, consequent gas formation and acid production by isolated Azotobacter
Carbon sources
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Sucrose
Starch
Rham nose
Glucose
Maltose
Lactose
Mannitol
Inositol
Malonate
Arabi nose
---------------------------------------------------------------------------------------------------------------------------g s
a g s
a g
s
a g
s a g
s
a
g
s
a
g
s
a
g
s
a
g
s
a g
s
Samples
A A1
+ + + - - +
+
+ +
+ +
+
+ A2
+ + + + + +
- - +
+
+ + +
+ +
+
+ A3
+ + + + +
+ +
+ + +
+
+ + +
+ + + +
+
- B1
+ - - +
+
+ + + + - B2
+ - - +
+
+ + +
- +
+
B B3
+ +
+ + +
- W +
+ + + + + + + +
+ B4
+ + + + +
+ + - - +
+
+ + + + + +
+
+ C1
+ + - - +
+
+ + +
- C C2
+ W - - +
+
+ + + + +
+
+ +
+
C3
+ + - - +
+
+ + + + +
+
+ +
+
D1
+ + + + +
+ + - - +
+
+ + W + + + + +
+
+ +
W
D D2
+ W +
+ + - - +
+
+ + +
+ + + + W W W +
+
D3
+ + +
+ +
- W +
+ + + + + + + + + +
- D4
+ + - - W W + + +
+
- E1
+ + - - W W + +
+ + + + +
+
+ E E2
+ + - - +
+
+ +
+ - +
+
E3
+ + - - +
+
+ + +
+ + + - +
+
F1
+ + +
+ +
- W +
+ + + + + + +
+ +
+
F F2
+ + - - +
+ +
+ + + +
+
+ +
F3
+ + +
+ W
- W +
+ + + + + + + + + +
+
+ +
G1
+ + +
+ + +
- W +
+ + +
+ + + + +
+ +
+
G G2
+ + - - +
+
+ + + + + +
+
+ +
G3
+ + - - +
+
+ + + + + - G4
+ + - - +
+
+ + + + + + +
+
H1
+ + +
W + +
- W +
+ + + + + + + +
+
+ +
+
H H2
+ + + + +
+ + - - +
+
+ + + + +
+
W H3
+ + - - +
+
+ + +
+ + + + +
+
+ +
+
I1
+ + +
+ + +
+ - +
+ _
_
+ + + +
+ +
+
I I2
+ + + + +
+ + - - +
+
+ + +
+ + + +
+
+ I3
+ + + + +
+ + - - + +
+ + + +
+
+ +
+
J1
+ + - - +
+
+
+
+ +
+
J J2
+ + + + + +
+ + - - +
+
+ + + + +
+
+ +
+
J3
+ + - - + +
+ + + +
+
+ +
+
A-J: Soil samples; A1-J3: Isolated N2-fixing bacteria; g: Growth of isolates; s: Gas formation; a: Acid production
572 a
+
+
+
+
+
+
+
+
+
+
+
Curr. Res. J. Biol. Sci., 4(5): 570-577, 2012
1
2
3
Fig. 1 : The PCR product of Azotobzcter chrococcum isolated from soil as follows: Lane 1: Negative control; Lane 2:
A. hrococcum isolate; Lane 3: DNA marker
electrophoresis was compared with that of reference
Azotobacter strains, the results showed that there was
no significance difference in banding pattern Fig. 1.
The PCR production was cared out for all tested
isolated cultures in order to check the presence of
nifH1, nifH2, nifH3, nifV, nifU and FV genes for
A. chrococcum. The PCR products for A. chrococcum
were nifH1 1102bp, nifH2 246bp, nifH3 128bp, nifU
930bp, nifV 1146bp and FV gene 594bp Fig. 1 lane 2,
these genes were detected on gel electrophoresis, while
these bands does not appear in the negative control
Fig. 1 lane 1. Therefore according to the classical
approach, all cultures that considered A. chrococcum
compared with that of molecular based method.
DISCUSSION
This research work firstly aimed to compare two
different methods reported in literature for the isolation
of Azotobacter from soil samples, in order to
individuate the most effective one. Different criteria
have been presented by several researchers to delimit
characteristic which can be used as a key for
identification of Azotobacter, generally included
morphological, cultural and biochemical characteristics,
highlighting the points which may be used as the
diagnostic indications.
Ten soil samples were processed through the colony
characteristics, morphology and biochemical testes,
using commonly used procedures for isolation N2fixing bacteria and identification Azotobacter species in
the soil using ASHM medium. All the samples show
presences of free living N2-fixing bacteria, i.e.,
Azotobacter. Varying in colony morphology of
Azotobacter species using selective media due to the
ability of such isolates to act as oligonitrophiles
(Knowles, 1982), represent one of the major problems
in isolating Azotobacter. The colony moephology on
ASHM medium is not clearly recognizable and the
media that used are not to be selective enough because
of presence different N2-fixing bacteria in soil samples.
Similarly, the enrichment broth media proved not to be
selective enough. In fact the pellicle on the enrichment
broth surface was manufactured not only for aerobic
N2-fixing bacteria, but also by bacteria unable to
573 Curr. Res. J. Biol. Sci., 4(5): 570-577, 2012
3721 ACCCGCTTCA TCAGCAGCAC TGGCAACGCC GGTCGCCGGT TTGCGCATCC GGTTGAGGGT
3781 GGCGGCTGCT CCGGTCTGAA ATACAGCCTG AAGCTGGAGG AGGCCGGTGC CGAAGGCGAT
3841 CAGCTGATCG ACTGCGACGG CATCACCCTG CTGGTCGACG ATGCCAGCGC CCCTCTGCTC
3901 AATGGCGTGA CCATGGACTT TGTGGAAAGC ATGGAAGGTA GCGGTTTCAC CTTCGTCAAT
3961 CCGAATGCCA GCAACAGCTG TGGTTGTGGC AAGTCTTTTG CTTGCTGATT AGGCAACCCT
4021 GAGGTCGCCG GCTGGGGCCC CAAAGACTCA CTGGGAGATG AAGCCGACAT GTGGGATTAT
4081 TCGGAAAAAG TCAAAGAGCA TTTTTACAAC CCCAAGAATG CCGGAGCCGT GGAAGGCGCC
4141 AACGCCATCG GCGACGTCGG ATCGCTGAGC TGCGGTGACC GGCTGCGTCT GACCCTGAAG
4201 GTGGATCCGG AAACCGACGT GATCCTGGAT GCCGGCTTCC AGACCTTCGG CTGTGGCTCC
4261 GCCATCGCTT CCTCCTCGGC ACTGACCGAG ATGGTCAAGG GGGCTGACCT GGACGATGCG
4321 CTGAAGATCA GCAACAGGGA CATCGCCCAT TTCCTCGGAC GGCTGCCGCG GGAGAAGATG
4381 CACTGCTCGG TGATGGGCCG CGAACGCCTG CAGGCCGCCG TGGCCAACTA CCGTG6CGAA
4441 GAGCTCAGGA CCGACCACGA GAAGCCGCAG CTGATCTGCA AGTCGTTCGC CATCGACGAA
4501 GTGATGGTCC GCGACACCAT TCGCGCCAAC AAGCTGTCCA CCGTCGAGGA CGTGACCAAA
4561 CACACCAAGC GCGGCGGCGG CTGCTCGGCC TGTCATGAGG GCATCGAGCG CGTGCTGAGC
4621 GAGGAGCTGG CGCCCGTGGC GAGGTCTTCG TCGTGCGCCG ACCAAGGCCA AGAAGAGGTC
4681 AAGGTGCTCG CCCCGAGCCG GCTCCGCTCG TGGCCGAGGA GACTCCGCGC CACGCCGAAG
4741 CTGAGCAACC TGCAGCGCAT CCGCCGCATC GAAACCGTGC TGGCGGCGAT CCGTCCGACC
4801 CTGCAGCGCG ACAAGGGTGA TGTCGAGCTG ATCGATGTCG ACGGCAAGAA CATCTACGTC
4861 AAGCTAACCG GCGCCTGCAC CGGCTGCCAG ATGGCCTCCA TGACCCTTGG CGGCATCCAG
4921 CAGCGCCTGA TCGAGGAACT CGGCGAATTC GTCAAGGTGA TCCCGGTCAG CGCCGGCCCA
4981 CGGCAGATGG AGGTCTGACA TGGCTGACGT CTATCTCGAC AACAACGCCA CCACCCGGGT
5041 GGACGACGAA ATCGTCGAGG CCATGCTGCC GTTCTTCACC GABCAGTTCG GCAACCCCTC
5101 GTCGCTGCAC AGCTTCGGCA ACCAGGTCGG CCTGGCGCTG AAGAGGGCGC GGCAGCAGCG
5161 TGCAGGCGTG CTCGGCGAGC ATGATTCGGA AATCATCTTC ACTTCCTGCG GCACCGAGTC
5221 GGATCACGCG ATCCTCTCGG CGCTCAGCCC AGCCCGAGCG CAAGACCTGA TCACCACCGT
5281 GGTCGAGCAC CCGGCAGTGC TGAGCCTGTG CGACTACCTC GCCAGCGAGG GCTACACCGT
5341 GCACAAGCTG CCGGTGGACA AGAAGGGCCG CCTGBATCTG GACCATTACG CCAGCCTGCT
5401 GAACGACGAC GTCGCCGTGG TGTCGGTGAT GTGGGCCAAC AACGAGACCG GCACCCTGTT
5461 CCCGGTCGAG GAAATGGCAC GCATGBCCGA CGAGGCCGGC ATCATGTTCC ACACCGACGC
5521 CGTGCAGGCC GTGCGCAAGC TGCCGATCGA CCTGAAGAAC TCGTCGATCC ACATGCTCTC
5581 GCTGTCGGGC CACAAGCTGC ATCGCAAGGG CGTCGGCGTG CTCTACCTGC GCCGCGGCAC
5641 CCGCTTCCGT CGCTGCTGCC GCGGCCACCA GGAGCGGCCC GCGGGCGGTA CCGAGAACGC
5701 TGCCTCGATC ATCGCCATGG GCTGGGCCGC CGAGCGCGCG CTGGCCTTCA TGGAGCACGA
5761 GAACACCGAG GTCAAGCGCC TGCGCGACAA GCTGGAGGCC GGCATCCTCG CCGTCGTGCC
5821 GCACGCCTTC GTCACCGGCG ACCCGGACAA CCGCCTGCCC AACACCGCCA ACATCGCGTT
5881 CGAGTACATC GAGGGCGAGG CCATCCTGCT GCTGCTGAAC AAGGTCGGCA TCGCCGCCTC
5941 CAGCGGCTCG GCCTGCACCT CCGGCTCCCT GGAGCCCTCC CACGTGATGC GCGCCATGGA
6001 CATTCCCTAC ACCGCCGCCC ACGGCACCGT GCGTTTCTCC CTGTCGCGCT ACACCACCGA
6061 GGAGGAGATC GACCGGGTGA TCCGCGAGGT GCCGCCGATT GTGGCCCAGC TGCGCAACGT
6121 GTCGCCCTAC TGGAGCGGCA ACGGTCCGGT GGAACATCCG GGCAAGGCCT TCGCGCCGGT
6181 CTACGGCTGA GCCGCCGCCT GCGGGAGCGC ATCCCGCAGG AAACCGCCTC GGGGAGCCCC
6241 GCCCGAGTTG TTGGAGAAAG CCATGGCTAG CGTGATCATC GACGACACCA CCCTGCGTGA
6301 CGGCGAGCAG AGTGCCGGGG TCGCCTTCAA TGCCGACGAG AAGATCGCCA TCCGGCGTGC
6361 GCTCGCCGAG CTGGGCGTAC CGGAGCTGGA GATCGGCATT CCCAGCATGG GCGAGGAGGA
6421 GCGCGAGGTG ATGCGCGCCA TTGCCGGCCT CGGCCTGTCG TCGCGCCTGC TGGCCTGGTG
6481 CCGGCTGTGC GACTTCGACC TCTCGGCCGC GCGCTCCACC GGGGTGACCA TGGTCGACCT
6541 GTCACTGCCG ATCTCCGACC TGATGCTGCG CCACAAGCTC AATCGTGATC GCGACTGGGC
6601 ACTGGGCGAG 6TCGCCCGGC TGGTCAGCGA GGCGCGCATG GCCGGGCTTG AGGTGTGCCT
6661 GGGCTGCGAG GACGCCTCGC GGGCGGATCA GGACTTCATC GTGCGGGTGG GGGCGGTGOC
6721 GCAGGCCGCG CGCCCGCCGC CTGCGTTCGC CGATACCGTC GGGGTGATGG AGCCGTTCGG
6781 CATGCTCGAC CGCTTCCGTT TCCTCCGCCA GCGCCTGGAC GTGGAGCTGG AGGTGCACGC
6841 CCACGACGAC TTCGGGCTGG CCACCGCCAA CACCCTGGCG GCGGTGATGG GCGGGGCGAC
6901 CCACATCAAT ACCACGGTCA ACGGGCTCGG CGAGCGCGCC GCCAACGCCG CGCTGGAAGA
6961 GTGCGTGCTG GCGCTCAAGA ACCTCCACGG CATCGACACC GGCATCGACA CCCGCGGCAT
7021 CCCGGCCATC TCGGCGCTGG TCGAGCGGGC CTCGGGGCGT CAGTGGCCTG GCAGAAGAGC
7081 GTGGTTGGCG CCGGTGTTCA CCCACGAGGC CGGCATCCAC GTCGACGGGC TGCTCAAGCA
7141 CCGGCGCAAC TACGAGGGAC TGAATCCCGA CGAGCTCGGC CGCAGCCACA GCCTGGTGCT
7201 GGGCAAGCAT TCCGGCGCGC ACATGGTGCG CAACAGCTAC CGCGAGCTGG GCATCGAGCT
7261 GGCCGACTGG CAGAGCCAGG CACTGCTCGG CCGCATCCGC GCCTTCTCCA CCCGCACCAA
7321 GCGCAGCCCG CAGGCTGCCG AGCTGGAGGA CTTCTATCGC CAGCTGTGCG AGCAGGGGAC
7381 TGCCGAACTG GCGGCAGGAG GAATGGCATG AGCCTGCTTG CGCAATGGCG TGAAGACATC
7441 CGCTGCGTGT TCGAGCGCGA TCCGGCGGCA CGCACCACCT TCGAGGTGCT GACCACCTAT
7501 CCGGGCTGCA CGCGATCATG CTCTACCGGC TCGC4CCATC GTCTGTGGCG GCCGAATGCG
7561 TTACCTCGCC CGGCTGCTGT CGTTCGCGCG CGCCTGGTGA GCAACGTCGA CATCCATCCC
7621 GGGGCCGTCA TCGGTGCGCG CTTCTTCATC GACCACGGCG CCTGCGTGGT GATCGGCGAG
7681 ACCGCCGAGA TCGGCCGGGA CGTGACTCTC TACCACGGCG TCACCCTGGG CGGCACCACT
Analysis of predicted nifU and nifV genes products:
Sequence gene No.of aminoacids
4068-4998
nifU (930) bp
310
6261-7407
nifV (1146) bp
382
Fig. 2: Sequence of the chromosomal region contains nifH1 gene, nifH2 gene and nifH3 gene in A. chroococcum. Sequence with
under lines is sites for primers nifH1-F, nifH1-R, nifH2-F, nifH2-R, nifH3-F and nifH3-R for amplification
fix nitrogen, because of present of a certain content of
fixed nitrogen and non-selective carbon sources i.e., the
streaking and incubation on NFJM andmannitol
medium permit a rapid individuation of slimy and
glistering Azotobacter like colonies. The important
observations that the isolates
classified
as
574 Curr. Res. J. Biol. Sci., 4(5): 570-577, 2012
ORIGIN
1 cagacacgaagaagccgggcccgtgacatgcccgccatggactgctgctccgtcgccgca
61 cgccacttcctgcaccagccggcatgaaccccggtaccacatgggaacggatcgccgcgg
121 cgttactaccggtacgccgccagcccgggacgacgcagatcgctgccgtccgactcccga
181 cacatgccatatgcagcatgaaatatcgctgaaaacatattactggtttttttatccaaa
241 aaacaaacaacatatgaaattcacatcttgatggcaccacccttgctccatcccctgcga
301 caccagtcaaacgccacgaatcaatggaggttccaagatggcattgcgtcagtgtgcaat
361 ttacggcaagggtggtatcggcaagtccaccaccacccagaacctggtcgccgcgctcgc
421 cgaggccggcaagaaggtgatgatcgtcggttgcgacccgaaagccgactccacccgcct
481 gatcctgcattccaaggcccagaacaccgtcatggagatggccgcatccgccggctcggg
541 tgaagacctcgagctggaagacgtgctgcagatcggctacggcggcgtcaagtgcgtcga
601 gtccggcggccctgagccgggcgtcggctgcgcgggccgtggcgtgatcaccgcgatcaa
661 cttcctggaagaggaaggcgcctacagcgacgacctggacttcgtgttctacgacgtgct
721 gggcgacgtggtgtgcggcggcttcgccatgccgatccgcgagaacaaggcccaggaaat
781 ctacatcgtctgctccggcgagatgatggccatgtacgccgccaacaacatcgccaaggg
841 catcgtgaagtacgcccactccggcagcgtgcgtctgggcgggctgatctgcaacagccg
901 caagaccgaccgcgaagacgagctgatcatggccctggccgcgaagatcggcacccagat
961 gatccactttgtgccgcgcgacaacgtcgtgcagcacgccgaaatccgccgcatgaccgt
1021 gatcgaatacgatccgaaagccaagcaggccgacgagtaccgtgccctggcccagaagat
1081 cctcaacaacaagctgctggtcatcccgaacccggcgagcatggaggacctcgaagagct
1141 gctgatggagttcggcatcatggaagccgaagacgagtccatcgtcggcaaggccggcgc
1201 cgagggctgatcccgccggcgcagtgtttgcggaggagcgtgcgtcgcgggctgtccgga
1261 atggcttctcgcggccggcacgccgccctcccttttgaatcgccccgaattctccaacct
1321 caggagctgaccctatggccatggccatcgacggctacgaatgcaccgtctgcggcgact
1381 gcaagccggtctgcccgaccggctcgatcgtcctccagggcggtatctacgtgatcgacg
1441 ccgacagctgcaacgagtgcgccgacctgggcgagccacgctgtctcggcgtctgccccg
1501 tggacttctgcatccagccgctcgatgactgaagactgaacgagccgcacccgcttgccg
1561 gcgacagagcatcccgccgctctgccaccggaccaccaaacggcgatcgctttcctcagg
1621 tcgccgttttttctctcctgaccgcacctt
Analysis of predicted nifH1, nifH2 and nifH3 genes products:
Sequence gene No. of amino acids
1-1102
nifH1 (1102) bp 367
1213-1459 nifH2 (246) bp
82
1522-1550 nifH3 (128) bp
42
Fig. 3: Sequence of the chromosomal region contains nifH1 gene, nifH2 gene and nifH3 gene in A. chroococcum. Sequence with
under lines are sites for primers nifH1-F, nifH1-R, nifH2-F, nifH2-R, nifH3-F and nifH3-R for amplification
A. chrococcum, when utilized lactose and produce of
gas but did not formed acid. While some other species
gas producing and acid forming. Utilization of xylose
by A. chrococcum and A. beijrenickii were
accompanied by both acid production and gas
formation. A. paspali produce gas but not forming acid.
Maltose pointed positive indication for both acid and
gas formation if utilized by A. chrococcum and
A. paspali. A. vinelandii utilize maltose, were produce
gas without acid formation and A. beijerinckii were
forming acid without gas production and all isolates
were capable of utilizing glucose, in A. chrococcum and
A. paspsli glucose utilization accompanied with gas
production and acid formation, while A. vinelandii were
positive for acid formation and negative to gas
production, A. beijrenickii were gas positive and acid
negative.
Concerning the utilization along with gas
formation and acid production of other sugars showed
that the isolates belonging to the same Azotobacter sp.,
were differ in gas formation and acid production. Same
results have also reported by other researchers (Parson,
2003; Tejera et al., 2005). A. chrococcum colonies
showed good growth on NFJM with insoluble brown or
brown black pigmentation and darken with the age
reaching maximum intensity after two weeks at 28oC,
while behave differently when grown on potato
dextrose agar. They produce some times yellow or
white pigment, depending on the ability of these
isolates to utilize organic acid which may be present in
potato (Brenner et al., 2005) and they differ in utilizing
other sugars. These features that observed on the
isolates are identical with feature of A. chrococcum
(Jensen, 1954; Norris and Chapman, 1968; Brenner et
al., 2005; Dworkin et al., 2006).
Indeed molecular characterization of the isolates
showed that all isolates were identified on ASHM and
NFJM media. As concerning assignation into species by
means of molecular basis, the utilization of used primer
complementary to well-conserved regions in the
bacterial genome, led to amplification of the nifgenes,
(nitrogenase genes) nifH1, nifH2, nifH3, nifV, nifU and
575 Curr. Res. J. Biol. Sci., 4(5): 570-577, 2012
FV genes for isolates belonging to genus Azotobacter
which identified as A. chrococcum by classical
approach. The nifH1 gene was 1102 bp, while nifH2
gene was 2461 bp, these two genes were separated by
flank of DNA of 111bp. The nifH3 gene 128bp was
separated from nifH2 gene by 63 bp. The genes
organization and their sequence are show in Fig. 2. The
nifU and nifV gene is clustered in a region of
A. chroococcumgenome spanning about 3339 bp Fig. 2.
The gene nifU which involved in maturation of Fe for
Fe-S cluster synthesis and repair was 930 bp long,
while nifV gene, which involved maturation of FeMocomplex, was 1146 bp, these two genes were separated
by 1263 bp and nifS is located between them. The
region of chromosome which contain nifK, nifD, nifM,
nifA, nifN, nifB, nifQ, nifZ, nifP, nifF, nifW, nifB, nifL
and nifY genes are located between the fragment of
chromosome which contain nifH1, nifH2, nifH3 and the
fragment contain nifV, nifS and nifU. Because
important of region of nifH1, nifH2 and nifH3, nifV and
nifU genes on chromosome they were selected for this
study, detected by PCR technique in each of A.
chroococcum cells Fig. 3. This allowed to compare and
better evaluate selectivity of the isolation strategies
tested, extending preliminary identification to the major
number of species possible.
Previous studies by Kirshtein et al. (1991), Ueda
et al. (1995), Zehr et al. (2003), Aquilantia et al.
(2004), Mary Ann and Virginia (2007) and Hamilton
et al. (2011), N-fixing bacteria were investigated by the
diversity of nitrogenase genes in different
environments, through amplification of nif genes, i.e.,
nifH, infD, nifV, nifK, nifU which encode nitrogenase
complex from cultivated organisms.
The two methods compared, although described as
selective for Azotobacter, led to the isolation of a large
number of soil bacteria unable to fix nitrogen. This lack
of selectivity, possibly due to the ability of such isolates
to act as oligonitrophiles (Knowles, 1982), represents
one of the major problems in isolating Azotobacter.
Similarly, the enrichment on ASHM solution proved
not to be selective enough. In fact, the pellicle on the
broth surface of the enrichment medium was formed
not only by aerobic N-fixing bacteria, but also by
anaerobic N-fixing microorganisms. Our conclusion in
present study is that the utilization of ASHM and NFJM
medium has to be reconsidered. Indeed, this medium
revealed to be differential more than selective and is
usefulness in the individuation of Azotobacteraceae,
followed by sugar tests and must be confirm by
molecular bases through amplification of nifgenes for
extracted DNA.
Presence of A. chrococcum in all soil samples in
Erbil city may be due to the ability of this species to
grow under different environmental conditions and it
may be more resistant to unfavorable conditions.
Mrakovacki and Milic (2001) found that the abundance
of A. chrococcum in different soil is always possible.
ACKNOWLEDGMENT
We wish to thank Dr. Aras Dashti from Agriculture
College, Salahaddin University for her helpful during
isolation of Azotobacter, we also wish to thank Dr.
Shanga A.K. in Charite Medical Faculty Humboldt
University, Berlin, Germany for their cooperation.
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