Zobia Dar
Middlesex County College
Dr. Gerben J. Zylstra, Rutgers University
Summer 2005
Diversity of the Alkane Hydroxylase (AlkB) Gene in Various Alkane Enrichments
Oil contamination is a great concern environmentally. Moreover, some of the
hydrocarbons that are spreading due to oil spills and runoff can be toxic for many living
organisms. Some of these toxic contaminants bioaccumulate and move up the food chain.
Alkanes are a major component of crude oil and are mostly chemically inert. Alkanes
form strong apolar sigma bonds between carbons and also form strong carbon-hydrogen
bonds (C-H). However, they are used as an energy source by most of the
microorganisms.
There are three types of alkanes: Cycloalkanes, n-alkanes and branched alkanes.
Left:
Cycloalkanes: Image from:
www.chemguide.co.uk/alkanes/cyclos.gif
Below:
n-alkanes or linear alkanes
Right: Branched alkanes
Image from:
www.ias.ac.in/.../chemistry/alkanes.gif
Alkane Hydroxylase (AlkB) Gene
Alkanes can be degraded aerobically by bacteria. During the course of the
summer, I examined the diversity of the Alkane Hydroxylase (alkB) gene when different
alkanes were present. Alkane degradation by bacteria, usually occurs through sequential
oxidation of one or both methyl groups of the molecule. AlkB catalyses the first step in
the alkane degradation pathway. The pathway generally followed is from the n-alkane, to
an alcohol, to an aldehyde, and then finally to a fatty acid, which is assimilated through
the β-oxidation pathway.
The following pathways show the degradation of an n-alkane.
Decane
CH3
Alkane hydroxylase
(AlkB)
OH
Alcohol
O
Aldehyde
H
O
O
H
Fatty Acid
Methods and Materials
To begin, the soil sample was obtained from a site that had a utility pole - this
sample was obtained before I joined the lab. The transformer on the top of the pole
leaked a little, thus contaminating the soil underneath. This soil sample was stored at 4ºC.
Foremost, Deoxyribonucleic Acid (DNA) had to be extracted from the soil sample
and purified, so that there would be no contamination in it. I did this by using a number
of DNA extraction (MoBio Ultrapure Soil extraction kit) and purification kits (Bio101
GeneClean kit). Secondly, I had to make sure that the DNA was amplifiable. After I
extracted the genomic DNA, a Polymerase Chain Reaction (PCR) was conducted with
degenerate primers designed to amplify the alkane hydroxylase gene, alkB. Although, I
did the PCR a number of times I did not get any results.
To make sure that the DNA was amplifiable I conducted PCR with 16S rDNA
gene primers. If bacterial DNA is present the 16S PCR is usually positive. As expected
the PCR worked, proving that the experiment I was conducting could work.
I set up four different enrichment cultures so I could continue with the
experiment. I used two medium length n-alkanes: Octane (C-8) and Dodecane (C-12),
one long length n-alkane: Hexadecane (C-16). The fourth enrichment culture was
glucose, which was used as a positive control, as all bacteria should be able to utilize
glucose. After two weeks, DNA was extracted from these cultures. I continued with the
PCR for the alkB gene and on the fifth try I got a positive result – the expected 549 base
pair PCR product. These bands were cut out and cloned into the pGEM-T cloning vector.
96 clones from each enrichment were sequenced. I did sequence analysis and came up
with a Phylogenetic tree.
Enrichment on GLU,
OCT, DOD, HXD
Sample site
Isolate DNA
&
PCR
Phylogenetic
Analysis
549 bp
pGEM-T
clone
Sequence
I did a cell count to see which alkane source was most favored by the bacteria. I
found out that octane was the most preferred carbon source for the bacteria. Although, I
expected the most cell count to be in glucose. Octane was followed by hexadecane, then
glucose and lastly, dodecane. Furthermore, I plated out these samples, after several
dilutions just so that I could see what bacteria would grow on alkanes on agar plates in
the lab, since a very small percentage of environmental microorganisms have been
known to grow on media in laboratories. I also wanted to see if we could isolate any
bacteria which contained an alkB gene which matched a sequence from one of our
libraries. After leaving the plates in an incubator for over two weeks, several different
bacteria and fungi grew on them.
Octane
Hexadecane
Dodecane
Results and Future Work
I have learned so much from the three months in Dr. Zylstra’s lab that I do not
think it might be possible to do the same in any class for the same period of time. Not
only have I learned experimental techniques, but also that no matter how fast you want to
finish the work, the best asset in research is patience. Moreover, I have learned how to
extract, PCR amplify and clone DNA. Most importantly, I learnt how to analyze results
and have understood what most of the results mean.
I have analyzed the culture kept with Octane and Dodecane. According to the
results I have been able to get until now, I can state that different alkanes select for
different populations of alkB genes and bacteria.
Contig
Base
Pairs
Gene
Acc. #
Organism
Match
29
498
Fatty acid desaturase Alkane-1
monooxygenase
ZP_00282029.1
ZP_00464279.1
Burkholderia fungorum LB400
Burkholderia cenocepacia H12424
155/166
141/166
30
498
Fatty acid desaturase Alkane-1
monooxygenase
ZP_00282029.1
ZP_00464279.1
Burkholderia fungorum LB400
Burkholderia cenocepacia H12424
161/166
141/167
31
498
Fatty acid desaturase alkane
hydroyxlase
ZP_00282029.1
CAC36356.1
Burkholderia fungorum LB400
Burkholderia cenocepacia
108/178
105/178
32
498
putative alkane hydroxylase
Fatty acid desaturase
CAC37048.1
ZP_00282029.1
CNM-group bacterium HXN600
Burkhloderia fungorum LB400
108/176
111/177
33
498
alkane hydroxylase
Fatty
acid desaturase
CAC36356.1
ZP_00282029.1
Burkholderia cepacia Burkholderia
fungorum LB400
109/175
108/163
34
498
putative alkane hyroxylase
putative alkane hydroxylase
CAC370481.1
AAY85982.1
CNM-group bacterium HXN600
Mycobacterium sp. CH-2
139/165
137/170
35
498
alkane hydroxylase
Fatty
acid desaturase
CAC3635.1
ZP_00423704.1
Burkholderia cepacia Burkholderia
vietnamesesis G4
109/178
107/178
36
498
Fatty acid desaturase Alkane-1
monooxygenase
ZP_00282029.1
ZP_00464279.1
Burkholderia fungorum LB400
Burkholderia cenocepacia H12424
118/164
118/164
In the future, I would like to continue analyzing the sequences, especially for the
hexadecane and glucose cultures, to get data that further solidifies the conclusion that I
have reached. Moreover, I would like to study the morphology of bacteria growing on the
plates, to see if anything interesting and different is growing on them.