Bacterial diversity and methanotrophic abundance in landfill soil

using PCR targeting 16s rDNA and pmoA gene fragments and
genetic analysis of microsatellite variation among captive South
American and pet store populations of the guppy (Poecilia
reticulata)
Biology 362 Laboratory

Submitted by: Stephanie Norman

Lab partner: Jill Irvine
Instructor: April Goebl
April 7, 2010

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Stephanie Norman
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Abstract
The bacterial diversity and the presence of methanotrophic bacteria in landfill soil
was assessed by generating clone banks for 16s rDNA genes from all bacterial species
and for pmoA, a functional gene specific to methanotrophs which encodes a subunit for
the methane monooxygenase enzyme. The analyses were performed with DNA
extracted directly from methane-enriched soil and from cultured methanotrophic
bacteria. The desired inserts for both 16s rDNA and pmoA was confirmed by colony
hybridization with chemiluminescent probes, sequenced and phylogenetic trees were
constructed. We determined there was a large diversity for the landfill soil bacteria and
confirmed the presence of methanotrophs. Individual variation among and between two
populations of guppies was also analyzed by amplifying a microsatellite locus using
labeled and unlabeled primers to examine the allele variation and heterozygosity
between pet store and captive South American guppies. The pet store guppies showed
six different alleles but were not in Hardy-Weinberg equilibrium whereas the captive
South American guppies showed only two alleles at the studied locus but were within
Hardy-Weinberg equilibrium. Opsin, a receptor gene found in rods and cones of the
retina, was also extracted from the guppy DNA, amplified, cloned and sequenced for
use as a protein-coding sequence for phylogenic analysis.
Introduction
Methanotrophic bacteria are a physiologically distinct group of microbes known
for their ability to rely on methane as their exclusive source of carbon and energy (Wise
et al. 1999). Methanotrophs are widespread in nature, found in a variety of
environments including rice fields, subarctic peat soils, estuaries, wetlands, freshwater,
groundwater and the sea (Liebner et al. 2009; Wartiainen et al. 2003). Ecologists and
microbiologists have long been focusing on methanotrophic bacteria, as they represent
the largest sink for methane in aerobic soils because of their key role in the global
methane cycle, where they oxidize methane into carbon dioxide (McDonald et al. 2005;
Wise et al. 1999). Methane is an end product of organic matter in aerobic
decomposition, thus, landfill soil seemingly provides favourable conditions for the
growth of methanotrophic bacteria (Wartiainen et al. 2003).
In view of the fact that that the global warming potential of methane over a time
horizon of 100 years is 23-fold that of carbon dioxide, the attraction of this group of
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bacteria based on their potential for bioremediation in these environments is of

importance (Liebner et al. 2009). Furthermore, methanotrophic bacteria could aid in the
biodegradation of xenobiotic pollutants due to their methane monooxygenase (MMO)
enzymes which oxidize numerous pollutants (DeJournett et al. 2006).
The methanotrophs consist of twelve genera that are divided into two major
groups based primarily on the structures of their internal membranes and their carbon
assimilation pathways: the type I methanotrophs in the -subdivision of the
Proteobacteria, and the type II methanotrophs in the -subdivision of the Proteobacteria
(McDonald et al. 2005). The type I methanotrophs have disc-shaped bundles of
intracytoplasmic membranes and use the ribulose monophosphate pathway for
formaldehyde assimilation. Type II methanotrophs enclose their membrane structures
along the outside edge of the cell and although they are also able to take in carbon,
they use a serine pathway in place of the ribulose monophosphate pathway at the
oxidation level of formaldehyde (Wise et al. 1999).
The breadth of methanotroph diversity has caught the interest of microbiologists
in the last decade because of the greenhouse gas effect of methane along with the
recent advances in the molecular biology and phylogeny of methanotrophs (McDonald
et al. 2005). The use of PCR primers to selectively identify and amplify specific bacteria
of interest is a potent tool in molecular biology (Wartiainen et al. 2003). In this study,
PCR primers targeted to pmoA along with primers specific for the 16s ribosomal
deoxyribonucleic acid (rDNA) gene, which is present in all bacteria, were used to
determine if methanotrophic bacteria are present in landfill soils and the diversity of the
methanotrophs if indeed they are present (McDonald et al. 2005). PmoA is a gene
encoding the methanotroph-specific methane monooxygenase, an enzyme that plays a
crucial role in the bacterial conversion of methane into carbon dioxide. Variation of the
pmoA gene is null between methanotrophs therefore its use in this study was to
determine the presence of methanotrophic bacteria in the soil to indicate whether the
landfill soil is capable of digesting the methane produced by the surrounding
decomposing organic matter. PmoA was also analyzed because it was the only protein3

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coding sequence isolated from the bacteria. However, during the procedure, it appeared
that pmoA was not amplifying in the reaction, so we amplified opsin, a genes encoding
membrane-bound receptors that are expressed primarily in rod and cone cells of the
retina, from the guppy (Poecilia reticulata), that would enable us to have an alternate
protein coding sequence for phylogenic analysis (Ward et a. 2008). Phylogenetic
analysis of 16s and pmoA was performed to show the diversity within the
methanotrophs. Simultaneously, cultured methanotrophic bacteria (Interium isolate)
were analyzed using identical techniques to act as a positive control for the examination
of the landfill soil bacteria.
The guppy (Poecilia reticulata) is a small freshwater fish native to the coastal
streams and rivers of Venezuela, Guyana, Surinam and several of the Lesser Antilles
including Trinidad and Tobago (Rosen and Bailey 1963). This live-bearing ornamental
fish is a model organism for the study of genetics because of its short life cycle, ease of
breeding and reproductive capacity (Nakajima and Taniguchi 2001).
In this study, the genetic diversity among two populations of guppies was
analyzed in order to gain insight into the processes of microevolutionary change.
Historical colonization events, population connectivity, adaption to local selection
pressures along with inbreeding in captivity can shape such diversity (Suk and Neff,
2009). Microsatellite markers are commonly used by microbiologists for analyzing the
relationships within and between populations of many species including humans (Lin et
al. 2010). Their abundant presence in genomes, high mutation rates, and multi-allelic
nature make microsatellites ideal for the analysis of population genetics and
microevolutionary change (Lin et al. 2010). These characteristics of microsatellites
allows for many allelic polymorphisms between individuals within a population, namely
at neutral microsatellites that do not exhibit reduced diversity for a selected phenotypic
trait (Bleakley et al. 2008). However, inbreeding of a population quickly brings
microsatellites to homozygosity allowing researchers to correlate the amount of
homozygosity at a microsatellite locus to the level of inbreeding that has occurred within
a population (Li et al. 2006).
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In this study, South American guppies that were collected and kept in captivity
since 2004 were genotyped at one microsatellite locus and analyzed for genetic
diversity. Their small population size in captivity renders the South American guppy
population more susceptible to inbreeding and the stochastic effects of random genetic
drift (Barson et al. 2009). Pet store guppies were also collected from the Hillside pet
store just prior to the study and analyzed at the same microsatellite locus in order to
compare the allelic diversity within and between the two populations of guppies.
The objective of this study was to determine the species diversity of the bacteria
in landfill soil from the Hartland landfill in Victoria, British Columbia using 16s rDNA as a
marker for biodiversity, and to use pmoA to determine the presence of methanotrophic
bacteria in the soil, thus indicating whether the landfill soil is capable of digesting
methane. It is hypothesized that methanotrophic bacteria will be present in the landfill
soil due to the methane-rich conditions of the landfill environment and that diversity of
the bacteria will be abundant.
The genetic differences within and between the pet store guppies and the South
American captive guppies was also a focus of this study, where microsatellites were
used as markers for allelic diversity within a population. It is hypothesized that the
captive guppies are inbred and thus show reduced polymorphisms on a polyacrylamide
sequencing gel as compared to the pet store guppies that are taken from a larger
population size.
Materials and Methods
Methanotrophic Bacteria
Collection of samples
Soil was collected from Hartland Landfill in Victoria, British Columbia, Canada on
December 2009. Methane levels were measured and the sample was enriched with
methane when the levels of methane decreased in order to promote the methanogenic

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bacterial survival and growth. Interium isolate, a known methanotroph, was collected in
January 2010 and grown in culture by Dr. Roy.
DNA extraction and purification
Total genomic DNA was extracted from both the soil and cultured samples using 0.1mm
zirconia/silica beads in 0.1M sodium phosphate buffer at pH 8 to enhance cell lysis.
Then 100mM NaCl, 500 mM Tris-HCl @ pH 8, 10% w/v SDS was added and the tubes
were shaken for 5 minutes on a mini-bead beater to enhance cell lysis, then centrifuged
at 12000 rpm for 3 minutes. Ammonium acetate at 7.5M was added to each supernatant
and incubated for 10 minutes on ice then centrifuged again at 12000 rpm for 3 minutes.
Then the soil sample was purified by running through an acid-washed PVPP spin
column, the PVPP was equilibrated in 20mM potassium phosphate buffer at pH 7.4. The
PVPP spin column was prepared by centrifuging the PVPP in the column at 2900 rpm
for 3 minutes, discarding the flow-through and repeating the centrifugation. Then 100 L
aliquots of the soil sample were applied to the PVPP and centrifuged for 3 minutes at
2900 rpm until 500 L was purified into 5 microcentrifuge tubes of decreasing DNA
concentrations. The cultured bacteria were not purified through PVPP because it would
account for too much loss of DNA and the cultured bacteria was clean enough.
The DNA from the cultured soil was extracted by boiling for 2 minutes, put on ice for 1
minute then centrifuged at 10,000 rpm for 5 minutes. The supernatant was stored at 20C.
The DNA concentration of all the samples was measured by use of the nanospec
spectrophotometer at an absorbance of 260nm. The A260/280 ratio was also taken,
which indicates the purity of the sample and should ideally lie between 1.8 and 2.0;
contamination results in lower ratios.
Polymerase Chain Reaction amplification
All methanotrophic PCR reactions were performed using a thermocycler. PCR reactions
contained (volume = 50L): 2x Ready Mix PCR (Sigma, Oakville, ON), 25 mM MgCl2,
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15M forward and reverse primers, and ~60ng of template DNA. The soil DNA was an
exception and used only 10ng template for the PCR reaction.
PCR reactions targeting the 16s rDNA gene for all bacteria were conducted using
primers 16s-f1 and 16s-r13, and reactions targeting the pmoA for methanotrophs used
the primers pmoA-f and pmoA-r (table 1). A touchdown PCR protocol was used to
amplify both the 16s and pmoA reactions, including an initial denaturing at 94C for 2
minutes followed by 20 cycles of denaturing at 94C for 45 seconds, annealing at 62C
for 1 minute with a decrease of 1C every second cycle, and extension at 72C for 2
minutes. This was followed by 10 cycles of denaturing at 94C for 45 seconds,
annealing at 52C for 1 minute, extension at 72C for 2 minutes, and a final extension at
72C for 6 minutes. A positive control with mitrichosporium DNA was used to ensure
correct reaction components and proper techniques were followed, and a negative
control with water was used to check for background contamination for the 16s rDNA
PCR reaction. PCR products were checked by electrophoresis on a 1.6% agarose gel
containing SYBR safe (used at a concentration of 1:10,000) and run against 1 Kb and
100 bp ladders (New England Biolabs, Pickering, ON) at 130 V for 45 minutes. The gel
was visualized with a VernierBlueview Transilluminator.
Target gene

Primer name

Expected Fragment
Length (bp)

Sequence

pmoA

pmoA-f

531

5-GGNGACTGGGACTTCTGG-3
5-GASGCNGAGAAGAASGC-3

pmoA-r
16s

16s-f1

1533

5-AGAAAGGAGGTGATCCAGCC-3

16s-r13
Guppy Microsatellite

Guppy opsin

FB1

5-GAGTTTGATCCTGGCTCAG-3

200-300

5-ACTTTATCACTTATTCACTT-3

FB2 reverse

5-GTGACCGAACGAAACCATA-3

F1A

Data not available

Rev 8

Data not available

T7 forward

Data not available

Sp6 reverse

Data not available

Table 1. PCR primer sequences used in this study.

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DNA purification
The soil and cultured PCR products were purified using the QIAquick Spin Kit according
to manufacturers protocol (QIAGEN, Mississauga, ON).
Construction of 16s and pmoA clone libraries
Gene sequences were ligated into pGEM-T Easy vectors according to manufacturers
protocol (Promega, Madison, WT), with an incubation time of 30 minutes. The ligation
products were then incubated with JM 109 cells on ice for 20 minutes then heat
shocked at 42C for 45 seconds and returned to ice for 2 minutes. SOC broth was then
added and the reaction was incubated for 1 hour at 37C. The transformed cells were
plated on Luria-Bertani (LB)+Ampicillin+100mM IPTG+50mg/ml X-Gal plates and
incubated at 37C overnight. A white colony (indicating a successful transformation)
was selected and placed into LB/AMP (50 g/ml) media and incubated at 37C in a
shaking incubator. The plasmid DNA of the transformed E. coli was purified by miniprep,
performed according to manufacturers protocol (QIAprep Spin Miniprep Kit, QIAGEN,
Mississauga, ON). A restriction digest of the mini-prepped plasmids was carried out
using EcoR 1 restriction enzyme and EcoR I buffer and left to digest at 37C for 1 hour.
The digest was analyzed by a 1% agarose gel electrophoresis (as above) and using a 1
kb ladder.

DNA sequencing
DNA sequencing of the 16s rDNA and pmoA genes was carried out from both strands
using the Beckman Coulter CEQ Dye Terminator Cycle Sequencing kit as per the
manufacturers protocol (Beckman Coulter, Mississauga, ON) using T7 forward primer
and Sp6 reverse primers. PCR was then carried out on the sequencing reactions using
the following profile: 96C for 3 minutes, then 30 cycles of: 96C for 30 seconds; 50C
for 20 seconds; 60C for 4 minutes 30 seconds and finally 4C for infinity. At 4C, 3 l
stop/loading buffer was added to each tube and sequencing was performed in the
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University of Victoria Centre for Biomedical Research using the CEQ 8000 Beckmancoulter capillary sequencer.
Colony Hybridization
The colony lift was performed by laying nylon membrane over the colonies for one
minute then lowering colony side up onto the puddle of denaturation solution (0.5M
NaOH, 1.5M NaCl) and incubating for 15 minutes at room temperature. The membrane
was then let to air dry for 1 minute on a dry sheet of Whatman 3MM paper then the
membrane was transferred to the Neutralization Solution (1.5M NaCl, 1.0M Tris-HCl, pH
7.4) and incubated for 15 minutes. The membrane was air-dried as above then
incubated in 2X sodium chloride sodium citrate (SSC) for 10 minutes then air-dried
twice more, successively on fresh pieces of Whatman 3MM paper. Methylocystis was
used as a positive control for the colony hybridization and 4 l and 8 l were added to
unpopulated areas of the membrane. The DNA was cross-linked to the membrane by
illuminating the discs with UV light for 5 minutes at an energy of 200 x 100 J/cm2 using
a UVP HL-200 hybrilinker.
Following cross-linking, the membranes were placed on an aluminum try and incubated
in proteinase K (>600U/mL, 18 mg/ml) diluted 1/10 in 2X SSC for 1 hour at 37C without
fan convection. The colonies were lifted by running a ruler over a wet sheet of Whatman
3MM paper placed on top of the membrane. This lift was repeated 4 times until all
debris was off the membrane, then the membranes were covered in 2X SSC.
DIG-labeled DNA was hybridized to the colony lifts using 16s probes prepared following
the protocol outlined in the DIG DNA Labeling kit from Roche (Mississauga, ON). The
probes were amplified using 16s-f1 and 16s-r13 primers (table 1) under the touchdown
PCR conditions listed for 16s and pmoA above.
The membranes were placed in a roller bottle, each separated by a mesh material. DIG
Easy Hyb Solution ( Roche, Mississauga, ON) was added and the membranes were

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prehybridized for 30 minutes in the closed bottle at the appropriate hybridization

temperature, 50C, calculated using the formula Thyb=Tm-(20-25C)
-Tm of hybrid = 49.82 + 0.41[%G + C] (600/L), where %G+C = GC content of labeled
probe, L = length (bp) of probe-target hybrid.
-Thyb = 20-25C below Tm of hybrid, where Thyb = optimal hybridization temperature (in
DIG-Easy Hyb).
The DIG-labeled probe (25 ng/ml) was denatured by boiling for 5 minutes and then
immediately placing on ice. The probes (at a concentration of 2l probe/ml hybridization
solution) were then hybridized to the membranes in the closed container at the
hybridization temperature and incubated for 90 minutes. The membranes were washed
with Low Stringency Wash Buffer (2X SSC = 0.1% SDS) twice for 5 minutes at room
temperature then washed with the High Stringency Wash Buffer (0.1X SSC + 0.1%
SDS) twice for 15 minutes at 68C. The membranes were then stored at 4C until ready
for detection.

Chemiluminescent Detection of Probe-Targeted Hybrids was achieved by washing the

membranes for 5 minutes at room temperature gently shaking in Washing Buffer (0.1M
Maleic Acid, 0.15M NaCl, pH 7.5; 0.3% (v/v) Tween 20). The membranes were blocked
by adding 20 ml of 1X Blocking Solution [10x Blocking Solution diluted 1/10 in Maleic
Acid Buffer (0.1M Maleic Acid, 0.15M NaCl, pH 7.5)] and shaken at room temperature
for 30 minutes. Anti-Digoxigenin-AP was diluted 1:10 000 in 1x Blocking Solution and
added to the membranes and incubated with shaking at room temperature for 30
minutes. The unbound antibodies were removed by washing the membranes twice with
Washing Buffer for 10 minutes each time then the membranes were equilibrated for 5
minutes with Detection Buffer (0.1M Tris-HCl, 0.1M NaCl, pH 9.5). The membrane was
transferred to a plastic transparency film, covered with 25mM chemiluminescent alkaline
phosphatase substrate (CSPD) diluted 1/ 100 with water, covered with a second
transparency, sealed and incubated for 5 minutes. Then the excess liquid was drained,
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the CSPD was activated by incubating the membrane at 37C for 10 minutes, then
exposed to a film cassette in a dark room for 25 minutes and developed.
South American captive and Pet store guppies
Sample Collection
Two guppy populations were analyzed for microsatellite variation. South American
guppies were collected and kept in captivity with no new fish added since 2004. Pet
store guppies were collected from the Hillside Pet Store in Victoria, Canada just prior to
the study. Nine guppies were randomly selected from the pet store population and ten
guppies were randomly selected from the South American captive guppies.
DNA extraction
The guppy was euthanized in Ethyl 1,3-aminobenzoate methylsulfonate salt (MS222,
Sigma A5040-5G) then the stomach contents were removed, which might contain DNA
from plants or other fish. Homogenization was carried out by chopping it with a razor
then using a micro-pestle and 500 l extraction buffer (100nM Tris-base, 100mM EDTA,
0.5% SDS, pH 7.8). To the homogenate, 20% SDS and proteinase K was added and
incubated in a 55C water bath for 15 minutes then stored on ice. DNA was isolated
with a Phenol:Chloroform:Isoamyl Alcohol (Sigma P3803), manual shaking for 5
minutes the centrifuged at maximum rpm for 2 minutes. The supernatant was isolated
and the Phenol:Choroform:Isoamyl Alcohol addition, shaking and centrifugation was
repeated. Chloroform was then added to the supernatant and the shaking,
centrifugation and supernatant transfer was repeated again. 99% cold ethanol was
added and mixed by inversion and centrifuged for 5 minutes at max rpm after which
70% cold ethanol was added to the pellet and removed without dislodging the pellet.
Once the ethanol had completely evaporated the DNA was re-suspended in water.
The DNA concentration was measured as described previously.
Polymerase Chain Reaction Amplification
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The guppy PCR reactions were performed using a thermocycler. PCR reactions
contained (volume = 20L): 5x iProof buffer (Bio-Rad, Mississauga, ON), iProof DNA
polymerase (Bio-Rad), 25 mM MgCl2, 10 mM dNTPs, 5% DMSO, 10 M forward and
reverse primers (1 M forward and reverse primers for the labeled reaction), and ~20ng
of template DNA. PCR reactions targeting the microsatellite (TTA) n locus were
conducted using the FB1 forward primer and FB2 reverse primer, and reactions
targeting opsin used an F1A forward primer and Rev8 reverse primer (table 1). A
labeled guppy microsatellite reactions was also carried out using labeled FB1 and FB2
primers. Poly A tails were added to opsin genes prior to ligation into pGEM-T vectors
using the A-tailing protocol (total reaction volume = 10 l): 10x reaction buffer, 25mM
MgCl2, 1 mM dATPs, Taq polymerase and ~60 ng purified opsin template. The reaction
mixture was incubated on ice for 10 minutes followed by 30 minutes at 70C.
PCR was used to amplify the guppy reactions, including an initial denaturing at 98C for
30 seconds followed by 35 cycles of denaturing at 98C for 5 seconds, annealing at
55C for 12 seconds, and an extension at 72C for 25 seconds, and a final extension at
72C for 5 minutes. A negative control with water and the F1A and Rev8 primers was
used to check for background contamination. PCR products were checked by
electrophoresis on a 1.6% agarose gel containing SYBR safe (used at a concentration
of 1:10,000) and run against 1 Kb and 100 bp ladders (New England Biolabs, Pickering,
ON) at 130 V for 45 minutes. The gel was visualized with a Vernier
Blueview Transilluminator.
Construction of opsin clone libraries, DNA sequencing and colony hybridization
Procedure for opsin clone libraries, DNA sequencing and colony hybridization was as
listed above for the 16s and pmoA.
Phylogenetic Analysis
The 16s, pmoA and opsin sequences were edited with BioEdit to cut out the vector and
primer sequences, then a BLASTn search was performed for all sequences to identify
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the nearest sequence and a lower-matching sequence (around the 10th most
homologous sequence) in GenBank (http://ncbi.nlm.nih.gov/) by using the FASTA
program. Sequences were optimally aligned with Clustal W, pmoA was toggletranslated, and MEGA was used to reconstruct maximum parsimony and genetic
distance-based phylogenies. The evolutionary history was inferred using the NeighborJoining method. Bootstrap analyses for 1000 resamplings were performed to provide
confidence estimates for the phylogenetic trees.

Results
Landfill soil bacterial diversity and the presence of methanotrophic bacteria in this soil
were tested in this study. The soil sample showed increasing methane consumption
over time, indicating a growing population of methanotrophic bacteria from the collection
to DNA extraction date. The spectrophotometric results of the extracted DNA is shown
in table 2.
Sample

A260

A260/280

DNA Concentration
(ng/l)

Soil bacteria

1.168

1.47

58.4

Cultured bacteria

0.837

1.84

41.9

Guppy

0.187

1.655

4675

Opsin

1.093

1.93

54.7

Table 2. Spectrophotometric results using the NanoDrop spectrophotometer of the soil sample and
cultured bacteria, the guppy genomic DNA and the opsin DNA. The first extract of the five taken from
the PVPP spin columns was used for spectrophotometric analysis. The guppy genomic DNA was diluted
by 1/500 using ddH2O before its spectrophotometry.

My amplification of the 16s, pmoA, and guppy genomic DNA revealed two overlapping
bands for each lane, most noticeable for the ladders. Lane two, the negative control,
showed no bands, lane three with the positive control for the 16s had the strongest
band which appeared just larger than 1.5kb and both the enriched soil and cultured
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methanotrophic 16s rDNA showed ample amplification. However, the pmoA sequences
showed no amplification for either bacterial sample. The genomic guppy had a long
continuous band throughout the lane.

1 kb
ladder

100bp
ladder

<3.0 kb
<1.5 kb

1,517 bp>
1,000 bp>

500/517 bp>
bpbp>

Figure 1. 1.6% agarose gel electrophoresis showing 16s and pmoA PCR products and guppy
genomic DNA. 16s rDNA was amplified successfully from both the enriched soil bacteria and
cultured methanotrophic DNA (lanes 4 and 5, respectively). Lane 2 shows a negative control
and lane 3 shows a positive control for 16s rDNA using Mitrichosporium DNA. PmoA showed no
bands on the gel for either the cultured (lane 6) or soil (lane 7) DNA. Guppy genomic DNA (lane
9) appeared as a long continuous streak down the lane. The bands were measured against 1kb
(lane 1) and 100bp (lane 8) ladders.

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ladder

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Figure 2. Agarose gel electrophoresis of the guppy opsin DNA. The gel ran properly and was stained
properly based on the ladder (lane 1), but the opsin samples (lanes 2-9) showed very faint bands. Arrows
are indicating the two bands produced by the PCR of the opsin.

The agarose gel from the restriction digest of our 16s insert in the pGEM-T vector
(Figure 3, lane 2) showed six bands; one at 3015bp, representing the vector, one larger
band and four smaller bands. Two bands were expected for the 16s digest, one at 3 kb
and one at 1.5 kb. The pmoA restriction digest (lane 4) and opsin digest (lanes 1 and 3)
produced some expected bands, pmoA should have a band at the 545 bp level as well
as the 3kb vector band.

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ladder

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1 Kb
ladder

3.0 Kb>

<3.0 kb

Figure 2. 1% agarose gel of the EcoR1 restriction enzyme digest of the pGEM-T Easy vector and insert.
The vector is 3015bp and is present in all lanes, lane 1 shows opsin, lane 2 was a 16s rDNA insert; two
bands were expected but six are visible. Lane 3 is opsin and it shows a clean vector band but a very faint,
small second band, lane 4 is pmoA and has 2 bands in the appropriate areas. Lane 5 is 16s and shows the
vector band with two other faint bands, one slightly larger and one slightly smaller than the 3.0 kb
vector.

The class successfully genotyped nineteen guppies showing six heterozygotes and had
seven different observed alleles at the (TTA)n locus. However, issues with labeling
made comparing the allelic diversity between the pet store and South American captive
guppies impossible, so the gel from the class of 2009 was used for genetic comparison
between the two populations.
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The class of 2009 followed the same protocol and genotyped guppies from the same
two populations as our class: the Hillside pet store and Dr. Taylors captive South
American guppies, thus, the results should be comparable to what our class would have
obtained if the labeling order had been maintained (Figure 3).

a.

b.

Figure 3a and 3b. Microsatellite polyacrylamide gel from captive South American and pet store guppies.
A) Our class gel, showing successful genotyping but incorrect labeling made testing impossible. B) The
class of 2009 gel, showing clean bands for all fish. Lanes 1-11 were the South American captive fish with
noticeably reduced allelic variation and increased homozygosity. Lanes 12-22 were the pet store guppies
with six different alleles and only two homozygotes at this locus. Stutter bands are evident in both gels.

Last years pet store population had six alleles at the microsatellite locus, and 7 of the 9
fish were heterozygotes. GenPOP analysis concluded that the pet store population was
not in Hardy Weinberg (HW) equilibrium (P-value = 0.03). The number of homozygotes
and heterozygotes was as predicted by HW equilibrium, but the genotype frequency
was not as predicted by the allele frequency. Last years South American captive
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guppies had only 2 alleles at the tested locus and 6 out of 10 fish were homozygotes,
However, GenPOP concluded the population was in HW equilibrium (P = 0.56). The Fst
values for the full population of the guppies was 0.1412 higher than the expected
heterozygosity in the total population.

Our 16s plates showed many colonies on the plate including both blue and white
colonies. There were approximately eight white colonies, indicating a successful
transformation. However, the colony hybridization resulted in no DIG-labeled colonies
when exposed to film (Figure 4a) but others in the class had successful hybridization
(Figure 4b), with many colonies DIG-labeled, including blue colonies, which did not have
the 16s insert.

a.

b.

Figure 4a and 4b. Colony hybridization from the cloning reaction with the 16s probe. The large black
circle is the nylon membrane and the dark and light spots are DNA from JM109 E. coli colonies, the black
spots are the colonies that strongly bound the DIG-labeled 16s probe. A) Our results showing no
colonies; the large dark spots are the blue staining from the galactosidase of the colonies containing
the vector with no insert and B) expected 16s results, showing many successful colonies.

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The pmoA plates (not shown) also resulted in DIG-labeled colonies, yet fewer colonies
were labeled. Controls for both the 16s and pmoA were present, leaving a larger
diameter spot for the 8L sample in comparison to the 4 L sample.
The phylogenic p-distance analysis of the 16s soil sample revealed that the VA sample
had high similarity to Burkholderia sartisoli (AM745942.1)(Figure 5). These sequences
have less than 0.025 substitutions per nucleotide position and 47% of the replicate trees
in the bootstrap analysis. The next closest relation was Burkholderia cepacia
(AY741358.1) which was branched off at this point with a 100% tree replication. The BM
16s sequence showed less similarity to the Genbank sequences than the VA sequence
but it was branched off of two clostridium sequences (Clostridium sp. BL-30,
DQ196622.2 and Clostridium frigoris, AJ506116.1)

14 16s V A

Burkholderia sartisoli AM745942.1

gi|148745932|emb|AM745942.1|B
gi|53774180|gb|AY741358.1|Bur
Burkholderia
cepacia AY741358.1
3 16s B M

Clostridium sp. BL-30 DQ196622.2

gi|194245267|gb|DQ196622.2|Cl
gi|22531593|emb|AJ506116.1|Cl
Clostridium frigoris AJ506116.1

Figure 5. P-distance analysis of six 16s sequences.

The optimal tree with the sum of branch length = 0.60198192 is shown. The percentage of
replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates)
are shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as
those of the evolutionary distances used to infer the phylogenetic tree. Phylogenetic analyses were
conducted in MEGA4.

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By analyzing the 16s sequences with Jukes-Cantor, some different results were
obtained (Figure 6). The VA sequence became more distantly related to the
Burkholderia sartisoli, and was branched off from the two Burkholderia sequences
100% of the time. The BM phylogeny changed very little.
Burkholderia sartisoli AM745942.1
gi|148745932|emb|AM745942.1|B
gi|53774180|gb|AY741358.1|Bur
Burkholderia cepacia AY741358.1
14 16s V A
3 16s B M
Clostridium sp. BL-30 DQ196622.2
gi|194245267|gb|DQ196622.2|Cl
gi|22531593|emb|AJ506116.1|Cl
Clostridium frigoris AJ506116.1

Figure 6. Jukes- Cantor analysis of six 16s sequences.

The optimal tree with the sum of branch length = 1.01943927 is shown. The percentage of
replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates)
are shown next to the branches The tree is drawn to scale, with branch lengths in the same units as
those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances
were computed using the Jukes-Cantor method and are in the units of the number of base
substitutions per site. All positions containing alignment gaps and missing data were eliminated only
in pairwise sequence comparisons (Pairwise deletion option). There were a total of 547 positions in
the final dataset. Phylogenetic analyses were conducted in MEGA4.

Analysis of p-distance trees using DNA sequences from pmoA genes showed that three
nearly identical sequences were obtained from the soil sample from both the Thursday
and Friday labs (Figure 7). These sequences were most closely related to uncultured
bacteria (AY50036.1 and AY372363.1). However, the phylogeny suggests that these
sequences are related to methanotrophs, the closest related known methanotroph to
the isolated soil samples being Methylocapsa acidiphila (AJ278727.1) and the most
distant being Methylomicrobium album BG8 (U31654.1). A non-methanogenic bacteria,
Nitrosomonas sp. AL212 (AF327918.1) was also a part of the phylogenic analysis, but
was distantly related. Both trees (Figure 7 and Figure 8) had extremely similar
20

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nucleotide substitutions per position values and their corresponding bootstrap values
were very similar.

6 pmoa J J

Uncultured
putative methanotroph AY50036.1
gi|40647409|gb|AY500136.1|Unc
5 pmoa A A

Uncultured bacterium AY372363.1

gi|34733050|gb|AY372363.1|Unc
15 pmoa M HallG
gi|145694851|gb|EF452675.1|Un
uncultured bacterium EF452675.1

unculturedbacterium
bacteriumFN393863.1
EF452675.1
gi|255696822|emb|FN393863.1|U
Uncultured
gi|238544139|emb|FN394110.1|U
Uncultured methanotroph FN394110.1
Uncultured methanotroph FN 394169.1
gi|238544257|emb|FN394169.1|U

Methylocapsa acidiphila AJ278727.1

gi|15072625|emb|AJ278727.1|Me
Methylocystis parvus AJ544097.1
gi|37699864|emb|AJ544097.1|Me
gi|7188930|gb|U31650.2|MTU316
Methylosinus trichosporium U31650.2
gi|21628644|emb|AJ431386.1|Me
Methylocystis sp. SC2 AJ431386.1
gi|21685158|emb|AJ459042.1|Me
Methylocystis parvus AJ459042.1
gi|21685074|emb|AJ459000.1|Me
Methylocystis echinoides AJ459000.1

Methylococcus capsulatus AF533666.1

gi|33329870|gb|AF533666.1|Met
Methylomonas methanica U31653.1
gi|1002720|gb|U31653.1|MMU316
gi|1002718|gb|U31654.1|MAU316
Methylomicrobium album BG8 U31654.1
gi|19310207|gb|AF327918.1|Nit
Nitrosomonas sp. AL212 AF327918.1

Figure 7. P-distance analysis of nineteen pmoA sequences.

The optimal tree with the sum of branch length = 1.44142373 is shown. The percentage of replicate
trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are
shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as
those of the evolutionary distances used to infer the phylogenetic tree. Codon positions included
were 1st+2nd+3rd+Noncoding. All positions containing gaps and missing data were eliminated from
the dataset (Complete deletion option). There were a total of 447 positions in the final dataset.
Phylogenetic analyses were conducted in MEGA4.

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6 pmoa J J
Uncultured putative
gi|40647409|gb|AY500136.1|Unc
methanotroph AY500136.1

5 Uncultured
pmoa A A

putative
methanotroph AY500136.1

Uncultured bacterium AY372363.1

gi|34733050|gb|AY372363.1|Unc

15 pmoa M HallG
gi|145694851|gb|EF452675.1|Un
uncultured bacterium EF452675.1
Uncultured bacterium FN393863.1
gi|255696822|emb|FN393863.1|U
Uncultured methanotroph FN394110.1
gi|238544139|emb|FN394110.1|U
Uncultured methanotrophic

gi|238544257|emb|FN394169.1|U
bacterium FN 394169.1

gi|15072625|emb|AJ278727.1|Me
Methylocapsa acidiphila AJ278727.1
gi|37699864|emb|AJ544097.1|Me
Methylocystis parvus AJ544097.1
gi|7188930|gb|U31650.2|MTU316
Methylosinus trichosporium U31650.2
gi|21628644|emb|AJ431386.1|Me
Methylocystis sp. SC2 AJ431386.1
Methylocystis parvus AJ459042.1
gi|21685158|emb|AJ459042.1|Me
Methylocystis echinoides AJ459000.1
gi|21685074|emb|AJ459000.1|Me
Methylococcus capsulatus AF533666.1
gi|33329870|gb|AF533666.1|Met
Methylomonas methanica U31653.1
gi|1002720|gb|U31653.1|MMU316
Methylomicrobium album BG8 U31654.1
gi|1002718|gb|U31654.1|MAU316
Nitrosomonas sp. AL212 AF327918.1
gi|19310207|gb|AF327918.1|Nit

Figure 8. Jukes-Cantor analysis of nineteen pmoA sequences.

The optimal tree with the sum of branch length = 1.86678146 is shown. The
percentage of replicate trees in which the associated taxa clustered together in the
bootstrap test (1000 replicates) are shown next to the branches. The tree is drawn to
scale, with branch lengths in the same units as those of the evolutionary distances used
to infer the phylogenetic tree. The evolutionary distances were computed using the
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Jukes-Cantor method and are in the units of the number of base substitutions per site.
Codon positions included were 1st+2nd+3rd+Noncoding. All positions containing gaps
and missing data were eliminated from the dataset (Complete deletion option). There
were a total of 447 positions in the final dataset. Phylogenetic analyses were conducted
in MEGA4.
Opsin was not used for phylogenic analysis as the sequences obtained did
not appear to in fact be true opsin genes, and only a very small sequence was viable
which had high phylogeny to many varied and unrelated sequences in Genbank.
Discussion
In this study we have confirmed the presence of methanotrophic bacteria in
soil collected from the Hartland landfill in Victoria, Canada by PCR amplification of 16s
rDNA and the methanotroph-specific pmoA gene. Cultured methanotrophs (Interium
isolate) were also analyzed following the same methods as the landfill soil and a colony
hybridization with a DIG probe was performed. The colony hybridization of our 16s
resulted in three colonies reacting with the 16s probe. It was expected that all bacterial
colonies on the plate would hybridize with the 16s probe and thus fluoresce as seen in
Figure 3b. Universal colony fluorescence is expected for the JM 109 E. coli cells since
16s rRNA is present in all bacteria.
A restriction digest of both 16s and pmoA fragments confirmed both fragments
were incorporated into pGEM-T vectors, sequenced, and tested for diversity. Phylogenic
analysis revealed a broad diversity of methanotroph-like sequences. These initial steps
to detect the microbes are vital in community analysis, but detailed investigation into
physiology is only possible when pure cultures are obtained (Wise et al. 1999). For this
reason, we were not able to classify all bacteria found. Furthermore, the methaneenriched conditions of the soil sample may have allowed opportunistic methanotrophic
bacteria to outcompete naturally dominant bacterial species, skewing the phylogenetic
tree to not be representative of the general population of the soil sample (Ward et al.
1995).

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Not as many sequences were obtained as we had hoped for; many of the sequences
were rejected before the phylogenetic analysis due to very short insert sequences or
primers annealing to each other due to sequence similarity and not incorporating an
insert. However, the pmoA sequence analysis resulted in three nearly identical
sequences derived from the bacteria on from both the Thursday and Friday class.
The 16s samples isolated were separated by high bootstrap values and more
closely related to other bacterial species than to each other (Figures 5 & 6). This could
be due to the 16s primers being non-specific for methanotrophs and thus isolating any
bacterial species. It is possible to create primers specific for methanotrophs, but since
all bacteria contain 16s rDNA, many bacterial species will be amplified by general
primers such as the ones used in this study (Wise et al. 1999).
The pmoA samples isolated had lower bootstrap values and were very similar
to each other as compared to the 16s samples. They were most closely related to
uncultured bacteria, yet the phylogenetic analysis strongly suggested that the
sequences obtained were methanotrophic. The high presence of methanotrophic
bacteria in the soil could be explained by the levels of methane produced by
decomposing organic matter within the landfill (Wartiainen et al. 2003).
Though P-distances were measured, they did not provide accurate amount of distances
so Jukes and Cantor tables were used. For pmoA, larger p distances were enhanced by
the Jukes and Cantor distances whereas smaller p distances were not largely affected
by the Jukes and Cantor equation. For example, number 1 compared to number 8 in the
pmoA p-distance table (supplementary figure 1) corresponded to a p distance of 0.424.
This large p distance number was greatly increased to a distance of 0.625 by the Jukes
and Cantor method whereas a p distance of 0.007 (the p-distance between 9 and 10)
was not increased at all by the Jukes and Cantor method.
The 2009 guppy analysis for the (TTA) n microsatellites produced expected
results. There are a total of at least 26 alleles at this locus in wild guppies from South
America, suggesting the captive guppies have been greatly inbred to lose all but two
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alleles likely due to a small captive population size maintained over the previous five
years, and bottle neck effects from when the population was reduced to eight individuals
during captivity. However, the six alleles that arose at this locus from the eleven guppies
tested from the pet store signify that the pet store guppies are more genetically diverse
than the captive guppies, likely due to the larger population size maintained within the
pet store suppliers guppy tank, and the guppy supply frequently being restocked with
fish from different sources. Microsatellites have high mutation rates with allows for many
allelic polymorphisms between individuals in a population (Lin et al. 2010; Bleakley et al.
2008). Because of such high mutation rates at a microsatellite locus, the probability of a
mutation occurring twice in the same location is reasonably high.
It was found that the captive South American guppies were in HW equilibrium yet
the pet store guppies were not. However, these results may be due to the small sample
size (n=10 from each population), and if more pet store guppies were sampled, it may
reveal that they are in fact, in HW equilibrium. That being said, achieving the necessary
criteria for the HW equilibrium in nature is rare (Sperlich and Frh 2009) and the lack of
equilibrium displayed in this sample does detract from the increased allele frequency of
the pet store guppies. The FST value from the collective population was 0.1412
indicating that there were more homozygotes in the population than expected. This is
due to the lost allelic diversity in the captive guppies.

References
Barson, N., Cable, J., and Oosterhoust, V. 2009. Population genetic analysis of
microsatellite variation of guppies (Poecilia reiculata) in Trinidad and Tobago:
evidence for a dynamic source-sink metapopulation structure, founder events and
population bottlenecks. Evol Biol. 22(2009): 485- 497. doi: 10.1111/j.14209101.2008.01675.x.

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Bleakly, B., Eklund, A., and Brodie, E. 2008. Are designer guppies inbred?
Microsatellite variation in five strains of ornamental guppies, Poecilia reticulate, used
for behavioral research. Zebrafish. 5(1):39-48. doi: 10.1089/zeb.2007.0513.
DeJournett, T., Arnold, W., and LaPara, T. 2006. The characterization and quantification
of methanotrophic bacterial populations in constructed wetland sediments using
PCR targeting 16S rRNA gene fragments. Applied Soil Ecology. 1(35): 648-659.
10.1016/j.apsoil.2006.09.006.
Li, R., Dong, G., Wu, X., Wang, P., Wang, X., Chen, Z. 2006. Microsatellite DNA
monitoring in inbred cultivation of outbred rats. Yi Chuan. 28(7):821-4. PMID:
16825169
Lin, H., Fan, H., Zhang, F., Huang, X., Lin, K., Shi, L., Hu, S., Chu, J., and Wang, D.
2010. Genetic relationships of ethnic minorities in southwest China revealed by
microsatellite markers. PLoS ONE. 5(3): e9895. doi:10.1371/journal.pone.0009895.
McDonald, I., Smith, K., and Lidstrom, M. 2005. Methanotrophic populations in
estuarine sediment from Newport Bay, California. FEMS Microbiology Letters.
1(250): 287-293. doi: 10.1016/j.femsle.2005.07.016.
Nakajima, M., and N. Taniguchi. 2001. Genetics of the guppy as a model for experiment
in aquaculture. Genetica. 1(111):279-289. doi: 10.1023/A:1013717516234.

Sperlich, D., and Frh. 2009. The Hardy-Weinberg-Distribution was discovered 100
years ago: A short biography of W. Weinberg. J. Zool. Syst. Evol. Res. 47(1), 26.
doi: 10.1111/j.1439-0469.2008.00518

Suk, H., and Neff, B. 2009. Microsatellite genetic differentiation among populations of
the Trinidadian guppy. Heredity. 1(102):425-434. doi: 0018-0677009.

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Ward, M., Churcher, A., Dick, K., Laver, C., Owens, G., Polack, M., Ward, P., Breden,
F., and Taylor, J. 2008. The molecular basis of color vision in colorful fish: Four long
wave-sensitive (LWS) opsins in guppies (Poecilia reticulata) are defined by amino
acid substitutions at key functional sites. BMC Evol Biol. 8(210). PMCID:
PMC2527612.

Ward, N., Rainey, F., Stackebrandt, E., and Schlesner, H. 1995. Unraveling the extent
of diversity withing the order Planctomycetales. Appl. Environ. Micorbiol. 61(6):
2270-2275. doi: 0099-2240/95/$04.0010.
Wartiainen, I., Hestnes, A., and Svenning, M. 2003. Methanotrophic diversity in high
arctic wetlands on the islands of Svalbard (Norway) denaturing gradient gel
electrophoresis analysis of soil DNA and enrichment cultures. Can. J. Microbiol.
1(49): 602-612. doi: 10.1139/W03-080.
Wise, M., McArthur, J., and Shimkets, L. 1999. Methanotroph diversity in landfill soil:
Isolation of novel type I and type II methanotrophs whose presence was suggested
by culture-independent 16S ribosomal DNA analysis. Appl. Environ. Microbiol.
65(11): 4887-4897. doi :0099-2240/99$04.00+0.

Supplementary Material
Title: : pmoAclustalsn
Description
No. of Taxa : 19
Data File : D:\Documents and Settings\slnorman\Desktop\pmoAclustalsn.meg
Data Title : : pmoAclustalsn
Data Type : Nucleotide (Coding)
Analysis : Pairwise distance calculation
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->Compute : Distances only

Include Sites : ==============================
->Gaps/Missing Data : Pairwise Deletion
->Codon Positions : 1st+2nd+3rd+Noncoding
Substitution Model : ==============================
->Model : Nucleotide: p-distance
->Substitutions to Include : d: Transitions + Transversions
->Pattern among Lineages : Same (Homogeneous)
->Rates among sites : Uniform rates
No. of Sites : 450
d : Estimate

[ 1] #gi|1002720|gb|U31653.1|MMU316 Methylomonas methanica

[ 2] #gi|1002718|gb|U31654.1|MAU316 Methylomicrobium album BG8

[ 3] #gi|33329870|gb|AF533666.1|Met Methylococcus capsulatus

[ 4] #gi|255696822|emb|FN393863.1|U partial pmoA gene for particulate methane
monooxygenase subunit A, clone p4c58.

[ 5] #gi|238544139|emb|FN394110.1|U Uncultured methanotrophic bacterium partial

pmoA gene for
particulate methane monooxygenase subunit A, clone AF31.

[ 6] #gi|238544257|emb|FN394169.1|U partial pmoA gene for

particulate methane monooxygenase subunit A, clone NF22.

[ 7] #6_pmoa_J_J
[ 8] #5_pmoa_A_A
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[ 9] #gi|40647409|gb|AY500136.1|Unc Uncultured putative methanotroph clone Evo72

particulate methane
monooxygenase (pmoA) gene, partial cds.

[10] #gi|34733050|gb|AY372363.1|Unc Uncultured bacterium clone Vlp12 particulate

methane monooxygenase alpha subunit (pmoA) gene, AY372363.1
[11] #15_pmoa_M_HallG
[12] #gi|145694851|gb|EF452675.1|Un = uncultured bacterium, EF452675.1
[13] #gi|15072625|emb|AJ278727.1|Me = Methylocapsa acidiphila AJ278727.1
[14] #gi|21685158|emb|AJ459042.1|Me = Methylocystis parvus AJ459042.1
[15] #gi|21685074|emb|AJ459000.1|Me = Methylocystis echinoides AJ459000.1
[16] #gi|21628644|emb|AJ431386.1|Me = Methylocystis sp. SC2 AJ431386.1
[17] #gi|7188930|gb|U31650.2|MTU316 = Methylosinus trichosporium U31650.2
[18] #gi|37699864|emb|AJ544097.1|Me = Methylocystis parvus AJ544097.1
[19] #gi|19310207|gb|AF327918.1|Nit = Nitrosomonas sp. AL212 AF327918.1

10

11

12

13 14

15

16

17

18

19

[ 1]
[ 2] 0.230
[ 3] 0.317 0.301
[ 4] 0.424 0.413 0.323
[ 5] 0.422 0.415 0.332 0.013
[ 6] 0.429 0.417 0.323 0.033 0.031
[ 7] 0.422 0.406 0.327 0.033 0.038 0.040
[ 8] 0.424 0.406 0.330 0.038 0.042 0.045 0.013
[ 9] 0.424 0.408 0.325 0.031 0.036 0.038 0.007 0.016
[10] 0.424 0.411 0.330 0.024 0.029 0.031 0.009 0.013 0.007
[11] 0.424 0.408 0.327 0.024 0.033 0.040 0.027 0.031 0.024 0.022
[12] 0.424 0.420 0.330 0.031 0.031 0.036 0.038 0.042 0.036 0.029 0.038

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[13] 0.420 0.400 0.296 0.232 0.238 0.232 0.245 0.238 0.245 0.243 0.232 0.241
[14] 0.402 0.364 0.321 0.305 0.312 0.310 0.303 0.310 0.303 0.305 0.303 0.312 0.283
[15] 0.408 0.371 0.327 0.307 0.316 0.310 0.305 0.312 0.305 0.307 0.305 0.316 0.281 0.027
[16] 0.402 0.373 0.321 0.316 0.323 0.316 0.310 0.316 0.310 0.312 0.314 0.325 0.287 0.069 0.071
[17] 0.426 0.388 0.303 0.301 0.307 0.301 0.303 0.305 0.303 0.301 0.303 0.310 0.258 0.134 0.145 0.118
[18] 0.397 0.377 0.278 0.281 0.285 0.283 0.278 0.278 0.278 0.281 0.281 0.294 0.274 0.252 0.254 0.243 0.227
[19] 0.417 0.440 0.392 0.468 0.474 0.468 0.479 0.479 0.477 0.472 0.472 0.468 0.443 0.445 0.443 0.439 0.445 0.437

P dis genetic distances pmoA nucleotides

Title: : pmoAclustalsn
Description
No. of Taxa : 19
Data File : D:\Documents and Settings\slnorman\Desktop\pmoAclustalsn.meg
Data Title : : pmoAclustalsn
Data Type : Nucleotide (Coding)
Analysis : Pairwise distance calculation
->Compute : Distances only
Include Sites : ==============================
->Gaps/Missing Data : Pairwise Deletion
->Codon Positions : 1st+2nd+3rd+Noncoding
Substitution Model : ==============================
->Model : Nucleotide: Jukes-Cantor
->Substitutions to Include : All
->Pattern among Lineages : Same (Homogeneous)
->Rates among sites : Uniform rates
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No. of Sites : 450

d : Estimate

[ 1] #gi|1002720|gb|U31653.1|MMU316
[ 2] #gi|1002718|gb|U31654.1|MAU316
[ 3] #gi|33329870|gb|AF533666.1|Met
[ 4] #gi|255696822|emb|FN393863.1|U
[ 5] #gi|238544139|emb|FN394110.1|U
[ 6] #gi|238544257|emb|FN394169.1|U
[ 7] #6_pmoa_J_J
[ 8] #5_pmoa_A_A
[ 9] #gi|40647409|gb|AY500136.1|Unc
[10] #gi|34733050|gb|AY372363.1|Unc
[11] #15_pmoa_M_HallG
[12] #gi|145694851|gb|EF452675.1|Un
[13] #gi|15072625|emb|AJ278727.1|Me
[14] #gi|21685158|emb|AJ459042.1|Me
[15] #gi|21685074|emb|AJ459000.1|Me
[16] #gi|21628644|emb|AJ431386.1|Me
[17] #gi|7188930|gb|U31650.2|MTU316
[18] #gi|37699864|emb|AJ544097.1|Me
[19] #gi|19310207|gb|AF327918.1|Nit

10

11

12

13 14

15

16

17

18

19

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[ 1]
[ 2] 0.275
[ 3] 0.412 0.385
[ 4] 0.625 0.600 0.422
[ 5] 0.620 0.605 0.438 0.013
[ 6] 0.635 0.610 0.422 0.034 0.032
[ 7] 0.620 0.585 0.430 0.034 0.039 0.041
[ 8] 0.625 0.585 0.434 0.039 0.044 0.046 0.013
[ 9] 0.625 0.590 0.426 0.032 0.037 0.039 0.007 0.016
[10] 0.625 0.595 0.434 0.025 0.030 0.032 0.009 0.013 0.007
[11] 0.625 0.590 0.430 0.025 0.034 0.041 0.027 0.032 0.025 0.023
[12] 0.625 0.615 0.434 0.032 0.032 0.037 0.039 0.044 0.037 0.030 0.039
[13] 0.615 0.571 0.377 0.277 0.287 0.277 0.297 0.287 0.297 0.293 0.277 0.290
[14] 0.575 0.498 0.418 0.392 0.403 0.399 0.388 0.399 0.388 0.392 0.388 0.403 0.355
[15] 0.590 0.511 0.430 0.395 0.411 0.399 0.392 0.403 0.392 0.395 0.392 0.411 0.352 0.027
[16] 0.575 0.515 0.418 0.411 0.422 0.411 0.399 0.411 0.399 0.403 0.407 0.426 0.362 0.072 0.075
[17] 0.630 0.547 0.388 0.384 0.395 0.384 0.388 0.392 0.388 0.384 0.388 0.399 0.317 0.147 0.161 0.128
[18] 0.566 0.524 0.348 0.352 0.359 0.355 0.348 0.348 0.348 0.352 0.352 0.373 0.341 0.307 0.310 0.293 0.271
[19] 0.610 0.662 0.555 0.733 0.751 0.733 0.763 0.763 0.757 0.745 0.745 0.733 0.670 0.676 0.670 0.660 0.676 0.654

pmoa jukes and cantor table

Supplementary Table 1. Estimates of Evolutionary Divergence between

Sequences.

The number of base substitutions per site from analysis between sequences is shown . All results
are based on the pairwise analysis of 19 sequences. Analyses were conducted using the JukesCantor method in MEGA4 [1, 2]. Codon positions included were 1st+2nd+3rd+Noncoding. All
positions containing alignment gaps and missing data were eliminated only in pairwise sequence
comparisons (Pairwise deletion option). There were a total of 450 positions in the final dataset.

32

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April Goebl

Stephanie Norman
V00482420

Title: : 16shelp
Description
No. of Taxa : 6
Data File : C:\Users\Owner\Desktop\362 Report\16shelp.meg
Data Title : : 16shelp
Data Type : Nucleotide (Coding)
Analysis : Pairwise distance calculation
->Compute : Distances only
Include Sites : ==============================
->Gaps/Missing Data : Pairwise Deletion
->Codon Positions : 1st+2nd+3rd+Noncoding
Substitution Model : ==============================
->Model : Nucleotide: Jukes-Cantor
->Substitutions to Include : All
->Pattern among Lineages : Same (Homogeneous)
->Rates among sites : Uniform rates
No. of Sites : 593
d : Estimate

[1] #16s_B_M
[2] #Burkholderiacaryophylli16Srib
[3] #ClostridiumspBL3016Sribosomal
[4] #Clostridiumfrigoris16SrRNAgen
33

Lab Section B01

April Goebl

Stephanie Norman
V00482420

[5] #BurkholderiaxenovoransstrainT
[6] #16s_V_A

6]

[1]
[2] 0.000
[3] 1.109 1.109
[4] 1.119 1.119 0.049
[5] 1.254 1.254 0.257 0.231
[6] 1.266 1.266 1.522 1.449 1.364
Title: : 16shelp
Description
No. of Taxa : 6
Data File : C:\Users\Owner\Desktop\362 Report\16shelp.meg
Data Title : : 16shelp
Data Type : Nucleotide (Coding)
Analysis : Pairwise distance calculation
->Compute : Distances only
Include Sites : ==============================
->Gaps/Missing Data : Pairwise Deletion
->Codon Positions : 1st+2nd+3rd+Noncoding
Substitution Model : ==============================
->Model : Nucleotide: p-distance
->Substitutions to Include : d: Transitions + Transversions
34

Lab Section B01

April Goebl

Stephanie Norman
V00482420

->Pattern among Lineages : Same (Homogeneous)

->Rates among sites : Uniform rates
No. of Sites : 593
d : Estimate

[1] #16s_B_M
[2] #Burkholderiacaryophylli16Srib
[3] #ClostridiumspBL3016Sribosomal
[4] #Clostridiumfrigoris16SrRNAgen
[5] #BurkholderiaxenovoransstrainT
[6] #16s_V_A

6]

[1]
[2] 0.000
[3] 0.579 0.579
[4] 0.581 0.581 0.048
[5] 0.609 0.609 0.217 0.199
[6] 0.611 0.611 0.651 0.641 0.628

35