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Journal of Student Research (2013)

Volume 2, Issue 1: pp.

9-16

Research Article

Department of Biology; Washington & Jefferson College; Washington; Pennsylvania; 15301; USA.

Corresponding Author: Anupama Shanmuganathan, ashanmuganathan@washjeff.

edu

Culture-dependent methods increase observed soil

bacterial diversity from Marcellus shale temperate forest

in Pennsylvania

Evan Lutton

, Roos Schellevis

, Anupama Shanmuganathan

Soil bacteria comprise a largely untapped resource for improvements in environmental and biomedical sciences, yet only 1-10%

are culturable in the laboratory (Hirsch and Valdés, 2009).

This poor representation has been attributed to the stringent growth

demands and low growth rate of some species (Torsvik et al.

, 1990).

Developing culture-dependent protocols that identify unique

bacterial operational taxonomic units (OTUs) is an important research topic in soil bacterial ecology.

Establishing new OTUs in

culture will permit the study of their morphology and physiology that may advance agriculture and pharmacology.

Culturability

may be improved by employing different media to satisfy inherent preferences of growth substrate utilization.

Therefore, soil-

extract agar, R-2A agar and 1% nutrient agar were used in this study.

Soil bacteria were isolated in the winter from Abernathy

Field Station, a Marcellus shale temperate forest in Washington, Pennsylvania.

Monitoring bacterial diversity in this ecosystem

can be used to assess the early environmental consequences of anthropological factors, such as hydraulic fracturing in the

Marcellus shale region.

For long term monitoring, this sample collection was analyzed in conjunction with previous years’

assessments.

Isolates were analyzed taxonomically and phylogenetically.

Unique OTUs were identified through comparative

analysis of 16S rDNA.

The Shannon-Weaver and Simpson’s diversity indices ranked isolates on soil-extract agar highest for

species richness.

Rarefaction analysis suggested that sampling saturation of OTUs identified on soil-extract agar had not yet been

reached.

Each medium studied supported isolates of four common phyla: Actinobacteria, Bacteroidetes, Firmicutes, and

Proteobacteria.

Soil-extract agar supported the greatest proportion of pigmented colonies including a cyanobacterium with intra-

16S rDNA polymorphism.

Each medium supported the growth of unique OTUs and genera with Bacillus, Flavobacterium,

Pseudomonas, Rhizobium, and Streptomyces found on each.

This study suggests that utilizing different media can increase the

culturability of soil bacteria, giving a wider representation of soil bacterial communities.

Keywords: 16S rDNA; Soil bacteria; Bacterial phylogeny

Introduction

The ecology of soil bacteria is becoming of increased

interest as an early indicator of potential changes within an

ecosystem.

Alterations in climate change, pollution levels,

and ecosystem inhabitants each impact the overall

biodiversity of an ecosystem (Hannah et al., 2002).

Responses

to such changes are first apparent in the dynamic bacterial

flora in the soil (Lundquist et al.

, 1999).

Specifically, changes

in soil bacterial diversity can be used to assess the impact of

anthropological factors on surrounding areas (Stan et al.

2011).

For this reason, long term ecological monitoring

studies can be established to predict the effects of human

behavior, such as hydraulic fracturing in the Marcellus shale

region, by first understanding the responses of the native

prokaryotes.

In the process of hydraulic fracturing, a fluid is injected

into the ground under high pressure.

This injection fractures

the underlying rock formation, and a propping agent is

administered to prevent the fractures from closing so that

natural gas can be collected (Cooke, 1975).

The injected fluid

consists of chemically enhanced water containing compounds

like polysaccharides and polyacrylamides and salts or organic

acids to crosslink these polymers.

These chemicals are

digested after fracking with breakers including enzymes and

radical peroxides (Harris et al., 1997).

Unfortunately, not all

compounds are removed from the rock, and some of the

chemicals may leak into the surrounding ground, thereby

contaminating the soil.

This invasive fracturing method may

also create unexpected fractures in the ground through which

the natural gas can escape to the surface.

All of these

chemicals might have an influence on the diversity of soil

bacteria present in the soil.

Overall, the effects of the recent

expansion of hydraulic fracturing, in the Marcellus shale

region of Pennsylvania are not well understood.

Therefore it

is ever more important to establish and maintain baseline

definitions of the region’s soil bacterial populations to better

depict the bacterial responses resonating from such activity

(Kohler et al., 2011).

While some soil bacteria are easily cultured in the

laboratory, there persist unidentified populations of

unculturable bacteria that are thus overlooked in routine

culture-dependent analyses (Janssen et al.

, 2002).

It is

estimated that only 1-10% of all soil bacteria are considered

to be culturable (Torsvik et al.

, 1990).

This incomplete picture

may be remedied by allowing bacteria to grow under different

conditions, perhaps to mimic their natural niches and allow

for the growth of certain populations over others.

The use of

methods that permit the growth of previously unculturable

bacteria is necessary to understand the physical and biological

properties of such bacteria beyond the sequences of their 16S

rDNA (Joseph et al., 2003; Sait et al.

, 2002).

Furthermore,

increasing bacterial culturability will more accurately reflect

bacterial diversity in the soil, thus giving a better

Journal of Student Research (2013)

Volume 2, Issue 1: pp.

9-16

Research Article

ISSN: 2167-1907

www.jofsr.

com

understanding of early prokaryotic responses to

environmental changes.

After obtaining soil samples from Abernathy Field

Station, a mixed-temperate forest in Washington,

Pennsylvania, three different culture media were utilized with

the intent of favoring different bacterial groups with inherent

differences in preferred growth substrate composition.

Previous work conducted by Joseph et al. (2003) suggests that

many unculturable groups of bacteria may be isolated in pure

culture with simple media.

Soil-extract agar, R-2A agar, and

1% nutrient agar have been reported to select for certain

bacterial populations based primarily on bacterial growth

characteristics.

It is crucial to consider the differing bacterial

growth rates because slow growing colonies are typically

overwhelmed by faster growing populations and are therefore

missed in culture-dependent diversity analyses.

By selecting

for different growth patterns between media, one can more

inclusively assess the bacterial diversity within a given soil

sample.

Bacterial diversity was assessed in terms of unique

operational taxonomic units (OTUs) (defined as having less

than 97% sequence similarity) which were identified via

comparative analysis of 16S rRNA genes amplified from

crude bacterial genomic DNA extracts (Sait et al., 2002).

This

method is generally accepted for the phylogenetic

classification of bacteria and has been used for the

identification of novel groups in previous studies (Joseph et

al., 2003; Sait et al., 2002; Janssen et al.

, 2002). Sequences

used for analysis are products of sample collection in 2011

(Papale et al., 2012) and 2012 (current study).

This study will

contribute to a better understanding of the microbial

ecosystem present in Abernathy Field Station including

seasonal shifts in bacterial populations and long term

responses to anthropological factors in the Marcellus shale

region.

Results

Figure 1.

Bacterial colony morphological diversity.

LSR3; Gray with a deep purple center, round with a smooth

margin, convex, 1 mm diameter.

B) LSR6; Off white and

glossy, irregular with smooth and rhizoid margins, convex, 4

mm diameter.

C) LSR7; Off white, filamentous, flat.

LSR8; Translucent, irregular with smooth/irregular margin,

flat, 1.

5 mm diameter.

E) LSR13; Marigold, round with

filamentous margins, convex, 2.

5 mm diameter.

F) ASR2;

Pale orange, irregular with filamentous margins, very convex,

0.5 mm diameter.

Bacterial culture gave morphologically distinct colonies

Plating diluted soil samples on 1% nutrient agar, R-2A

agar, and soil-extract agar gave rise to many morphologically

distinct colonies between and within media types (Figure 1).

Macroscopically unique colonies were subcultured on nutrient

agar to establish pure cultures for further experimentation.

While morphologically distinct colonies were isolated on

each culture medium studied, different proportions of

pigmented colonies were observed across media types (data

not shown).

Pigmentation was arbitrarily defined as colony

coloration with the exceptions of clear/translucent, white, and

off white, which were deemed not pigmented.

Sixty-seven

percent of soil-extract agar isolates were pigmented compared

to 42% on R-2A agar and only 34% on 1% nutrient agar.

All

culture media supported the growth of waterborne bacteria

without substantial differences.

(Waterborne isolates were

those obtained from the streambeds of Abernathy Field

Station.

)

PCR of 16S rDNA confirmed genomic DNA extraction

Following genomic DNA extraction and PCR

amplification of 16S rDNA, PCR products were confirmed

via gel electrophoresis with amplified 16S rDNA appearing as

a band at 1600 base pairs (bp).

Visualizing the successful

amplicons provided an unexpected distinction for one isolate,

ASR12 (Figure 2A).

This bacterium exhibited two amplicons

for 16S rDNA amplification which persisted when tested in

duplicate (Figure 2B).

PCR products were sequenced for

those isolates that gave bands for 16S rDNA (data not

shown).

Figure 2.

Unique results observed through gel

electrophoresis confirmation of bacterial 16S rDNA

amplification.

A) Green pigmented bacterial colonies

(ASR12; round, irregular margin, 0.

5 mm diameter) isolated

on soil-extract agar from streambed soil in Abernathy Field

Station, Washington, Pennsylvania.

B) 16S rDNA is indicated

by a band at 1600 bp.

ASR12 exhibited two amplicons for

16S rDNA amplified by PCR.

Only the lower molecular

weight band was successfully sequenced and characterized as

genus Chlorophyta.

Journal of Student Research (2013)

Volume 2, Issue 1: pp.

9-16

Research Article

ISSN: 2167-1907

www.jofsr.

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16S rDNA sequences analysis suggests increased culturability

with different media

After compiling sequence data for 2011 (Papale et al.,

2012) and 2012 isolates, bacterial diversity indices and OTU

counts were determined using MOTHUR, a microbial

community analysis platform (Schloss et al., 2009).

As shown

in Table 3, different indices conveyed mixed conclusions.

The

Shannon-Weaver and Simpson’s indices showed the most soil

bacterial diversity obtained on soil-extract agar, followed by

R-2A agar and 1% nutrient agar.

The ACE and Chao1 indices

showed the highest diversity obtained from R-2A agar,

followed by soil-extract agar and 1% nutrient agar.

Table 1.

Bacterial diversity indices.

R-2A

SEA

Total

Sequences analyzed

182

OTUs

OTU to sequence ratio

0.39

0.38

0.67

0.13

Shannon-Weaver Index

2.47

3.14

3.34

2.62

Simpson’s Index

0.08

0.06

0.017

0.09

ACE

20.75

93.76

60.86

31.57

Chao1 Index

17.5

Sequences were obtained in 2011 (Papale et al., 2012) and 2012 from isolates cultured on 1% nutrient agar (NA), R-2A agar, and

soil-extract agar (SEA).

All diversity indices were calculated from sequence data using MOTHUR (v.

1.24.1) (Schloss et al.,

2009).

Shannon-Weaver diversity index=-Σ[(n

/N)ln(n

/N)] where n is the number of individuals in the ith OTU and N is the total

number of individuals; Simpson’s index=[Σn

-1)]/ N(N-1) where n is the number of OTUs with i individuals and N is the total

number of individuals; Chao1 index= S

obs

+{[n

-1)]/2(n

+1)} where S

obs

is the number of species observed, n

is the number of

OTUs with only 1 sequence and n

is the number of OTUs with only two sequences; ACE values were determined as described

by MOTHUR (Schloss et al., 2009).

All isolates were classified under the domain Bacteria.

Bacterial isolates predominantly belonged to four common

phyla for each culture medium studied (Figure 3).

Classifications with greater than or equal to 80% sequence

identity as determined by RDP Classifier (Wang et al.

, 2007)

were considered to be accurate.

ASR12, the unique bacterium

isolated on soil-extract agar, belongs to the phylum

Cyanobacteria and is the first of its phylum and 16S rDNA

banding pattern to be cultured from soil of Abernathy Field

Station.

Observed phyla and their percent contributions to the

whole were as follows: Actinobacteria (15%), Bacteroidetes

(30%), Cyanobacteria (0.

6%), Firmicutes (12%), and

Proteobacteria (44%).

Bacterial isolates belonged to 40

genera, 24 families, and 13 orders.

Figure 3.

Taxonomic distribution of isolated soil bacteria.

Depicted isolates were obtained in winter 2011 (Papale et al.

, 2012)

and 2012.

Each culture medium gave isolates belonging to phyla Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria.

(NA = 1% nutrient agar; SEA = soil-extract agar)

Journal of Student Research (2013)

Volume 2, Issue 1: pp.

9-16

Research Article

ISSN: 2167-1907

www.jofsr.

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The use of three culture media increased soil bacteria

culturability as supported by the presence of unique isolates

obtained on each medium (Figure 4).

While the OTU level

overlap displayed increased similarity between colonies

isolated on each medium compared to the genus level overlap,

each culture medium demonstrated the ability to support

unique bacterial isolates of less than 97% sequence similarity.

The five common genera observed on each medium were

Bacillus, Flavobacterium, Pseudomonas, Rhizobium, and

Streptomyces.

Figure 4.

Venn diagrams depicting overlap of bacterial isolate classifications.

Venn diagrams displaying overlap at the OTU

(A) and genus (B) levels for bacteria isolated on 1% nutrient agar (NA), R-2A agar, and soil-extract agar (SEA).

Representative isolates from each OTU observed were

plotted in a phylogenetic tree based on multiple sequence

alignments of 16S rDNA (Figure 5).

The represented

phylogeny of the bacterial isolates cultured in this study

conform to the evolutionary history accepted of domain

Bacteria.

Journal of Student Research (2013)

Volume 2, Issue 1: pp.

9-16

Research Article

ISSN: 2167-1907

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Figure 5.

Phylogenetic distribution of isolated soil bacteria.

Multiple alignments of 16S rDNA sequences and phylogenetic

trees were constructed using the platform phylogeny.

fr (Dereeper et al.

, 2008) [settings “Advanced Mode” (MUSCLE v.

3.7) for

multiple alignment, GBlocks (v.

0.91b) for alignment refinement, and PhylMl (v.

3.0) for phylogeny using the maximum

likelihood method with 500 replicates for bootstrap values].

After analysis, phylogenetic trees were assembled using the TreeDyn

platform (v.

1.3.1) (Chevenet et al.

, 2006). One bacterial genus representing each OTU identified is depicted.

The use of 1% nutrient agar, R-2A agar, and soil-extract

agar for bacterial isolation yielded heterogeneous bacterial

colonies.

Rarefaction curves, constructed with data obtained

from MOTHUR (Schloss et al.

, 2009) suggest that sampling

saturation had not been reached for R-2A agar and soil-extract

agar (Figure 6).

Given an unsaturated sample, continued

isolation could identify a greater number of OTUs.

Figure 6.

Rarefaction analysis estimation of observed bacterial diversity.

Rarefaction curves were generated using MOTHUR

(v.1.24.

1) by a randomized re-sampling procedure (frequency=5) without replacement (Schloss et al.

, 2009).

The average

numbers of OTUs of less than 97% similarity found per number of bacteria isolated on each culture medium are depicted.

(NA:

1% nutrient agar; SEA: soil-extract agar)

Discussion

Hydraulic fracturing has raised many concerns regarding

the long term effects it may cause for entire ecosystems

(Rozell and Reaven, 2011).

Understanding the bacterial

responses to such effects is fundamental to predicting the

changes that may occur within the ecosystem as a whole.

Through the use of culture media with a range of nutritional

components and selective properties, it was hypothesized that

different bacterial populations would be obtained from the

same initial soil sample.

This increased culturability will more

accurately represent the soil bacterial diversity of Abernathy

Field Station and the local Marcellus shale region, an area that

may soon reflect the impact of local fracking in its soil.

The soil-extract agar used in this study appears to be

competent in obtaining morphologically distinct colonies and

may favor the growth of slow-growing pigmented colonies as

suggested by the relatively high proportion observed on this

medium.

Like soil-extract agar, R-2A agar permits the growth

of bacterial colonies traditionally repressed or unnoticed in

culture.

R-2A agar is reported to favor the growth of

pigmented bacteria (Carter et al., 2000) as well as waterborne

species (Harding et al., 1989).

Such slow growing colonies

are typically overwhelmed by faster growing populations and

are therefore missed in culture-dependent diversity analyses.

One colony isolated from soil-extract agar, ASR12, illustrates

the need to observe the primary bacterial cultures over an

extended period of time.

After two weeks of incubation at

room temperature, this small green colony (round, irregular

margin, 0.

5 mm diameter) was observed on soil-extract agar

plated with a diluted soil sample obtained from a streambed in

Abernathy Field Station.

As recommended by Carter et al.

(2000), prolonged incubation periods may facilitate the

identification of slow growing pigmented bacteria.

Findings

of the present study supported this claim.

Furthermore,

Hamaki et al.

(2005) used soil-extract agar to discover

potentially novel species of Acrocarpospora and

Streptosporangium, among others.

Soil-extract agar was noted

to support a greater diversity of bacteria than nutrient agar

(Taylor, 1951) and may offer growth rate advantages for

certain bacterial groups that are particularly well-suited to the

nutritional environment of their soil habitat.

Upon PCR amplification of 16S rDNA, ASR12 proved

to have unique properties beyond its green pigment.

Certain

mycobacteria (Ninet et al., 1996), as well as human pathogens

Bartonella henselae (Viezens and Arvand, 2008) and

Journal of Student Research (2013)

Volume 2, Issue 1: pp.

9-16

Research Article

ISSN: 2167-1907

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Campylobacter helveticus (Linton et al., 1994) have been

reported to yield two amplicons from a single PCR reaction

for 16S rDNA, a phenomenon attributed to the existence of

multiple rrn operons with intra-16S rDNA polymorphism

(Moreno et al., 2002).

ASR12 represents the first example of

this particular banding pattern for a bacterium isolated from

Abernathy Field Station.

However, unlike the aforementioned

examples, ASR12 was classified through the Ribosomal

Database Project (Wang et al., 2007) as phylum

Cyanobacteria, genus Chlorophyta. Cyanobacteria, like many

bacterial groups, have been reported to exhibit multiple rrn

operons and paralogous 16S rRNA gene copies.

One genus in

particular, Lyngbya, presented 16S rRNA gene copies with

sequence divergence of a greater magnitude than the 3%

difference threshold typically employed to distinguish

bacterial OTUs (Engene and Gerwick, 2011).

Since only the

lower molecular weight 16S rDNA band was successfully

sequenced for the Chlorophyta spp.

isolated in this study, it

would be of great interest to continue investigating the

functional properties and sequence identity of this bacterium.

Bacterial diversity was assessed between culture media

using diversity indices calculated by MOTHUR (Schloss et

al., 2009).

The Shannon-Weaver and Simpson’s indices

showed the most soil bacterial diversity obtained on soil-

extract agar, while the ACE and Chao1 indices show the

highest diversity with R-2A agar.

These discrepancies arise

from the different approaches taken to calculate each diversity

index.

The Shannon-Weaver and Simpon’s indices take into

account the number of unique OTUs as a function of the total

number of isolates in a sample set.

Since soil-extract agar

gave the highest ratio of OTUs per number of sequences

analyzed, it was calculated to display the greatest bacterial

diversity.

The ACE and Chao1 indices are based on the whole

number value of OTUs observed and therefore attribute the

highest level of bacterial diversity to R-2A agar.

As displayed

in Table 2, observed OTUs varied between culture media.

When the OTUs in the overall sample were assessed by

combining the sequences from the individual sample sets, the

number of observed OTUs was found to be lower than the

sum of the separate groups.

This can be explained by the fact

that when OTUs are calculated the result is entirely dependent

on the “neighbors” or other sequences involved in the

analysis.

Increasing the number of sequences for the

combined analysis increased the likelihood of sequence

similarity thus decreasing the overall number of observed

OTUs.

Of the bacterial phyla observed in this study, most

isolates belonged to Bacteroidetes and Proteobacteria at 30%

and 44%, respectively.

High levels of these oligotrophic

bacteria contradict the sampling outcomes of Kohler et al.

(2011) for soil collection in Abernathy Field Station during

the summer.

A study conducted by Manganelli et al. (2009)

reported greatly increased proportions of γ-Proteobacteria in

aquatic environments during winter months, contributing a

greater number of isolates from this group.

It must be noted

that bacterial populations fluctuate with seasonal changes, and

nutrient levels tend to diminish in the winter months with

increased soil moisture (Bardgett et al., 1999).

examination of bacterial genera obtained between culture

media illustrates the ability of each medium to select for

unique genera not seen on the other media studied.

This

supports the need for increased culturability in soil bacterial

diversity studies.

This study has supported claims that selective media and

prolonged incubation can drastically improve the bacterial

diversity detected in studies of soil microbial ecology

(Hamaki et al., 2005).

This improvement is particularly

important in understanding how bacterial populations change

over time and in response to changes in the environment.

accordance with the observations of Carter et al. (2000), R-

2A agar supported a greater percentage of pigmented colonies

than did 1% nutrient agar while both showed proportionately

fewer pigmented colonies than soil-extract agar.

Utilizing

each culture medium and experimenting with the selective

properties of others should be aims of future bacterial

diversity studies.

Experimental Procedures

Soil sample collection and media preparation

Soil-extract was prepared by autoclaving 200 g soil

suspended in 400 ml tap water.

The suspension was allowed

to settle, and fluid was decanted and centrifuged for 10

minutes at 3,400 rpm.

Supernatant was stored at -20 °C

overnight.

The thawed solution was filtered through a paper

towel with the filtrate being the final soil extract.

Soil-extract

agar was prepared, with slight modifications, according to a

previously established protocol (Rao, 1977).

Briefly, soil-

extract agar contained D-(+)-glucose (Sigma) (0.

1%),

potassium phosphate dibasic (Sigma) (0.

02%), Bacto-Agar

(Difco) (1.

5%), soil-extract (26%), and tap water to 1 L.

Medium was autoclaved for sterilization.

Soil samples of approximately 50 grams were collected

from Abernathy Field Station, a mixed temperate forest in

Washington, Pennsylvania, on February 9, 2012 from three

separate sampling sites, including one land (N 40° .

07.912 W

080° .

10.984) and two stream bed locations.

Leaf litter was

cleared and soil was collected at a depth of about 5 cm into

sterile 50 ml conical tubes.

Soil was also collected at the land

site for preparation of the soil-extract agar culture medium.

Two streambed soil samples were collected in the same

manner nearby in streams (N 40° .

07.883 W 080° .

11.010; N

40° .08.

046 W 080° .11.

059). Sequences used in analysis

were also obtained by Papale et al. (2012).

Culturing of soil bacteria

Stream bed samples were centrifuged at 3,200 rpm for 2

minutes, and the supernatant was discarded.

Suspensions were

made for samples from each of the three collection sites by

agitating 1 wet gram of soil in 100 ml sterile deionized water

at 200 rpm for 15 minutes.

Each suspension was serially

diluted to 10

-6

, and dilutions were mixed at 50 rpm for 10

minutes prior to plating.

200 μl of each dilution were spread

onto two plates of each 1% nutrient agar (Difco), R-2A agar

(Fluka), and soil-extract agar.

One plate of each pair was

incubated at room temperature and one at 4 °C.

Plates were

first examined for bacterial growth after 24 hours of

incubation.

Unique colonies, as determined by differences in

color, shape, margin, and elevation characteristics, were

subcultured on nutrient agar and incubated at room

temperature.

Subculturing continued as more macroscopically

unique colonies were identified, up to three weeks after the

initial cultures were established.

Journal of Student Research (2013)

Volume 2, Issue 1: pp.

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Research Article

ISSN: 2167-1907

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Bacterial DNA isolation

Bacterial DNA was extracted from colonies via

mechanical lysis according to a previously established

protocol (Sait et al., 2002).

Briefly, bacterial colonies from

pure culture were suspended in 200 μl sterile water with 0.

mm glass beads and shaken in a mini-bead-beater (Biospec

Products) for 1 minute.

Samples were then boiled for 10

minutes followed by 10 minutes of incubation on ice.

Samples

were centrifuged at 14,000 rpm for 10 minutes.

Supernatants

containing genomic DNA were collected and transferred to

sterile tubes.

The crude lysates were used immediately in PCR

or stored at -20 °C for later use (Kohler et al., 2011).

PCR of 16S rDNA

Genomic DNA was used to amplify 16S rDNA by PCR.

A 25 μl PCR reaction was prepared with 2X PCR Master Mix

(Fermentas), 0.

6 μM UnivF primer [GAG TTT GAT YMT

GGC TC], 0.

6 μM UnivR primer [GYT ACC TTG TTA CGA

CTT], and 3 μl genomic DNA extract.

PCR was performed

(PCR System 2700, Applied Biosystems) at 95 °C for 5

minutes followed by 32 cycles of 94 °C for 1 minute, 55 °C

for 2 minutes, and 72 °C for 3 minutes.

PCR products were

purified using the QIAquick PCR purification kit (Qiagen)

and confirmed through agarose gel electrophoresis.

The

concentrations of purified samples were determined using the

NanoDrop 2000c (Thermo Scientific).

In cases where multiple 16S rDNA bands were obtained

after PCR, the PCR product was run on a 2% agarose gel, and

the individual bands were cut out with a sterile scalpel.

DNA

was extracted using the QIAquick gel extraction kit (Qiagen).

Sequence analysis of 16S rDNA

Successful samples were sequenced at the Genomics

Core Facility, West Virginia University.

Returned sequences

were trimmed at the 5’- and 3’-ends for quality and selected

for further analysis based on nucleotide quality value and

probability of error using Sequence Scanner Software (v.

1.0

Applied Biosystems).

Trimmed sequences were characterized

using the Ribosomal Database Project (RDP).

Classifier tool

for 16S rDNA (Wang et al., 2007), MOTHUR, a microbial

community analysis platform (Schloss et al., 2009), was used

to analyze unique OTUs and provide indices of bacterial

diversity with an OTU defined as a sequence with greater than

3% difference from its neighbors.

Multiple alignments of 16S

rDNA sequences were made against a known 16S rDNA gene

database (Pruesse et al., 2007).

Representative 16S rDNA

sequences for unique OTUs were used to build a phylogenetic

tree using the platform phylogeny.

fr (Dereeper et al., 2008)

[settings “Advanced Mode” (MUSCLE v.

3.7) for multiple

alignment, GBlocks (v.

0.91b) for alignment refinement, and

PhylMl (v.

3.0) for phylogeny using the maximum likelihood

method with 500 replicates for bootstrap values].

After

analysis, phylogenetic trees were constructed using the

TreeDyn platform (v.

1.3.1).

Acknowledgments

This study was funded by the Undergraduate Science

Education Program Grant No.

52006323 from the Howard

Hughes Medical Institute to Washington & Jefferson College.

The authors acknowledge K.

Steider, A.

Del Sordo, N.

Beer,

and L.

Spilgies for aiding in sample collection and isolate

organization and A.

Kondas, P.

Leehan, and J.

Papale for

providing the 2011 data.

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