An Initial Inventory of Bacteria Found within the Soils
and Waters of Great Smoky Mountains National Park
Seán P. O’Connell, Emily A. York, Melissa B. Collins,
Derren T. Rosbach, Kristina Reid Black, and Weaver B. Haney
Southeastern Naturalist, Volume 6, Special Issue 1 (2007): 35–72
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1Department of Biology, 132 Natural Science Building, Western Carolina University,
Cullowhee, NC 28723. 2Current address - University of Alberta, Department of Medical
Microbiology and Immunology, 1-41 Medical Sciences Building, Edmonton, AB
T6G 2H7. 3Current address - Virginia Tech University, Department of Environmental
Design and Planning, Blacksburg, VA 24061. 4Current address - Dynamac Corporation,
Mail Code DYN-3, Kennedy Space Center, FL 32899. *Corresponding author
An Initial Inventory of Bacteria Found within the Soils
and Waters of Great Smoky Mountains National Park
Seán P. O’Connell1,*, Emily A. York1, Melissa B. Collins1,2,
Derren T. Rosbach1,3, Kristina Reid Black1,4, and Weaver B. Haney1
Abstract - Soils and waters were collected from Great Smoky Mountains National
Park (GSMNP), and bacteria were cultured or DNA cloned from the samples. Oconaluftee
Visitors Center and Kephart Prong Trail were sampled to examine distributions
of heterotrophs inhabiting streams and riparian soil. Soil from All Taxa Biodiversity
Inventory plots at Albright Grove, Cataloochee, and Purchase Knob were also
sampled. A total of eleven phyla were detected, of which six were only found via
culture-independent techniques. Overall, 69 genera were documented, with differences
in their detection in soil and water and by methodology. Firmicutes dominated
cultures from soil, while Acidobacteria dominated clone libraries; Bacteroidetes
was the dominant phylum in water. Three classes of the phylum Proteobacteria were
commonly seen as isolates or clones. Prokaryotic diversity is extraordinary; this is
the first inventory examining non-photosynthetic bacteria inhabiting GSMNP and it
lays the groundwork for investigations exploring the true breadth of diversity in the
park and what this diversity means to the broader ecosystem.
The extent of bacterial diversity on Earth has been of great interest to
taxonomists, microbial ecologists, and theoretical biologists over the last
two decades (Curtis et al. 2002, DeLong and Pace 2001, Gans et al. 2005,
Hong et al. 2006, Hughes et al. 2001, Torsvik et al. 1990, Whitman et al.
1998). With estimates ranging as high as ten million species in ten grams
of pristine soil (Gans et al. 2005), only a few thousand species globally
have been well classified (Janssen 2006). It is clear that much work remains
to catalog and understand the role of this diversity in natural ecosystems
(Handelsman 2004, Madsen 1998, Zak et al. 2006). Additionally, there is a
lack of consensus as to how to define a bacterial species (Rosselló-Mora and
Amann 2001) as well as evidence indicating that bacteria evolve quickly
(Cohan 2001); both concerns complicate the understanding and assessment
The Great Smoky Mountains National Park All Taxa Biodiversity Inventory:
A Search for Species in Our Own Backyard
2007 Southeastern Naturalist Special Issue 1:57–72
58 Southeastern Naturalist Special Issue 1
There are two chief ways to assess bacterial diversity in natural
samples: either by cultivating species using a growth medium followed by
isolation of single species (Janssen et al. 2002, Joseph et al. 2003, Sait et
al. 2002) or by using cultivation-independent techniques that detect species
by characteristic DNA sequences, most often via PCR amplification
of ribosomal genes (Dunbar et al. 2002, Hugenholtz et al. 1998, Janssen
2006). The benefit of culturing organisms is that one can measure various
metabolic, biochemical, and physiological parameters for an organism
once it is isolated from others in its community. This assessment of parameters
can lead to better understanding of ecosystem functions performed
by microorganisms (Madsen 1998). However, cultivation often selects
for organisms that are rare or perhaps ecologically unimportant in the
environments from which they were sampled (Hugenholtz et al. 1998,
Janssen 2006). Methods which detect DNA from numerically abundant
microorganisms should, theoretically, give a less-biased picture of diversity
in a given environment (Janssen 2006). A drawback to these molecular
techniques, however, is that they do not usually yield information about
ecological functions carried out by these species. Recent advances have
led to theoretical predictions of microbial roles in their environment via
sequencing of unknown genes extracted from samples (Handelsman 2004,
Schleper et al. 2005) and have provided the information necessary to cultivate
unusual species (Könneke et al. 2005). Creative techniques to narrow
the gap between the diversity observed in culture-independent and culturedependent
approaches are leading to many discoveries of novel species
heretofore known only by their DNA sequences from the environment
(Ferrari et al. 2005, Joseph et al. 2003). More complete culture collections
will allow microbial ecologists to elucidate the tasks performed by the majority
of species in various aquatic and terrestrial environments.
Due to the overwhelming number of individual cells and species of
bacteria in most natural environments, it is necessary to take a step back to
assess microbial diversity in macrobiotic ecosystems. Qualitative data can
be gathered by examining bacterial taxa at a level above that of species.
There are at least 52 established or proposed phyla in the domain Bacteria
(Schloss and Handelsman 2004), but only a small number of these phyla
are routinely encountered in culture-based studies (Jansen 2006). Instead,
most of the bacterial phyla are detected using only direct DNA-sequence
retrieval techniques (Dunbar et al. 2002, Jansen 2006). By first examining
the microbial community at the phylum or immediately lower levels,
predictive hypotheses may be generated about the relationships between
terrestrial or aquatic environments and the microorganisms that drive
their biogeochemistry. Such studies have been reported for soil (Barns et
al. 1999, Fierer and Jackson 2006, Zhou et al. 2002), streams (Hullar et al.
2006), and marine ecosystems (Rappé and Giovannoni 2003). The purpose
of this study was to initiate inventorying bacteria from soils and waters of
GSMNP and included comparing cultured and uncultured species in three
different forested habitats.
2007 S.P. O’Connell et al. 59
Annual sampling of Oconaluftee and Kephart Prong sites for heterotrophic
Samples from soil and water were taken as part of the Western Carolina
University Methods of General Microbiology (BIOL 414/514) class from
two sites in GSMNP during the fall semesters from 2002–2005. The sites
are near the Oconaluftee Visitor Center (UTM 17S 0291211, 3931770) and
Kephart Prong trail (UTM 17S 0286057, 3940540), and samples included
aseptically gathered soil and stream water. These locations were selected because
of their proximity to the Western Carolina University campus and they
serve as long-term study sites for examining bacterial distribution patterns.
Individual soil samples were prepared by removing loose leaf litter and then
homogenizing the upper 12–15 cm of soil using a propane torch-fl amed
spade. A smaller, sterile trowel was then used to subsample the mixed soil
to fill 50-mL sterile polypropylene centrifuge tubes. Stream-water samples
were collected by immersing a sterile centrifuge tube in the water and filling
to 80% volume. Each sample was collected upstream of the previous sample
to minimize collecting sediments stirred up from the bottom. All samples
were placed into a cooler on ice packs and returned to the lab where they
were kept at 4 °C until culture work began within 5–7 days.
Cultures were obtained by serially diluting environmental samples in
sterile water and spread-plating the dilutions on R2A media plates. Plates
were inverted, incubated in the dark at room temperature (≈25 °C), and
assessed for growth after one week, at which time each student selected a
colony for isolation and further study. Each colony was streaked for purity,
and this process was repeated 4–6 times until a pure culture comprised of
one species had been obtained. Students then characterized the isolate using
growth-based, biochemical, and microscopic techniques before obtaining a
portion of its ribosomal DNA sequence to identify it (n = 130 total isolates
for the four years).
DNA was obtained from the isolates by using a Mo Bio UltraClean Microbial
DNA Isolation kit (Mo Bio, Inc., Solana Beach, CA). Polymerase
chain reaction (PCR) conditions to amplify ca 550 bp of the 16S rDNA
from each isolate consisted of the following: 50 μL total volume in nuclease-
free water with final concentrations of 0.05% IgePal (Sigma, Inc., St.
Louis, MO), 1.5 mM Mg2+, 1X Taq buffer (Promega, Inc. Madison, WI or
Eppendorf, Inc., Westbury, NY), 0.25 μM of bacterial specific 341F and
universal 907R primers (Operon, Inc., Huntsville, AL; Casamayor et al.
2000), 2.5 U of Taq polymerase (Promega or Eppendorf), 0.20–0.25 mM
dNTPs (Promega or Eppendorf), and genomic DNA from the isolate. PCR
was conducted using a “touchdown” approach (Casamayor et al. 2000) on
a Mastercycler Personal thermal cycler (Eppendorf) with initial denaturation
for 5 min at 94 °C, followed by 30 PCR cycles consisting of: 1 min
60 Southeastern Naturalist Special Issue 1
denaturation at 94 °C; 1 min annealing at decreasing temperature (beginning
at 65 °C for two cycles, dropping 1 °C at each cycle for ten cycles,
and ending at 55 °C for 18 cycles); 3 min elongation at 72 °C; and a final
elongation for 7 min at 72 °C with a sample hold at 4 °C. Genomic DNA
extracts and PCR products were screened in 1% agarose gels, and PCR
products were cleaned using Montage spin filters (Millipore, Inc., Bedford,
MA) prior to DNA sequencing (see below).
Comparison of bacteria from Albright Grove, Cataloochee, and Purchase
Culturable bacteria. Soil samples were collected as above from three
established All Taxa Biodiversity Inventory (ATBI) study plots: Albright
Grove (old-growth forest in Tennessee), and Cataloochee and Purchase
Knob (second-growth forests in North Carolina). These sites are on the
eastern side of the GSMNP and were chosen because they have been classified as different forest types (Sharkey 2001) and it was hypothesized that
microbial communities would refl ect the differences in vegetation. Samples
were collected in 2002 (February), 2004 (September), and 2005 (February).
Isolates were obtained on R2A medium for the 2002 samples, 10% tryptic
soy agar (TSA) for the 2004 samples, and 0.1% nutrient broth (DDNB) for
the 2005 samples. DDNB cultures were either plated from samples that had
been frozen at -70° C or samples that had been diluted when fresh to 10-8
strength and incubated at room temperature (≈25 °C) for 3 months. All other
isolates were obtained via spread-plating of diluted soil samples within a
few days of sampling; DNA extractions and PCR conditions were implemented
as described in the previous section. All data from isolates obtained
from these four cultivation techniques were pooled (n = 81 total isolates) for
comparison to clone libraries generated, as described next.
Molecular clones. Paired soil samples were collected with the 2005
samples from the three ATBI plots described above, frozen in the field on
dry ice, and stored in the lab at -70 °C. These samples were taken to obtain
culture-independent 16S rDNA sequences from soil to compare to the sequences
obtained by isolating species on the heterotrophic growth media.
A Mo Bio PowerSoil DNA Isolation kit was used with the alternate lysis
method and 1 min of bead beating at 2500 RPM (Mini BeadBeater, BioSpec,
Inc., Bartlesville, OK) to obtain genomic DNA from the soil. Approximately
1500 base-pair fragments of the 16S rDNA from the mixed bacterial species
were amplified using bacterial primers 27F and 1492R (Corinaldesi et al.
2005, Ferrari et al. 2005). Genomic DNA extracts were all diluted to 10%
and added to a total volume of 50 μL containing the same PCR reagents and
concentrations as for the isolates above. Thermal cycler conditions were as
follows: 3 min of initial denaturation at 94 °C, 30 cycles of PCR consisting
of 1 min of denaturation at 94 °C, 1 min of annealing at 55 °C, 2 min of
elongation at 72 °C, a final elongation of 10 min at 72 °C, and a sample hold
2007 S.P. O’Connell et al. 61
at 4 °C. The resulting 1500 bp PCR products were cloned into Escherichia
coli (JM109 strain, Promega) using the pGEM-T-Easy Vector System (Promega),
and PCR inserts were reamplified by performing whole-cell PCR
(O’Connell et al. 2003) using primers M13F and M13R. Clones that had
the appropriate-sized inserts were then screened using restriction-fragment
length polymorphism (RFLP) analysis with the restriction enzymes RsaI
and MspI (Promega; RM Lehman, USDA, Brookings, SD, pers. comm.). A
total of 91, 66, and 22 unique clones were obtained from Albright Grove,
Cataloochee, and Purchase Knob, respectively, and sequenced (as described
below). One hundred and fifty clones from each site were targeted, but due
to cloning inefficiencies, we were only able to recover the above number of
unique sequences; however, in only a handful of cases did we find the same
RFLP pattern twice.
DNA sequencing and DNA sequence analysis
PCR using primers 341F/907R was run on the clone inserts from unique
RFLP banding patterns to amplify ca 550 bp of the product (the same size
and region of the 16S rDNA gene used for all of the isolates). All shortfragment
16S rDNA PCR products (isolates and clones) were then amplified
in a sequencing PCR reaction using primer 907R with a BigDye Terminator
Version 3.0 or 3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City,
CA). The PCR products were cleaned using AutoSeq Sephadex G-50 spin
columns (Amersham Biosciences, Piscataway, NJ) and analyzed by either
an ABI 377 gel sequencer or a 3130/3130xl automated capillary DNA sequencer
Sequences were compared to sequences of clones and isolates contained
in the Ribosomal Database Project II (RDP II) using both the “Classifier”
and “Sequence Match” programs (Maidak et al. 2001). Clone sequences
were additionally analyzed using the Greengenes suite of software including
the Classifier program (DeSantis et al. 2006) to obtain additional phylogenetic
inferences. Sequences were classified to the level of phylum, class (for
the Proteobacteria), and genus. All sequences were checked for chimeras by
using the Bellerophon program (Huber et al. 2004) and also by first aligning
them with ClustalW using the Vector NTI program (Invitrogen, Inc., Carlsbad,
CA) followed by Mallard software (Ashelford et al. 2006). Sequences
called into question by these programs were screened more thoroughly
using the Pintail computer program (Ashelford et al. 2005). No chimeric
sequences were discovered.
A total of eleven phyla were identified from riparian soil and water
samples from the Kephart Prong and Oconaluftee sites and from the soils
at the Albright Grove, Cataloochee, and Purchase Knob ATBI plots
(Table 1). These included Actinobacteria, Firmicutes, Bacteroidetes,
62 Southeastern Naturalist Special Issue 1
Table 1. Bacterial genera grouped by phylum, candidate phylum, and class (for the
Proteobacteria) and categorized by detection in culture collections or clone libraries
and presence in soil or water for all sites and sampling methodologies (number
after phylum or class shows how many sequences were analyzed; X = genus found
in sample type; -- = genus not detected). Classifications are based on the best available
sequence matches to the RDP II for cultured isolates and RDP II Classifier and
Greengenes Classifier for the cloned samples.
Genus Sample type
Phylum Cultured Cloned Soil Water
Acidimicrobineae -- X X --
Arthrobacter X -- X --
Curtobacterium X -- X --
Dermacoccus X -- X --
Kitasatospora X -- X --
Leifsonia X -- X --
Micrococcus X -- -- X
Nocardia X -- X X
Rhodococcus X -- X X
Streptomyces X -- X --
Terrabacter X -- -- X
Anaeroglobus -- X X --
Bacillus X -- X X
Paenibacillus X -- X --
Staphylococcus X -- X X
Acidobacterium -- X X --
Verrucomicrobium -- X X --
Isosphaera -- X X --
Planctomyces -- X X --
Unclassified genus -- X X --
Chitinophaga X X X --
Chryseobacterium X -- X --
Cytophaga X -- X --
Flavobacterium X -- -- X
Flexibacter X -- X --
Pedobacter X -- X X
Sphingobacterium X -- X X
Taxeobacter X -- -- X
Thermonema X -- -- X
Acidisphaera -- X X --
Agrobacterium X -- X --
Blastochloris -- X X --
Bradyrhizobium -- X X --
Inquilinus X -- X --
Magnetospirillum -- X X --
Methylosinus -- X X --
2007 S.P. O’Connell et al. 63
Table 1, continued.
Genus Sample type
Phylum Cultured Cloned Soil Water
Odyssella -- X X --
Phenylobacterium -- X X --
Rhizobium X -- X --
Rhodoplanes -- X X --
Roseomonas -- X X --
Sphingomonas X -- -- X
Tistrella -- X X --
Acidovorax X -- -- X
Aquaspirillum X -- X --
Burkholderia X X X --
Collimonas X -- X --
Caenibacterium -- X X --
Herbaspirillum X -- X --
Janthinobacterium X -- -- X
Tepidiphilus -- X X --
Variovorax X -- -- X
Zoogloea X -- X X
Desulfomonile -- X X --
Alkalispirillum -- X X --
Buttiauxella X -- -- X
Enterobacter X -- -- X
Frateuria X -- X --
Isochromatium -- X X --
Klebsiella X -- X --
Pantoea X -- -- X
Pseudomonas X -- X X
Rhodanobacter X -- X --
Rickettsiella -- X X --
Serratia X -- X X
Thiorhodospira -- X X --
Xanthomonas X -- X --
Yersinia X -- -- X
Deinococcus X -- -- X
Termite Group 1 (1) -- X X --
OP10 (2) -- X X --
Proteobacteria, Deinococcus-Thermus, Acidobacteria, Verrucomicrobia,
Planctomyces and Gemmatimonadetes. The first two phyla are the grampositive
bacteria, and the others are representatives of gram-negative bacteria.
Candidate phyla OP10 and Termite Group 1 were also detected in
the clone libraries from the ATBI sites. Because Proteobacteria is such a
large phylum and contains a large number of metabolically and genetically
diverse species, this phylum is broken down into four classes for further
comparison, including Alpha-, Beta-, Delta-, and Gammaproteobacteria.
While we did not recover enough clones and isolates from the ATBI plots
64 Southeastern Naturalist Special Issue 1
to make discrete comparisons between our study sites, some general
trends are reported below. For the purpose of discussion, we have also
combined data from waters at Oconaluftee and Kephart Prong and soils
from these sites and will treat the results as a master list of genera detected
in GSMNP to date (Table 1).
The eleven phyla recovered included DNA sequence data from 390 organisms
(211 bacterial isolates and 179 clones), which can be placed into
69 genera (Table 1). Of the 390 sequences, 324 originated from soil and 66
from water samples. At the level of phylum, molecular cloning detected
representatives from ten phyla and the four classes of Proteobacteria, while
culturing techniques yielded representatives from five phyla and three of
the proteobacterial classes. The latter also produced the only record of a
Deinococcus-Thermus representative. Cultures and clones accounted for 45
and 26 genera respectively, with only two genera, Chitinophaga (Bacteroidetes)
and Burkholderia (Betaproteobacteria) found using both methods.
Forty-six genera were found exclusively in soil samples, while waters produced
14 unique genera; nine genera overlapped between the two sample
types. With the relatively small number of sequences obtained from water
samples and the lack of clones from these samples, we cannot make strong
conclusions from these data. However, it is worth noting again the number
of unique genera from these samples and the occurrence of Deinococcus,
the sole representative of its phylum we found. Additionally, waters were
dominated by species from Bacteroidetes (34.8% of sequences) and Betaproteobacteria
(22.7% of sequences), while soils had more cultured representatives
from Firmicutes and Gammaproteobacteria, with 37.5% and
21.9% of sequences identified to these phyla, respectively, from the Kephart
Prong and Oconaluftee sites.
Six phyla and Deltaproteobacteria were represented only in the clone libraries.
Of these, only Acidobacteria (132 clones), Planctomyces (9 clones),
and Verrucomicrobia (5 clones) produced more than two clones (Table
1). For the other groups, only Alphaproteobacteria were more commonly
cloned than cultured (14 clones versus 10 isolates), and the highest number
of clones for any other group was six for Gammaproteobacteria. The number
of genera per phylum reveals some other interesting trends including
that the most sequence-rich group detected, Acidobacteria, was represented
by only a single genus. Alphaproteobacteria, with 24 sequences, accounted
for 14 genera and included the most cloned genera (10). Gammaproteobacteria
records show 14 genera as well, but with 41 sequences, and most of
these originating from cultures. Ten betaproteobacterial genera were detected
from 41 sequences and most were from isolated bacteria (eight versus
three genera from clones). Actinobacteria and Bacteroidetes were similar
in being represented largely by cultures (ten and nine genera, respectively),
with only one genus detected each in clone libraries. Lastly, Firmicutes was
relatively genera-poor (four genera) but sequence-rich with 65 records, with
all but one sample obtained from culturing techniques.
2007 S.P. O’Connell et al. 65
Table 2 shows the differences in sequence detection of cultures and
clones from the three ATBI plots. Interestingly, there is almost no consensus
between phyla and proteobacterial classes recovered using the two means of
assessing diversity. Only in the case of Proteobacteria does there appear to
be some similarity at this higher level of taxonomy, but as can be seen in Table
1, little overlap actually exists toward the level of species. Isolates from
the soils of Albright Grove, Cataloochee, and Purchase Knob largely fall
into one of three groups: Firmicutes, Betaproteobacteria, or Actinobacteria.
Firmicutes accounted for 44.4% of the sequences, Betaproteobacteria 21%,
and Actinobacteria 12.3%. Acidobacteria overshadowed all other sequence
data and represented 73.7% of the soil clones, with Alphaproteobacteria
(8.4%) and Planctomyces (5%) of secondary abundance.
Little to no overlap was seen between the bacteria inhabiting streams and
soils (Table 1) or between methods assessing diversity from three forests of
different histories (Table 2). The data in this study have been reported at the
level of genus rather than species; however, in only a few cases (less than 3%
of all samples) have we observed the same DNA sequence on more than one
occasion. A species definition for bacteria has not been unanimously established,
but ideally a combination of DNA sequence and ecological similarity
are criteria for taxonomic placement (Rosselló-Mora and Amann 2001). In
the instances where we have retrieved identical 16S rDNA sequences (from
Table 2. Percent of diversity attributed to each bacterial phylum, candidate phyla
(Termite Group 1 and OP10), and class (for the Proteobacteria) broken down by the
approach taken to obtain 16S rDNA sequences from soils from the Albright Grove,
Cataloochee, and Purchase Knob ATBI study plots (data are combined for the three
sites). Classifications are based on the best available sequence matches to the RDP II
database for cultured isolates (n = 81 isolates) and RDP II Classifier and Greengenes
Classifier for the cloned samples (n = 179 clones).
Phylum Cultured Cloned
Actinobacteria 12.3% 0.6%
Firmicutes 44.4% 0.6%
Acidobacteria - 73.7%
Verrucomicrobia - 2.8%
Planctomyces - 5.0%
Gemmatimonadetes - 0.6%
Bacteroidetes 9.9% 1.1%
Proteobacteria 33.3% 14.0%
Alphaproteobacteria 3.7% 8.4%
Betaproteobacteria 21.0% 1.7%
Deltaproteobacteria - 0.6%
Gammaproteobacteria 8.6% 3.4%
Termite Group 1 - 0.6%
OP10 - 1.1%
66 Southeastern Naturalist Special Issue 1
isolated bacteria), we have been able to measure some other characteristic
that could differentiate the two organisms (e.g., metabolic trait or other
growth parameter). What our data suggest is that even from a few small
samples from GSMNP, we are only beginning to be able to document the
bacterial diversity therein. Further complicating matters is that many of
the species we have detected overlap little in 16S rDNA sequence to their
closest relatives in the sequence databases that are available for comparison.
This diversity of bacteria in natural environments is not surprising as others
have shown similar results, especially from soils (Curtis et al. 2002, Gans et
al. 2005, Hong et al. 2006). Understanding the meaning of this diversity is
Soils are incredibly complex at multiple scales (Fierer and Jackson
2006, Zhou et al. 2002) and infl uence and are infl uenced by the plant and
microbial communities that they are associated with (Dunbar et al. 2002,
Grayston and Prescott 2005, Hackl et al. 2004, Smalla et al. 2001). Floyd
et al. (2005) reported on the most easily cultured bacteria from soils as
contained in the American Type Culture Collection (ATCC; Manassas,
VA), and their summary shows that the gram-positive bacteria are dominant
in soils, with Actinobacteria accounting for over 35% of all isolated type
strains and Firmicutes making up another 12.4%. In the former phylum,
the genus Streptomyces is an important source of antibiotics and therefore
may be intentionally over-sampled from soil, while the latter group contains
spore-forming bacteria that may also be over-represented in culture. This
possibility has been suggested by other work (Janssen 2006) and also likely
holds true in our study. Firmicutes were dominant in the cultures from soil at
Kephart Prong and Oconaluftee as well as at Albright Grove, Cataloochee,
and Purchase Knob (Tables 1 and 2). Actinobacteria were present in the
same samples, but not as commonly cultivated since we did not specifically
target this phylum. Proteobacteria comprised nearly 10% of the records in
the ATCC, while the Bacteroidetes accounted for 1.2% (only genera with
greater than 1% representation in the collection were reported). Proteobacteria
and Bacteroidetes were likewise commonly cultured in GSMNP, but at
greater levels (Tables 1 and 2).
Hugenholtz et al. (1998) analyzed over 8000 16S rDNA isolate and clone
sequences in public databases and estimated that 65% of all Proteobacteria
records were of cultured species, 80% each of Actinobacteria and Firmicutes
were represented by cultures, and 40% of Bacteroidetes entries were
from cultures. This contrasted with only 1% of the Acidobacteria and 10%
of Verrucomicrobia records from culture. The other records came from clone
libraries. In our study, the majority of soil bacteria have been assigned to the
phylum Acidobacteria (Table 2). This is not unusual, since Acidobacteria
have been shown to be dominant or co-dominant in many environments and
this group also shows great taxonomic diversity, although they are represented
by few genera (Barns et al. 1999, Dunbar et al. 2002, Hugenholtz et
2007 S.P. O’Connell et al. 67
al. 1998, Janssen 2006). What is surprising is how large a proportion of our
clone library this group comprises (73.7% of all clones; Table 2). Others
have shown Acidobacteria accounting for 40–50% of the clones from arid
soils (Dunbar et al. 2002) and 20% of the diversity of soils globally (Janssen
2006). It remains to be seen what roles the species in this phylum play, since
so few have been cultured; however, their sheer number and phylogenetic
breadth suggest their importance in soil ecosystems (Janssen 2006).
Other phyla commonly encountered in clone libraries from soil include
Proteobacteria, Actinobacteria, Verrucomicrobia, and Bacteroidetes, with
other groups such as Planctomyces and Firmicutes less common (Dunbar et
al. 2002 and Janssen 2006). Every soil sample reported by Janssen (2006)
contained representatives of the Alpha-, Beta-, and Gammaproteobacteria,
and the proteobacterial classes made up a mean of 40% of all soil clones
worldwide. Proteobacteria were commonly cloned in GSMNP sites as
well, comprising 14% of the clones from Albright Grove, Cataloochee, and
Purchase Knob (Table 2). Alphaproteobacteria were more abundant in the
clone libraries than in cultures and represent the group with the most genera
detected (Table 1), none of which are refl ected in our culture collection and
bear more investigation.
Unlike in the culture collection where Firmicutes accounted for a large
proportion of all DNA sequences obtained, this group was represented by
only a single clone. This is interesting since at Albright Grove, this phylum
accounted for nearly 80% of all isolates. The species within Firmicutes are
likely to be readily cultured from numerous spores. However, owing to their
relatively massive cell walls and spores, these species (and Actinobacteria)
may resist DNA extraction efforts and therefore, may not be detected
via molecular cloning (Janssen 2006). Although we tried to account for as
much diversity as possible in our molecular cloning efforts, e.g., using bead
beating as part of the DNA extraction step (Miller et al. 1999), we cannot
completely rule out inefficiencies in our DNA recovery methods.
Other groups that were readily cultured but not well-represented in the
clone libraries included Actinobacteria and Bacteroidetes (Table 2), perhaps
suggesting their relative scarcity or unimportance in soil ecosystems, since
molecular cloning should refl ect numerically abundant organisms (Actinobacteria
as discussed above) and gram-negative species such as those in
Bacteroidetes (Janssen 2006). Lastly, Planctomyces and Verrucomicrobia
represent phyla found globally most often in clone libraries. This was the
case in our work as well, with the former producing nine clones, and the latter
group producing five unique DNA sequences from soil.
The large gap between organisms which are readily cultivated and
those dominating in clone libraries has been narrowing somewhat thanks
to creative culturing efforts. These either mimic in situ conditions in
the lab or field or use dilution and long incubation times to select for
oligotrophs, which are the norm in nature (Ferrari et al. 2005, Kaeberlein
68 Southeastern Naturalist Special Issue 1
et al. 2002, Zengler et al. 2002). Recent literature has shown improvements
for culturing Acidobacteria (Davis et al. 2005, Sait et al. 2006,
Stevenson et al. 2004), Verrucomicrobia (Davis et al. 2005, Janssen et al.
2002, Joseph et al. 2003, Stevenson et al. 2004), Planctomyces (Wang et
al. 2002), and Acidobacteria, Actinobacteria, Alpha-, Beta-, and Gammaproteobacteria
simultaneously (Janssen et al. 2002, Joseph et al. 2003,
Sait et al. 2002). Our goal is to employ similar methods in an attempt to
culture the many species represented by the clones in our libraries, which
are numerous in the native soil and likely ecologically important. Once
we have these species in culture, their roles in the environment may be
more readily elucidated (e.g., specific nutrient turnover processes, symbioses,
pathogenic interactions, etc.).
Potential ecological functions these species participate in, other than in
heterotrophic processes (all of the isolates and many of the clones likely act
as decomposers), include a huge range of physiologies in the Proteobacteria.
These include the chemolithotrophs (methane, sulfur, iron, manganese,
hydrogen, ammonia, and nitrate oxidizers), nitrogen-fixers, sulfur-reducers,
and bacterial predators (e.g., Bdellovibrio; Perry et al. 2002, Madigan and
Martinko 2006). Bacteroidetes include animal gut symbionts and complex
polymer degraders and likely play a role in soil biogeochemistry. Actinobacteria
are producers of antibiotics, which may regulate population sizes of
bacteria and fungi in soils. This phylum also contains nitrogen-fixers. Some
Firmicutes are sulfur reducers, and cultured representatives of Verrucomicrobia
act as protist symbionts, protecting their host from protist predators.
Acidobacteria and Planctomyces certainly have roles as heterotrophs, but
the great taxonomic diversity within Acidobacteria, which rivals the betterstudied
Proteobacteria, suggests many niches yet to be discovered by microbial
ecologists (Janssen 2006).
This preliminary inventory of bacteria inhabiting the soils and waters
of GSMNP sets the stage for future investigations. The main finding of
this work is that different methodologies produced significantly different
views of bacterial diversity, even when examining the same samples. Other
than for the cyanobacteria (Johansen 2007), no bacterial inventories have
been undertaken in the GSMNP. We plan to continue to examine the three
forested ecosystems represented in this study by cultures and clones. The
long-term study plots established for soils and waters at Kephart Prong and
Oconaluftee will be sampled each fall for many years to come to document
species distribution patterns over time. Additionally, clone libraries should
be generated for these soil and water samples to further elucidate the patterns
of and participants in bacterial diversity in the GSMNP. Estimating the
total number of bacterial species in GSMNP is improbable using conventional
models, especially considering recent estimates that place the number
of species in a few grams of soil in the millions (Curtis et al. 2002, Gans
et al. 2005). However, with repeated sampling of the same sites, sampling
2007 S.P. O’Connell et al. 69
multiple sites, using standardized methods, and combining data from culture
collections and clone libraries, a greater proportion of the total species pool
can be detected. Some have called for more intensive cloning of environmental
samples to fill out the bacterial phylogenetic tree (Curtis 2006); this
would serve to especially detect the numerous and culture-resistant groups
and would be a good use of resources. Based on the clone libraries we have
generated from GSMNP, we believe the Acidobacteria to be quite important
in the forest soil. Understanding this group, in which only a few members
have been cultivated, would go a long way to aid in discovering important
processes occurring in the GSMNP as well as globally.
The authors would like to acknowledge Western Carolina University and Discover
Life in America for funding this work, and Keith Langdon, Paul Super (National
Park Service), and Jeanie Hilten (DLIA) for logistical support and advice. Most of
the DNA sequences were generated using support from a grant from the National
Science Foundation (DBI-0521334). We would also like to acknowledge the 132 students
from eight sections of the BIOL 414/514 classes who isolated and characterized
bacteria from Kephart Prong and Oconaluftee, and Philip Drummond for invaluable
lab assistance. Lastly, the authors thank three reviewers whose comments greatly
improved the quality and scope of this manuscript.
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