Filamentous "Epsilonproteobacteria" Dominate Microbial Mats from Sulfidic Cave Springs

Abstract

Hydrogen sulfide-rich groundwater discharges from springs into Lower Kane Cave, Wyoming, where microbial mats dominated by
filamentous morphotypes are found. The full-cycle rRNA approach, including 16S rRNA gene retrieval and fluorescence in situ
hybridization (FISH), was used to identify these filaments. The majority of the obtained 16S rRNA gene clones from the mats
were affiliated with the “Epsilonproteobacteria” and formed two distinct clusters, designated LKC group I and LKC group II, within this class. Group I was closely related
to uncultured environmental clones from petroleum-contaminated groundwater, sulfidic springs, and sulfidic caves (97 to 99%
sequence similarity), while group II formed a novel clade moderately related to deep-sea hydrothermal vent symbionts (90 to
94% sequence similarity). FISH with newly designed probes for both groups specifically stained filamentous bacteria within
the mats. FISH-based quantification of the two filament groups in six different microbial mat samples from Lower Kane Cave
showed that LKC group II dominated five of the six mat communities. This study further expands our perceptions of the diversity
and geographic distribution of “Epsilonproteobacteria” in extreme environments and demonstrates their biogeochemical importance in subterranean ecosystems.

Full text

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 2003, p. 5503–5511 Vol. 69, No. 9
0099-2240/03/$08.00⫹0 DOI: 10.1128/AEM.69.9.5503–5511.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.
Filamentous “Epsilonproteobacteria” Dominate Microbial Mats from
Sulfidic Cave Springs
Annette Summers Engel,
1
* Natuschka Lee,
2
Megan L. Porter,
3
Libby A. Stern,
1
Philip C. Bennett,
1
and Michael Wagner
4
Research Group for Microbial Geochemistry, Department of Geological Sciences, University of Texas at Austin, Austin,
Texas 78712
1
; Lehrstuhl fu¨r Mikrobiologie, Technische Universita¨t Mu¨nchen, D-85350 Freising, Germany
2
;
Department of Integrative Biology, Brigham Young University, Provo, Utah 84602
3
; and Department of Microbial
Ecology, Institute of Ecology and Conservation Biology, Vienna University, A-1090 Vienna, Austria
4
Received 6 February 2003/Accepted 2 May 2003
Hydrogen sulfide-rich groundwater discharges from springs into Lower Kane Cave, Wyoming, where mi-
crobial mats dominated by filamentous morphotypes are found. The full-cycle rRNA approach, including 16S
rRNA gene retrieval and fluorescence in situ hybridization (FISH), was used to identify these filaments. The
majority of the obtained 16S rRNA gene clones from the mats were affiliated with the “Epsilonproteobacteria”
and formed two distinct clusters, designated LKC group I and LKC group II, within this class. Group I was
closely related to uncultured environmental clones from petroleum-contaminated groundwater, sulfidic
springs, and sulfidic caves (97 to 99% sequence similarity), while group II formed a novel clade moderately
related to deep-sea hydrothermal vent symbionts (90 to 94% sequence similarity). FISH with newly designed
probes for both groups specifically stained filamentous bacteria within the mats. FISH-based quantification of
the two filament groups in six different microbial mat samples from Lower Kane Cave showed that LKC group
II dominated five of the six mat communities. This study further expands our perceptions of the diversity and
geographic distribution of “Epsilonproteobacteria” in extreme environments and demonstrates their biogeo-
chemical importance in subterranean ecosystems.
Caves containing hydrogen sulfide-rich springs represent
less than 10% of all known caves globally (42). However, these
caves serve as access points into sulfidic groundwater aquifers,
typically associated with geothermal regions and oil-field ba-
sins, which play an important role in global sulfur cycling. The
microbial communities colonizing sulfidic cave habitats have
recently received attention due to their chemolithoautotrophic
metabolism that can sustain complex ecosystems in the subsur-
face (48) and their geomicrobiological impact due to acid pro-
duction (14, 60). Filamentous bacteria dominate subaqueous
cave microbial mats, and from phylogenetic analyses, stable
isotope evidence, and aqueous geochemistry surveys, popula-
tions are considered to be chemolithoautotrophic, aerobic to
microaerophilic sulfur-oxidizing bacteria (5, 19, 48, 59). Re-
cently, two cultivation-independent studies based on the phy-
logeny of bacterial community 16S rRNA genes characterized
filamentous microbial mats from the Sulphur River of Parker
Cave, Kentucky, and Cesspool Cave, Virginia (5, 14). In both
16S rRNA gene libraries, most clones were affiliated with un-
characterized environmental groups within the “Epsilonpro-
teobacteria,” but the actual abundance and the morphotype(s)
of the respective organisms were not established. Additionally,
cultivation of any of these filamentous bacteria from sulfidic
cave mats has been unsuccessful to date. The only sulfur bac-
teria isolated from these caves are gram-negative, rod-shaped
thiobacilli (14, 60).
The most commonly studied genera of the “Epsilonpro-
teobacteria,” Helicobacter and Campylobacter, are often associ-
ated with the gastrointestinal tract of animals as pathogens (13,
40). Other major phylogenetic groups within the “Epsilonpro-
teobacteria” include Arcobacter,Wolinella,Sulfurospirillum, and
Thiovulum, commonly found in natural settings as living cells
or in symbiotic association with animals (13, 40, 51). Recently,
members of the Arcobacter were also detected in activated
sludge from wastewater treatment plants (53). Members of the
Thiovulum phylogenetic group have been described from many
natural habitats, some of which are considered extreme envi-
ronments, including caves (5, 14), springs (47), groundwater
associated with oil (17, 22, 64), marine water and muds (29,
58), deep-sea hydrothermal vent sites (9, 26, 36, 45), and vent-
associated metazoans (8, 27). However, relatively little is
known about the ecology or physiology of most of these “Ep-
silonproteobacteria,” as cultured representatives for most of the
detected environmental clone groups are missing. Moreover,
there are few detailed studies describing the occurrence of
“Epsilonproteobacteria” from terrestrial environments com-
pared to the relatively large number of such investigations
from marine habitats.
In this study we investigated bacterial communities from
filamentous microbial mats associated with aphotic sulfidic
springs in Lower Kane Cave, a system located in north-central
Wyoming. Using the full-cycle rRNA approach, including the
construction of 16S rRNA gene clone libraries and quantitative
fluorescence in situ hybridization (FISH), we report on the
occurrence of two distinct epsilonproteobacterial filament
groups within the microbial mats in the cave. Quantitative
FISH with the newly designed 16S rRNA-targeted oligonucle-
* Corresponding author. Mailing address: Department of Geologi-
cal Sciences, University of Texas at Austin, 1 University Station C1100,
Austin, TX 78712-0254. Phone: (512) 471-5413. Fax: (512) 471-5766.
E-mail: aengel@mail.utexas.edu.
5503

otide probes for both groups revealed that members of one of
the groups dominated the mat communities analyzed.
MATERIALS AND METHODS
Study site and sample acquisitions. Lower Kane Cave is located in the north-
central portion of the Bighorn Basin on the western flank of the Bighorn Moun-
tains (12). It is forming within the Madison Limestone. Two major and two minor
anaerobic, hydrogen sulfide-bearing springs discharge into Lower Kane Cave
along a fracture zone (Fig. 1). Each spring is associated with an orifice pool and
outflow channel. Groundwater pH, temperature, dissolved oxygen, and conduc-
tivity were measured in the field with specific probes. Dissolved hydrogen sulfide
and low concentrations of dissolved oxygen were measured directly in the field
with the methylene blue and rhodazine D colorimetric methods, respectively,
following the manufacturer’s guidelines (Chemetrics Inc., Calverton, Va.).
Samples for DNA extraction were obtained from microbial mats associated
with three of the springs, and samples were taken on several occasions during
2000 and 2001. We report here the clone library results from a subset of six
samples from Lower Kane Cave (Fig. 1): from August 2000, Fissure Spring
orifice white filament bundles (sample 21), Upper Spring orifice white filament
bundles (samples 57 and 58); from March 2001, Upper Spring orifice white
filament bundles (sample 114); from August 2001, Upper Spring thin white
filaments (sample 190), Lower Spring orifice white filament bundles (sample
199), and Lower Spring yellowish-white mat (sample 198). Mat samples used for
FISH were collected in December 2001 from the Fissure Spring orifice, Lower
Spring orifice, the white mat from the Lower Spring, Upper Spring orifice, and
three white filamentous mat samples from the Upper Spring stream channel
(white mat 1, 2, and 3). Samples white mat 1 and white mat 2 were separated by
4 m of stream flow; white mat 2 and white mat 3 were separated by 5 m.
DNA extraction, PCR amplification, and cloning. Approximately 0.2 to 0.5 ml
of microbial mat was aseptically collected in the cave and transferred into DNA
extraction buffer containing 10 mM Tris-HCl (pH 8), 100 mM EDTA, and 2%
sodium dodecyl sulfate. Total community DNA was isolated with an extraction
protocol similar to the commercially available Purgene DNA extraction kits
(Gentra Systems, Minneapolis, Minn.), with the following modifications: 9 ␮lof
proteinase K (20 mg/ml) was added to each DNA extraction buffer prior to
digestion; a freeze-thaw (three times at ⫺80°Cto65°C) series was used to aid in
the physical disruption of the mat structure; samples were incubated at 55°C
overnight to digest cellular material; RNase was added to the digests and incu-
bated at 37°C for up to 1 h; proteins were precipitated in 10 M ammonium
acetate; and nucleic acids were precipitated in isopropanol overnight at ⫺20°C
and washed in 70% ethanol.
Nearly full-length 16S rRNA gene sequences were PCR amplified with the
primer pairs 27f (forward, 5⬘-AGAGTTTGATCCTGGCTCAG-3⬘) and 1492r
(reverse, 5⬘-GGTTACCTTGTTACGACTT-3⬘), according to the protocol de-
scribed by Lane et al. (23). Amplification was performed with a Perkin Elmer
9700 thermal cycler under the following conditions: denaturation at 95°C for 1
min, primer annealing at 42°C for 1 min, and chain extension at 72°C for 1.5 min.
Fifty PCR cycles were used. A control tube containing sterile water instead of
DNA was used as a negative control.
Amplified PCR products were purified with the GeneClean II kit (Bio101,
Inc., Vista, Calif.), as recommended by the manufacturer. Purified PCR products
were cloned with the Topo TA cloning kit (Invitrogen, San Diego, Calif.),
following the manufacturer’s instructions.
Sequencing of 16S rRNA genes and phylogenetic analysis. Clones to be ana-
lyzed were lysed in 50 ␮l of buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0) for
10 min at 96°C. Forty-four clones were selected for sequencing, whereby se-
quence inserts were PCR amplified from lysed cells with plasmid-specific primer
pairs M13(⫺20) (5⬘-GTAAAACGACGGCCAGT-3⬘) and M13(⫺24) (5⬘-AAC
AGCTATGACCATG-3⬘) and the following PCR conditions: denaturation at
94°C for 1 min, primer annealing at 55°C for 1 min, and chain extension at 72°C
for 3 min, for 35 cycles. PCR products were purified with Sephadex columns and
sequenced with an ABI Big-Dye Ready Reaction kit (Perkin Elmer) with primers
27f and 1492r in conjunction with internal primers 907r (reverse, 5⬘-CCGTCA
ATTCCTTTRAGTTT-3⬘[where R is G or A]) and 704f (forward, 5⬘-GTAGC
GGTGAAATGCGTAGA-3⬘). Automated DNA sequencing was done on an
ABI Prism 377XL sequencer (Perkin Elmer).
DNA sequences were submitted to the CHECK-CHIMERA program of the
Ribosomal Database Project II at Michigan State University (http://rdp.cme.msu
.edu/html/) (30). Clone sequences were subjected to BLAST searches within the
GenBank database (http://www.ncbi.nlm.nih.gov/) to determine 16S rRNA gene
sequence similarities to culturable and not yet cultured organisms.
The retrieved nucleotide sequences were initially aligned with Clustal X (56)
and then manually adjusted based on conserved primary and secondary config-
uration. Phylogenetic analyses were done with maximum likelihood, minimum
evolution, and maximum parsimony criteria in PAUP* (55) and Bayesian infer-
ence (20). For minimum evolution, Bayesian inference, and maximum likelihood
searches, a model of evolution was chosen based on likelihood ratio tests (20), as
implemented in Modeltest 3.06 (44). Heuristic searches were run for either 100
(maximum likelihood), 500 (minimum evolution), or 1,000 (maximum parsi-
mony) replicates with random sequence addition and the tree-bisection and
reconnection branch-swapping algorithm. Bayesian inference searches were run
for 10
6
cycles at least two times to check for convergence and then combined,
deleting the first 1,000 trees from each search. As an indication of nodal support,
bootstrap analyses were performed for maximum likelihood (100 replicates),
minimum evolution (500 replicates), and maximum parsimony (1,000 replicates)
criteria with full heuristic searches. For Bayesian inference analyses, posterior
probabilities were used for nodal support. All trees were rooted with Desulfo-
capsa thiozymogenes (X95181) and Hydrogenophaga pseudoflava (AF078770) as
outgroups.
16S rRNA oligonucleotide probes. Oligonucleotide probes specific for two
epsilonproteobacterial clone groups from Lower Kane Cave (group I and group
II) were designed with the PROBE DESIGN tools from the ARB software
package (http://www.arb-home.de) and probe designations according to Alm et
FIG. 1. Location of Lower Kane Cave, near Lovell, Wyo. The inset plan-view cave map, modified from Egemeier (12), shows the cave entrance
and three spring sites. The Wyoming map is from http://fermi.jhuapl.edu/states/.
5504 ENGEL ET AL. APPL.ENVIRON.MICROBIOL.

al. (3). Probe LKC59 (S-*-eProt-0059-a-A-18) is specific for group I clones, and
probe LKC1006 (S-*-eProt-1006-a-A-18) targets group II clones. Probe speci-
ficity was verified with the RDP PROBE-MATCH function (30) and the
PROBE-MATCH tool of the ARB software package, and a 16S rRNA data set
including all publicly available sequences from “Epsilonproteobacteria.”Se-
quences and optimal hybridization conditions for probes LKC59, LKC1006, and
all other probes used are listed in Table 1, including probe EPS710, designed to
target environmentally retrieved clones within the “Epsilonproteobacteria”(64)
and the cultured strain YK-1 proposed as Sulfuricurvum kujiense gen. nov., sp.
nov. (22; Y. Kodama and K. Watanabe, presented at the International Sympo-
sium on Subsurface Microbiology, Copenhagen, Denmark, 2002). More detailed
information about the probes can be found at probeBase (http://www.microbial-
ecology.net/probeBase) (28).
Sample fixation, FISH, microscopy, and quantification. For FISH, microbial
mat samples were collected in December 2001, shipped on dry ice, and fixed in
two ways within 48 h of collection: (i) with 4% (wt/vol) paraformaldehyde for 3 h
as described by Manz et al. (32), and (ii) with 50% ice-cold ethanol according to
Roller et al. (46). Fixed sample material was spotted onto Teflon-coated slides
and air-dried overnight before dehydration by sequential washes in 50, 80, and
100% (vol/vol) ethanol for 3 min each.
The oligonucleotide probes were synthesized and directly labeled with the
monofunctional, hydrophilic, sulfoindocyanine dyes indocarbocyanine (Cy3) and
indodicarbocyanine (Cy5) or with FluosPrime (5,6-carboxyfluorescein-N-hy-
droxysuccinimide ester), purchased from Hybaid-Interactiva (Ulm, Germany).
Hybridization and washes were performed as described by Manz et al. (32). The
salt concentration in the wash buffer was adjusted to the formamide concentra-
tion in the hybridization buffer according to Manz et al. (32). Washes were
performed at 48°C.
Optimal hybridization stringency for probes EPS710, LKC59, and LKC1006
was determined by increasing the formamide concentration of the hybridization
buffer in increments of 5 or 10% while maintaining a constant hybridization
temperature of 46°C. Due to the nonavailability of suitable reference cells, the
optimal hybridization stringency for the three probes evaluated was defined by
the highest stringency allowing unambiguous visual detection of probe target
cells in fixed samples of white filamentous microbial mats from two different
sampling locations (Upper Spring white mat 1 and Upper Spring white mat 3;
Fig. 1).
To determine the percentage of all cells detected with the bacterial probe set,
EUB338I-III mix-hybridized mat samples were additionally stained with 10 ␮lof
a 10,000-fold-diluted SYBR Green I (FMS Bioproducts, Rockland, Maine)
working solution in the dark for 10 min at room temperature. Slides were then
washed briefly with double-distilled H
2
O and air-dried. Before examination,
samples were covered with the antifading agent Citifluor AF1 (Chemical Labo-
ratory, Caterbury, England). An LSM510 scanning confocal microscope (Zeiss,
Oberkochen, Germany) equipped with an Ar ion laser (450 to 514 nm) and two
HeNe lasers (543 and 633 nm) was used to visualize FISH results. All images
were recorded with a Plan-Apochromat 63x (1.4; oil immersion) objective. Image
processing was performed with the LSM510 software package (version 1.6).
Quantification of probe-detected cells was achieved with the Carl Zeiss Vision
KS400 software package in conjunction with the R.A.M (Relative Area Mea-
surement) macro, as described by Schmid et al. (49).
Nucleotide sequence accession numbers. The 16S rRNA gene sequences de-
termined in this study have been submitted to GenBank with accession numbers
from AY191466 to AY191497.
RESULTS
Mat distribution and structure. Water coming into the cave
had no detectable dissolved oxygen. Dissolved sulfide averaged
32 ␮mol liter
⫺1
in the three orifice pools and decreased to
nondetectable levels beyond the terminus of the mats. Along
the Upper Spring outflow channel (Fig. 2a), dissolved oxygen
concentrations progressively increased to 45 ␮mol liter
⫺1
, cor-
responding to a decrease in dissolved sulfide. Cave water had
a temperature of ⬇22°C and pH 7.2. All spring orifices had
long white filament bundles suspended in discharging water.
Some filamentous structures stretched up to1minlength. An
associated microbial mat formed in spring outflow channels,
having an average thickness of 5 cm in water 8 to 10 cm deep
(Fig. 2b). During all the sampling periods, the mats in the
Fissure Spring and Upper Spring streams were nearly 20 m in
total length, while the Lower Spring extended for only a meter
(Fig. 1).
Phylogenetic analysis of clone sequences. From six samples,
44 clones were randomly selected for sequencing in order to
conduct a broad survey of the microorganisms present in mat
communities. Nearly full-length 16S rRNA gene fragments
from the clones were amplified and sequenced. None of the
sequences were identified as chimeras from RDP analysis. All
clone sequences belonged to the Proteobacteria phylum, with
TABLE 1. Probe sequences used to screen cave mat microbial populations
Probe Target group Probe sequence (5⬘33⬘) Target site
a
FA
b
(%) Reference
EUB338 Eubacteria GCT GCC TCC CGT AGG AGT 16S (338) 0–40 10
EUB338-II Planctomycetes GCA GCC ACC CGT AGG TGT 16S (338) 0–40 10
EUB338-III Verrucomicrobia (and others) GCT GCC ACC CGT AGG TGT 16S (338) 0–40 10
NonEUB Negative control ACT CCT ACG GGA GGC AGC 16S (338) 0 62
ALF968 Alphaproteobacteria GGT AAG GTT CTG CGC GTT 16S (968) 20 38
BET42a Betaproteobacteria GCC TTC CCA CTT CGT TT 23S (1027) 35 32
GAM42a Gammaproteobacteria GCC TTC CCA CAT CGT TT 23S (1027) 35 32
HGC69a Actinobacteria TAT AGT TAC CAC CGC CGT 23S (1901) 25 46
LGC345A Many Firmicutes (together with two
other LGC345A probes)
TGG AAG ATT CCC TAC TGC 16S (354) 20 33
LGC354B Same as LGC345A CGG AAG ATT CCC TAC TGC 20 33
LGC354C Same as LGC345A CGG CGT CGC TGC GTC AGG 20 33
CF319a Some members of the “Flavobacteria”TGG TCC GTG TCT CAG TAC 16S (319) 35 31
G123T
c
Thiothrix CCT TCC GAT CTC TAT GCA 16S (697) 40 21
EPS710 “Epsilonproteobacteria”-Thiovulum
groundwater subgroup
CAG TAT CAT CCC AGC AGA 16S (710) 30
d
64
LKC59 Epsilonproteobacterial group I from
Lower Kane Cave
TCC TCT CAT CGT TCG ACT 16S (59) 30 This study
LKC1006 Epsilonproteobacterial group II from
Lower Kane Cave
CTC CAA TGT TTC CAT CGG 16S (1006) 30 This study
a
E. coli 16S rRNA position (6).
b
Formamide (FA) percentage in the FISH hybridization buffer.
c
Used in conjunction with a competitor probe, G123T-C (5⬘-CCTTCCGATCTCTACGCA-3⬘) (21).
d
This formamide concentration differs from the one suggested in the original publication (64) because we applied a different hybridization temperature.
VOL. 69, 2003 NOVEL “EPSILONPROTEOBACTERIA”FROM SULFIDIC CAVES 5505

85% of the clones affiliated with the “Epsilonproteobacteria,”
11% of the clones belonged to the Betaproteobacteria, and 4%
of the clones clustered with the Gammaproteobacteria.
Within the “Epsilonproteobacteria,”the Lower Kane Cave
clones clustered into two different clades (Fig. 3), referred to as
LKC group I and LKC group II, with high bootstrap values
supporting their phylogenetic position. The closest relatives to
both LKC group I and group II were two environmental
clones, sipK119 and sipK94, obtained from microbial aggre-
gates with a string-of-pearls-like morphology in sulfidic springs
at the Sippenauer Moor, Regensburg, Germany (35, 47).
Group I clones, identified from Fissure and Upper Spring
orifice samples, clustered closely (98 to 99% similar in nucle-
otide sequence) with clone sipK119 and Cesspool Cave clone
CC-4 (14). The closest cultured representative of LKC group I
sequences is Sulfuricurvum kujiense (22). LKC group II clones,
obtained from Upper and Lower Spring orifice and white mat
samples, were closely related to the sipK94 clone (99% similar
in nucleotide sequence), and more distantly to various marine
and hydrothermal vent clones (91 to 94% similarity), as well as
to epibionts of the polychaetous annelid Alvinella pompejana
(89% similarity) (18), ectosymbionts of the shrimp Rimicaris
exoculata (90% similarity) (43), and Parker Cave clones (92 to
94% identical; SRang names on Fig. 3) (5).
Fluorescence in situ hybridization. FISH probes were used
to identify and to quantify specific microorganisms in natural
white filamentous microbial mat samples from Lower Kane
Cave. For each of the two LKC clone groups clustering with
the “Epsilonproteobacteria,”a specific probe was designed.
Probes LKC59 and LKC1006 targeted clone sequences from
group I and group II, respectively (Table 2). Although LKC
group I is closely related to other environmental clones from
groundwater and caves (Fig. 3), probe LKC59 has at least one
mismatch with these and all other rRNA gene sequences in the
database. Probe LKC1006 also did not target any other se-
quences in the database, including clone sipK94 which has 99%
16S rRNA gene sequence similarity with LKC clone group II.
However, it should be mentioned that three of the LKC group
II clones possess a single mismatch within the target site of
probe LKC1006 and might not be detectable by this probe
(Table 2). These mismatches either indicate actual genetic
microheterogeneity or originate from PCR and/or sequencing
artifacts.
Optimal hybridization stringency was determined for the
newly designed probes LKC59 and LKC1006, as well as for the
previously published probe EPS710 (64) that targets environ-
mental clones within the “Epsilonproteobacteria”and strain
YK-1 (22). Since no cultured strains possessing the target sites
for probes LKC59 and LKC1006 are available, optimal hybrid-
ization stringency was inferred by visual comparison of fila-
ment fluorescence from cave samples. For all three probes,
mat filaments showed bright fluorescence if hybridization buff-
ers with up to 30% formamide were used. At more stringent
conditions, signal intensity decreased sharply (results not
shown).
In the six samples analyzed, between 68% and 88% of the
cells stainable with a general nucleic acid dye could be detected
with the bacterial probe set (Table 3). Of the nine different
group- and genus-specific probes applied (Table 1), positive
hybridization signals were only observed with the probes
BET42a, GAM42a, and G123T. Probe BET42a labeled small
FIG. 2. (a) Photograph of cave passage showing microbial mats
growing in sulfidic stream channel formed downstream of the Upper
Spring orifice in Lower Kane Cave (light area at lower right). The stake
in the center of the view is approximately 25 cm high. (b) Filamentous
microbial mats in cave stream. Scale bar, 10 cm.
TABLE 2. Difference alignment of the target region of the 16S
rRNA for LKC-specific probes
Probe and target Target sequence
a
LKC59 probe sequence (5⬘-3⬘)TCCTCTCATCGTTCGACT
Target sequence AGUCGAACGAUGAGAGGA
LKC group I clones
b
------------------
Uncultured Cesspool Cave clone
CC-4 (AF207530)
-------------U----
Uncultured groundwater clone
1023 (AB030610)
-------------U----
LKC1006 probe sequence (5⬘-3⬘)CTCCAATGTTTCCATCGG
Target sequence CCGAUGGAAACAUUGGAG
Most LKC group II clones
c
------------------
LKC1.114_5 (AY191480) ---G--------------
LKC1.199_5 (AY191494) -U----------------
LKC1.199_6 (AY191495) ---------U--------
a
—, identical to the probe sequence.
b
Based on nine LKC group I clones with 16S rRNA gene sequences at this
position.
c
Based on 15 LKC group II clones.
5506 ENGEL ET AL. APPL.ENVIRON.MICROBIOL.

FIG. 3. 16S rRNA gene-based phylogenetic tree showing the phylogenetic position of 32 clones from Lower Kane Cave (designated LKC1 and
having sequences ⬎1,120 nucleotides long) within the “Epsilonproteobacteria.”The topology of the tree was inferred from the results of the
maximum-likelihood analysis, and the phylogenetic affiliations of the LKC clones were confirmed by comparison with different reconstruction
methods (data not shown). Clones are labeled in bold with corresponding sample and clone numbers. Reference sequences (with GenBank
accession numbers) were chosen from the RDP to represent the diversity of “Epsilonproteobacteria”members and specifically the Thiovulum
phylogenetic group. The specificity of the “Epsilonproteobacteria”FISH probes applied in this study is shown. The tree was rooted with the
sequences of Desulfocapsa thiozymogenes and Hydrogenophaga pseudoflava, shown as an arrow labeled “to outgroups.”Numbers at branch
intersections refer to bootstrap values for each node from maximum likelihood, maximum parsimony, minimum evolution, and Bayesian inference
posterior probabilities (values below 50% or where only one method supports a node are not shown).
5507

rods specifically but weakly in all the mat samples, while long
filamentous cells hybridized strongly with probes GAM42a and
G123T, indicating the presence of Thiothrix spp. in some samples.
The three probes targeting subgroups within the “Epsilon-
proteobacteria”were used in different combinations. All probes
exclusively hybridized to filamentous microbes and conferred
very bright signals to their target cells, indicating high rRNA
contents (4). Filaments detected by probe LKC59 had an av-
erage diameter of 1 ␮m and appeared kinked or twisted, while
straighter, longer, and slightly thicker filaments were detected
by LKC1006 (Fig. 4). Simultaneous hybridization with the ep-
silonproteobacterial probes LKC59 and EPS710 showed that
they stained the same filamentous cell morphotypes (Fig. 4,
row I), and as expected, neither probe EPS710 nor probe
LKC59 overlapped with probe LKC1006 (Fig. 4, rows II and
III).
FIG. 4. Fluorescence in situ hybridization of Lower Kane Cave microbial mat samples with probes EUB338I-III mix, newly designed probes
LKC59 and LKC1006, and probe EPS710. Rows: I, Fissure Spring orifice filaments; II, Upper Spring white mat 1; III, Upper Spring white mat
2. Columns: A, EUB338I-III mix (labeled with FluosPrime, colored in green); B and C, epsilonproteobacterial LKC group probe or EPS710 probe
(labeled with Cy3 and colored in red, or labeled in Cy5 and colored in light blue); D, overlap of columns B and C. If Cy3- and Cy5-labeled probes
overlap, the filaments appear pink. Scale bar, 20 ␮m.
TABLE 3. Quantification of epsilonproteobacterial filaments of LKC groups I and II with specific FISH probes
a
Group
Relative biovolume, % (SE)
Fissure Spring
orifice
Upper Spring
b
Lower Spring
White mat 1 White mat 2 White mat 3 Orifice White mat
EUB338I-III mix/SYBR Green I ratio 88 (2) 87 (3) 77 (3) 80 (3) 74 (1) 68 (4)
LKC group I/EUB338I-III mix ⬍8
c
(2) ⬍10
c
(1) ⬍1⬍1 2 (1) ⬍1
LKC group II/EUB338I-III mix 64 (5) 67 (4) 70 (2) 57 (2) 50 (1) 4 (1)
a
Relative biovolumes are given as percentages, and the number in parentheses is the standard error.
b
White mat 1 and white mat 2 were separated by 4 m, and white mat 2 and white mat 3 were separated by 5 m.
c
This value is most likely overestimated due to relatively weak filament signal intensity and high background.
5508 ENGEL ET AL. APPL.ENVIRON.MICROBIOL.

Epsilonproteobacterial filaments belonging to LKC group II
dominated five of the six microbial mats examined and made
up to 70% of the biovolume of those cells detectable by FISH
with the bacterial probe set (Table 3). Only in the Lower
Spring white mats, which were dominated by Thiothrix spp.
(data not shown), epsilonproteobacterial filaments occurred at
relatively low numbers (4% of the bacterial biovolume). In
contrast, epsilonproteobacterial filaments of LKC group I were
below the detection limit in three of the six samples investi-
gated, and made up less than 10% of the bacterial biovolume
in the other three samples.
DISCUSSION
Defining the composition of microbial communities can aid
in our understanding of biogeochemical cycling that occurs in
remote and difficult-to-characterize habitats. Particularly for
the terrestrial subsurface, however, microbial community
structures are poorly understood due to a limited number of
investigations done on such systems. In this study we applied
the full-cycle rRNA approach to characterize microbial mats
from sulfidic cave springs and to assess dominant microbial
populations integral to biogeochemical cycling in this system.
The majority of 16S rRNA gene clones were assigned to two
evolutionary lineages within the “Epsilonproteobacteria,”des-
ignated LKC groups I and II, neither of which possesses closely
related cultured representatives. However, each of the clone
groups also contains a molecular clone, either sipK119 or
sipK94 (Fig. 3), from microbial communities growing in a
string-of-pearls-like morphology (35, 47). Several 16S rRNA
sequences retrieved from groundwater at an underground pe-
troleum storage cavity (64) and S. kujiense, an anaerobic sulfur
oxidizer recently isolated from this habitat (22), cluster with
LKC group I. Two clone groups (CC-4 and CC-9) from the
small sulfidic cave, Cesspool Cave (14), are also closely related
to group I (Fig. 3). In contrast, Lower Kane Cave group II
forms a monophyletic grouping with the 16S rRNA gene se-
quence of an uncultured filamentous epibiont associated with
the vent annelid Alvinella pompejana (7), while clone groups
from phylogenetic studies of Parker Cave, another sulfidic cave
system, are found in sister clades (5) (Fig. 3).
Quantitative FISH analysis with two newly developed 16S
rRNA-targeted oligonucleotide probes specific for LKC group
I and II revealed that both groups are filamentous bacteria.
Filaments for LKC group I were detected in rather low num-
bers in three of the six mats analyzed and were below detection
in other mat samples. In contrast, filaments of LKC group II
dominated five of the six mats and represented 50 to 70% of
the bacterial biovolume in these communities. Bright FISH
signals observed for both filament groups suggest that these
microorganisms were physiologically active in the mats.
While high in situ abundance of free-living and eukaryote-
associated “Epsilonproteobacteria”have been described from
many marine environments, including hydrothermal vent com-
munities (1, 7–9, 18, 25–27, 29, 36, 37, 43, 45, 58), there is
generally no evidence available that members of the “Epsilon-
proteobacteria”are also numerically important in terrestrial
systems. The only exceptions currently known are from two
engineered systems and sulfidic cave microbial mats in which
“Epsilonproteobacteria”exist in significant numbers. Watanabe
et al. (64) report between 12 and 24% of the total prokaryotic
cells in petroleum-contaminated groundwater could be identi-
fied via FISH as epsilonproteobacterial curved rods, and rela-
tively low abundances of Arcobacter (4%) were identified from
activated sludge in municipal wastewater treatment plant with
FISH (53).
Epsilonproteobacterial abundance estimates from 16S
rRNA clone libraries in Parker and Cesspool Caves, at 73%
and 47% “Epsilonprotoebacteria,”respectively, are similar to
the FISH biovolume values for LKC group I and group II (5,
14). Although the microbial aggregates in a string-of-pearls-
like morphology harbor “Epsilonproteobacteria”closely related
to those in Lower Kane Cave, FISH analysis revealed that the
string-of-pearls are dominated not by “Epsilonproteobacteria,”
but by Thiothrix spp. filaments and novel Archaea (35, 47).
Consequently, the mats from the sulfidic springs in Lower
Kane Cave represent the first nonmarine natural system that is
demonstrably driven by the activity of “Epsilonproteobacteria.”
Since the filamentous “Epsilonproteobacteria”dominating
the mats from Lower Kane Cave have not yet been cultured,
we can only speculate about the ecophysiology of these micro-
organisms. However, because most microbial mats in sulfide-
containing marine and freshwater environments are domi-
nated by sulfur bacteria that form the bulk of the mats as
filaments or webs and veils, including Beggiatoa (39), Thiothrix
(35), Thiomargarita (50), Thioploca (41), and Thiovulum (15,
36, 52), it is tempting to suggest that the two cave filamentous
groups are associated with sulfur cycling. Consistent with this
hypothesis, all “Epsilonproteobacteria”presently isolated are
associated with sulfur metabolism, specifically the oxidation of
reduced sulfur compounds with oxygen (27, 29, 43), nitrate (17,
22, 57), various sulfur species (16), or metals (51, 54) as elec-
tron acceptors. Some epsilonproteobacterial strains also grow
anaerobically by reducing sulfur to sulfide (2, 7, 34).
In Lower Kane Cave the two groups of “Epsilonproteobac-
teria”may be associated with anaerobic sulfur oxidation, as
incoming sulfidic groundwater is anoxic and dissolved oxygen
concentrations were generally low along the stream channel
where the mats occurred. LKC group I had highest abundances
in orifice filament bundles and proximal mats, and the phylo-
genetic affiliation of LKC group I to anaerobic groundwater
clones (63, 64) and S. kujiense (22) is consistent with anaerobic
sulfur oxidation. However, while the availability of alternative
electron acceptors, particularly sulfate, in the anaerobic to
disaerobic water in Lower Kane Cave is high, the concentra-
tion of nitrate (the electron acceptor used by S. kujiense)is
generally low or below detection (P. C. Bennett, unpublished
results). LKC group II filaments dominated all but one mat
sample and may also oxidize sulfur compounds anaerobically.
Interestingly, the abundance of LKC group II filaments in the
mats decreased with increasing distance from the orifice (Table
3), which might suggest that these microorganisms prefer more
anaerobic and microaerophilic conditions. At elevated dis-
solved oxygen concentrations, LKC group II may be outcom-
peted by Thiothrix (Table 3; white mats from Lower Spring), a
typical aerobic, sulfur-oxidizing bacterium frequently found in
freshwater habitats and activated sludge (21, 52, 61).
In addition to sulfur cycling, carbon fixation by chemolitho-
autotrophic microorganisms is of major importance for the
microbial mat communities in Lower Kane Cave because
VOL. 69, 2003 NOVEL “EPSILONPROTEOBACTERIA”FROM SULFIDIC CAVES 5509

chemolithoautotrophs would provide an energy source for
their respective ecosystem. The phylogenetic relatives to the
LKC clones, such as isolates of A. pompejana epibionts (7), T.
denitrificans (57), and S. kujiense (22), grow as autotrophs.
Because the concentration of available dissolved organic car-
bon is low (⬍80 ␮mol liter
⫺1
, including dissolved methane gas)
in the incoming spring water, the filamentous “Epsilonpro-
teobacteria”in Lower Kane Cave are presumably also chemo-
lithoautotrophs. Future characterization of the filamentous
populations with stable and radiolabeled isotopes and cultur-
ing work will address this issue, for instance by combining
fluorescence in situ hybridization with microautoradiography
to elucidate both sulfur and carbon metabolism (24).
The ecological significance of “Epsilonproteobacteria”is be-
coming more apparent, as these microorganisms are found
associated with cycling carbon and sulfur compounds in ex-
treme environments, including the sulfidic and oligotrophic
groundwater in Lower Kane Cave. In conclusion, this study
expands the known diversity of “Epsilonproteobacteria”in the
terrestrial subsurface and provides information about the dis-
tribution of these microbes relative to habitat geochemistry,
which can be used to aid future cultivation attempts of these
environmentally important bacteria.
ACKNOWLEDGMENTS
Special thanks to the Bureau of Land Management, Cody office, for
cooperation in permitting this research. We thank S. Engel, T. Dog-
wiler, and R. Payn for field assistance and K. Crandall for laboratory
support and critical insights.
This research was supported by a National Science Foundation
LExEn grant (EAR-0085576) and in part by Brigham Young Univer-
sity and the Geology Foundation of the University of Texas at Austin.
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