1 community structure of subsurface biofilms in the thermal sulfidic caves of 1 acquasanta terme
TRANSCRIPT
1
Community structure of subsurface biofilms in the thermal sulfidic caves of 1
Acquasanta Terme, Italy 2
3
D. S. Jones1, D. J. Tobler
1†, I. Schaperdoth
1, M. Mainiero
2, and J. L. Macalady
1* 4
5
1Pennsylvania State University, Dept. of Geosciences, University Park PA, 16802 USA 6
2Studio Geologico Mainiero, Via Francesco Podesti 8, 60122 Ancona, Italy 7
8
*Corresponding author. Mailing address: Department of Geosciences, Pennsylvania State 9
University, University Park, PA 16802, USA. Phone: 814-865-6330. Fax: 814-86x-xxxx. 10
Email: [email protected]. 11
12
†Present address: Department of Geographical and Earth Sciences, University of 13
Glasgow, Glasgow G128QQ, UK 14
15
16
Running title: Subsurface hot spring biofilms 17
18
19
Copyright © 2010, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.00647-10 AEM Accepts, published online ahead of print on 16 July 2010
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Abstract 1
2 We performed a microbial community analysis of biofilms inhabiting thermal (35-50°C) 3
waters more than 60 m below the ground surface near Acquasanta Terme, Italy. 4
Groundwater hosting the biofilms has 400-830 µM sulfide, <10 µM O2, pH 6.3 to 6.7, 5
and specific conductivity 8500 to 10,500 µS/cm. Based on 16S rRNA gene cloning and 6
fluorescent in situ hybridization (FISH), the biofilms have low species richness, and 7
lithoautotrophic (or possibly mixotrophic) Gamma- and Epsilonproteobacteria are the 8
principle biofilm architects. Deltaproteobacteria sequences retrieved from the biofilms 9
have <90% 16S rRNA similarity to their closest relatives in public databases, and may 10
represent novel sulfate reducing bacteria. The Acquasanta biofilms share few species in 11
common with Frasassi cave biofilms (13°C, 80 km distant), but have a similar 12
community structure, with representatives in the same major clades. The ecological 13
success of Sulfurovumales-group Epsilonproteobacteria in the Acquasanta biofilms is 14
consistent with previous observations of their dominance in sulfidic cave waters with 15
turbulent water flow and high dissolved sulfide/oxygen ratios. 16
17
Introduction 18
19
Despite rapid progress in the past decade, the deep subsurface remains one of the least 20
explored microbial habitats on earth. Recent studies illustrate the presence of significant 21
spatial heterogeneity (10, 53) and the strong influence of mineralogy and fluid flow on 22
subsurface microbial biodiversity (9, 16, 31, 61). Data obtained by drilling are 23
complemented by an increasing number of studies that exploit subsurface passages 24
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navigable by humans (17). These subsurface passages include caves (14, 46) and mines 1
(22, 32, 47, 52, 57). Approximately 10% of known caves (49) and perhaps more (33) are 2
formed where reduced, sulfidic groundwaters interact with oxidized water descending 3
from surface environments. Limestone dissolution in these groundwater mixing zones 4
results in deep caves that receive few organic inputs from the surface. Due to the 5
presence of both sulfide and oxidants where the groundwaters mix, lithoautotrophic 6
microorganisms thrive and supply the primary productivity for food chains that may 7
include invertebrate and vertebrate animals (12, 23). Interest in these isolated terrestrial 8
chemosynthetic microbial communities is fueled by their potential as model systems for 9
microbial biogeography and as analogs for oxygen-poor, sulfur-rich environments 10
prevalent early in Earth history or on other planets. 11
Sulfidic caves studied by microbiologists to date include Lower Kane Cave in 12
Wyoming (15), Cueva de Villa Luz in Mexico (5, 23), Movile Cave in Romania (7, 28), 13
Parker Cave in Kentucky (4) and the Frasassi Caves in Italy (38, 39). Average water 14
temperatures of previously studied sulfidic caves range from 12-28oC. In contrast, 15
groundwater in the Grotta Nuova di Rio Garrafo and associated caves near Acquasanta 16
Terme (Italy) reaches temperatures up to 50oC (M. Mainiero, unpublished results). The 17
Acquasanta caves host conspicuous microbial biofilms that have not been previously 18
investigated, presenting an opportunity to compare subsurface environments with similar 19
energy resources but large differences in temperature. 20
A reconnaisance of the Acquasanta caves (Figure 1) showed that they contain 21
biofilm types also reported in other sulfidic caves, including viscous snottites on walls 22
above the water table (Figure S1a, b, and e), reddish clay-rich deposits ("ragu") similar to 23
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those on walls in Cueva de Villa Luz (Figure S1e), and white biofilms covering 1
sediments below the water table (Figure S1d and f). As in Cueva de Villa Luz, subaerial 2
surfaces in areas with high sulfur gas concentrations are covered with elemental sulfur 3
deposits (Figure S1c). Here we describe the diversity and community structure of 4
biofilms in the thermal Acquasanta groundwater. In addition, we investigate whether the 5
Acquasanta stream biofilms have a structure consistent with a simple ecological niche 6
model developed for sulfur-oxidizing clades inhabiting non-thermal sulfidic caves (37). 7
The niche model considers aqueous sulfide and oxygen concentrations and hydrodynamic 8
shear, and suggests that in turbulently flowing (high shear) waters with high 9
sulfide/oxygen ratios, Epsilonproteobacteria should outcompete filamentous 10
Gammaproteobacteria such as Beggiatoa and Thiothrix. We find that Acquasanta cave 11
stream biofilms share very few phylotypes in common with other sulfidic caves studied to 12
date, and are dominated by Epsilonproteobacteria as predicted by the niche model. 13
14
15
Materials and methods 16
17
Site description and field geochemistry 18
19
Grotta Nuova di Rio Garrafo (Figure 1) is located approximately 2 km south of 20
Acquasanta Terme, Italy (13o25’ E, 42
o45’ N). The cave entrance is 15 meters above the 21
western bank of the Rio Garrafo (Garrafo River), and the sulfidic water table in the cave 22
is approximately 50 meters below the level of the perched river. Grotta Nuova di Rio 23
Garrafo contains more than 1 km of passages with strong vertical development in marly 24
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Scaglia Rossa limestone (18, 21). The water table is accessible via vertical caving routes. 1
Sampling teams were equipped with gas monitors (O2, CO2, SO2 and CH4) for safety 2
reasons. Gas masks were necessary to prevent inhalation of toxic concentrations of sulfur 3
gasses while sampling. Sulfidic groundwater in the cave has conductivity values as high 4
as 10.5 mS/cm and contains up to 825 µM H2S (aq). The waters are near-neutral (pH 6.3-5
6.8) and the major ion constituents are Na+, Ca
2+, Cl
-, HCO3
- and SO4
2- (18). Ammonium 6
concentrations are ≤ 400 µM, dissolved organic carbon concentrations are ≤ 4.7 mg/L, 7
and dissolved Fe, Mn, and nitrate are below 1 µM. 8
Biofilms were collected at the head of the cave stream (site AS1, Figure 1) in 9
2005, 2007, and 2008. Each year, two samples were collected 1-3 m apart from each 10
other along the flow path of the stream (e.g. AS08-2 and AS08-3 in 2008). Biofilms were 11
sampled into sterile tubes using sterile plastic pipettes, stored on ice, and processed 12
within 8-12 hours of collection. Subsamples of approximately 0.25-0.5 grams dry weight 13
were fixed in 3 volumes of freshly prepared 4% (wt/vol) paraformaldehyde in 1x 14
phosphate buffered saline (PBS) for 3 to 4 h, and stored in a 1:1 PBS/ethanol solution at -15
20°C for FISH analyses. Samples for clone library construction were preserved in 4 parts 16
RNAlater (Ambion) to 1 part sample (v/v). Water samples were filtered (0.2 micron) at 17
the collection site into acid-washed polypropylene bottles and stored at 4°C until analysis. 18
Specific conductivity, pH, and temperature of the water were measured in the field using 19
a 350i multimeter (WTW, Weilheim, Germany) with multiple sensors. Dissolved sulfide 20
and oxygen concentrations were measured at the sample site using a portable 21
spectrophotometer (Hach Co., Loveland, CO) using methylene blue for total sulfides 22
(Hach method 690) and indigo carmine for oxygen (Hach method 8316). Duplicate 23
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sulfide analyses were within 5% of each other. Replicate oxygen analyses were within 1
20% of each other. No sulfide and oxygen concentration data were obtained in 2005 due 2
to a spectrophotometer malfunction. Nitrate, nitrite, ammonium and sulfate were 3
measured on water filtered (0.2 µm pore size) into sterile bottles at the point of collection 4
and transported on ice. Measurements were made at the Osservatorio Geologico di 5
Coldigioco Geomicrobiology Lab using a portable spectrophotometer within 12 hours of 6
collection according to the manufacturer’s instructions (Hach Co., Loveland CO). Light 7
microscopy was performed on live biofilm samples (stored 4 °C) and RNAlater-preserved 8
samples within 24 hours of collection on a Zeiss Model 47-30-12-9901 optical 9
microscope (1250x) at the Osservatorio Geologico di Coldigioco Geomicrobiology Lab. 10
The presence of elemental sulfur particles and inclusions was assayed using an alcohol 11
dissolution test as described in (35). 12
13
Fluorescent in situ hybridization (FISH) 14
15
Clones retrieved in 16S rRNA gene libraries were checked against the probe sequences in 16
Table 2 to ensure probe specificity and membership in target groups. FISH experiments 17
were carried out as described in (3) on 10-well teflon coated slides using the probes listed 18
in Table 2. Oligonucleotide probes were synthesized and labeled at the 5’ ends with 19
fluorescent dyes (Cy3, FITC) at Sigma-Genosys (USA). Cells were stained after 20
hybridization with 4’,6’-diamidino-2-phenylindole (DAPI), mounted with Vectashield 21
(Vectashield Laboratories, USA) and viewed on a Nikon E800 epifluorescence 22
microscope. Images were collected with a Nikon CCD camera using NIS Elements AR 23
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2.30 image analysis software. Fluorescence coming from DNA probes in Figure S2 is 1
shown in false color. An auto-levels function was applied to each image shown in Figure 2
S2. For population estimates based on FISH, the biofilm matrix was easily disrupted by 3
shaking the sample tube. At least 3 slide wells were examined for each probe 4
combination. At least ten images were collected for each sample and probe combination, 5
taking care to represent the sample variability, and a total DAPI-stained area of at least 3 6
x 104 mm
2 (equivalent to the area of 5 x 10
4 E. coli cells) was analyzed in each case. The 7
presence of cells ranging from small rods to large filaments precluded simple counting 8
that does not take into account differences in biomass between the cell types. A 9
comparison between visual area estimates and area counts obtained using the object count 10
tool of NIS Elements AR 2.30 image analysis software indicated that visual estimation 11
gave the same results within error in less time. Since semi-quantitative area counts are 12
sufficient to support the conclusions obtained in this study, a visual estimation approach 13
was employed. 14
15
Clone library construction 16
Environmental DNA was extracted, amplified, and cloned using archeal and bacterial 17
domain-specific primers as described in (37). Briefly, environmental DNA was extracted 18
from approximately 0.5 grams of biofilm using phenol-chloroform extraction. Each 50 19
µL PCR reaction mixture contained: environmental DNA template (1-150 ng), 1.25 U 20
ExTaq DNA polymerase (TaKaRa Bio Inc., Shiga, Japan), 0.2 mM each dNTPs, 1X PCR 21
buffer, 0.2 µM 1492r universal reverse primer and 0.2 µM 27f bacterial domain primer 22
(34). Thermal cycling was as follows: initial denaturation 5 min at 94 °C, 25 cycles of 94 23
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°C for 1 min, 50 °C for 25 sec and 72 °C for 2 min followed by a final elongation at 72 1
°C for 20 min. For archaeal PCR reactions, primers were 21f (5'-2
TTCCGGTTGATCCYGCCGGA) and 977r (5'-YCCGGCGTTGAMTCCAATT), and 3
thermal cycling was performed as described above except that the annealing temperature 4
was 58 °C. PCR products were cloned into the pCR4-TOPO plasmid and used to 5
transform chemically competent OneShot MACH1 T1
E. coli cells (TOPO TA cloning 6
kit, Invitrogen, Carlsbad, CA). Plasmid inserts were screened using colony PCR with 7
M13 primers. Colony PCR products of the correct size were purified using the QIAquick 8
PCR purification kit (Qiagen Inc., USA). 9
10
Sequencing and phylogenetic analyses 11
12
Clones were sequenced at the Penn State University Biotechnology Center with Sanger 13
technology (ABI Hitachi 3730XL DNA Analyzer with BigDye fluorescent terminator 14
chemistry) using T3 and T7 plasmid-specific primers. Sequences were assembled with 15
Phred base calling using CodonCode Aligner v.1.2.4 (CodonCode Corp., USA) and 16
manually checked for ambiguities. The nearly full-length gene sequences (all > 1400 bp) 17
were compared against sequences in public databases using BLAST (1) and submitted to 18
the online analyses CHIMERA_CHECK v.2.7 (8) and Bellerophon 3 (24). Putative 19
chimeras were excluded from subsequent analyses. Sequences were aligned to the 7682-20
character Hugenholtz alignment using the NAST aligner at greengenes (13). NAST-21
aligned sequences were loaded into an ARB database (36) containing over 230,000 full-22
length sequences. Alignments were edited manually in ARB using the ARB_Edit4 23
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sequence editor. Only sequences greater than 1350 base pairs were used for phylogenetic 1
analyses, with the exception of short Gammaproteobacteria clones from Parker Cave's 2
Sulfur River (4). Analyses included top BLAST hits, the top five closest relatives in the 3
ARB database, and representatives of major divisions within each of the targeted groups 4
(e.g. Sulfurovumales), with an emphasis on sulfidic cave sequences. Alignments were 5
trimmed so that all sequences were of equal length (final alignment length 1153, 1420, 6
and 1257 positions for Figures 3, 4, and 5, respectively), and nucleotide positions with 7
less than 50% base-pair conservation were masked (calculated separately from all 8
sequences in each analysis, exclusive of outgroups). Shorter length sequences (Parker 9
Cave clones AF047617 and AF047623, Fig. 4) were included in phylogenetic analyses 10
with their missing data coded as missing and treated according to PAUP* defaults for 11
each analysis type (59). Maximum likelihood, maximum parsimony, and neighbor joining 12
analyses were performed using PAUP* v.4b10 (59). Maximum likelihood analyses used 13
the general time reversible (GTR) substitution model with gamma distributed among-site 14
variation, at least five random-addition-sequence replicates, and tree bisection-15
reconnection (TBR) branch swapping. Base frequency, substitution rates, and shape 16
parameter were estimated from the data. Maximum parsimony bootstrap analyses (2000 17
replicates) were performed via heuristic search with 100 random-addition-sequence 18
replicates and TBR branch swapping. Neighbor joining bootstrap analyses (2000 19
replicates) were performed with Jukes-Cantor (JC) corrected distance matrix. 20
21
Nucleotide sequence accession numbers 22
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The 16S rRNA gene sequences determined in this study were submitted to GenBank and 1
were assigned accession numbers GU390708-GU390880. 2
3
Diversity analyses 4
5
Rarefaction analyses were calculated based on the frequency of OTUs defined by the 6
program DOTUR v.1.53 (54). OTUs were defined at 98% sequence similarity, using the 7
DOTUR ‘furthest neighbor’ algorithm, based on ARB distance matrices. 8
9
Results 10
11
Field observations and geochemistry 12
13
Water and biofilm samples were collected at site AS1 (Figure 1) in 2005, 2007, and 2008. 14
The stream at site AS1 was fast flowing and turbulent for the entire length of the passage, 15
and nearly every surface in the channel was covered with biofilms of ‘streamer’ 16
morphology (Figure S1d and f). Other biofilm morphologies were rare and limited in 17
areal extent. Streamers were 1-2 cm in length on average (Figure S1d and f), and covered 18
both fine gray sediment and exposed limestone surfaces underwater. Microscopic 19
examination of both live and RNAlater-preserved streamers within 24 hours of collection 20
revealed that none of the cells contained intracellular sulfur inclusions. All biofilm 21
samples had abundant elemental sulfur particles outside cells, in the biofilm matrix. 22
Streamers collected in 2007 and 2008 were dominated by filaments, with rod-shaped and 23
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coccoid cells also present. Filaments were less common in 2005 samples (AS05-2 and 1
AS05-3), which were dominated by long, thin rods. 2
Geochemical data for waters collected at sample site AS1 (Table 1) reflected 3
changes in the degree of dilution of the thermal groundwater by surface water recharge 4
(18). Conductivity was substantially lower in 2005 compared to 2007 and 2008. 5
Conductivity and major ion concentrations were similar for samples collected in 2007 6
and 2008, but hydrogen sulfide concentrations in both the stream and cave atmosphere 7
were much higher in 2007 (Table 1). These differences in geochemistry were not 8
accompanied by any noticeable changes in the density or macroscopic morphology of the 9
microbial streamers covering the stream sediments. 10
11
16S rRNA clone libraries 12
13
Attempts to amplify archaeal 16S rRNA genes from the biofilm samples were 14
unsuccessful, whereas positive control DNA yielded archaeal PCR products of the 15
expected length in the same PCR runs (data not shown). A total of 174 nearly full length 16
bacterial 16S rRNA gene sequences were retrieved from samples AS05-3 (80 clones) and 17
AS07-7 (94 clones) (Figure 2a). Rarefaction analyses (Figure 2b) suggested that the 18
major bacterial populations present in the biofilms have been adequately sampled. The 19
most abundant phylotype in both libraries fell within the Sulfurovumales clade in the 20
Epsilonproteobacteria. Sulfurovumales clones (Figure 3) constituted 40% and 67% of 21
libraries AS05-3 and AS07-7, respectively. Most of the Sulfurovumales sequences were 22
98% similar to clones AS053-B2 and AS077-B27 (Figure 3) and formed a clade with a 23
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single Frasassi cave clone. The AS05-3 library also included several clones (6.3%) of a 1
second Sulfurovumales phylotype. The second most abundant phylotype in both libraries 2
(10.6% in AS05-3 and 26.3% in AS07-7) fell within a clade containing the isolates 3
'Candidatus Thiobacillus baregensis' and Thiofaba tepidophilum in the 4
Gammaproteobacteria (Figure 4). Deltaproteobacteria sequences in the libraries were 5
very distantly related (<90%) to sequences in public databases (Figure 5). Both libraries 6
contained representatives of Bacteroidetes, Elusimicrobia and MVP-15, in addition to 7
rare phylotypes (Figure 2). Bacterial diversity in both samples was very low, even 8
compared with sulfur-oxidizing biofilms from other sulfidic cave environments (Figure 9
2). 10
11
Epifluorescence microscopy 12
Fluorescent in situ hybridization (FISH) was used to evaluate relative abundances of 13
major microbial populations in the biofilms (Table 1). FISH data were consistent with 14
clone libraries, suggesting little PCR or DNA extraction bias for major populations. No 15
hybridization to archaeal-specific probe ARCH915 was detected. Greater than 95% of 16
DAPI-stained cells hybridized with the bacterial-specific probe EUBMIX in all samples. 17
EUBMIX-negative populations were small cocci that did not appear to hybridize with 18
any probes used in this study (Figure S2). No nucleated cells (i.e. protists or fungi) were 19
observed. Large holdfast structures uniting many filaments such as those observed for 20
Thiothrix spp. or filamentous Epsilonproteobacteria "rosettes" in Frasassi cave streamers 21
(37, 39) were not observed in the Acquasanta biofilms. 22
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Samples AS07-6, AS07-7, AS08-2 and AS08-3 were dominated by EP404-1
positive filaments (Table 1, Figure S2). Samples AS05-2 and AS05-3 contained long, thin 2
EP404-positive rods, as well as a population of large EP404-positive cocci (Figure S2). 3
GAM42a-positive cells were present in all samples, and were short rods with uniform 4
morphology across samples (Figure S2). A newly designed FISH probe developed to 5
identify close relatives of 'Candidatus Thb. baregensis' in biofilms from other caves 6
hybridized to the same set of short rods visualized using GAM42a (data not shown). 7
Consistent with the results of FISH experiments, Gammaproteobacteria sequences 8
retrieved in both Acquasanta clone libraries consisted of a single phylotype closely 9
related to 'Candidatus Thb. baregensis' (Figure 4). FISH experiments using SRB385 and 10
Delta495 probes yielded similar area estimates and cell morphologies in all samples. 11
SRB385- and Delta495-positive cells were predominantly large rods, with smaller 12
populations of smaller rods and cocci (Figure S2). 13
14
15
Discussion 16
17
Biofilm community composition 18
19
Based on a full cycle rRNA approach, Acquasanta stream biofilms are dominated by 20
Sulfurovumales (Epsilonproteobacteria) populations, along with important and 21
sometimes equally large populations of Gammaproteobacteria related to 'Candidatus 22
Thb. baregensis' (Table 1 and Figures 2 and S2). Minor populations include 23
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Deltaproteobacteria, Bacteroidetes, Spirochaetales, Elusimicrobia, MVP-15, and rare 1
taxa. Although archaea were not detected using FISH or PCR, small cells that did not 2
hybridize with either arcaheal or bacterial domain-specific probes made up 5% of the 3
community and may prove to be archaeal cells. 4
The Sulfurovumales (Figure 3) are a monophyletic clade with few isolates and 5
large numbers of environmental sequences, and have been retrieved from sulfidic caves 6
and springs worldwide (6). Based on FISH experiments, the most abundant 7
Epsilonproteobacteria populations are filamentous in 2007 and 2008 biofilms, and 8
nonfilamentous in 2005 biofilms. Cells in these populations have indistinguishable 9
morphologies (long, thin rods) but different arrangements (Figure S2). Because the most 10
abundant Epsilonproteobacteria clones are nearly genetically identical in 2005 and 2007 11
(Figure 3, clones AS053-B2 and AS077-B27), we hypothesize that they represent highly 12
related species capable of both filamentous and non-filamentous habits. We recognize 13
that this hypothesis is speculative and remains to be tested using strain-specific probes. 14
Filamentous Sulfurovumales have not been cultured or investigated using metagenomics 15
to date, and thus there are few unassailable constraints on their metabolism. The genome 16
of Sulfurovum sp. NBC37-1, distantly related to Acquasanta clones (< 91.5% similarity) 17
but their closest cultivated relative, has recently been sequenced (44). Sulfurovum sp. 18
NBC37-1 is a lithoautotrophic hydrothermal vent symbiont that can use hydrogen and 19
reduced sulfur species as electron donors. Based on the predominance of sulfur-based 20
lithotrophic metabolisms in hydrothermal vent Epsilonproteobacteria (6, 43) and the 21
importance of Epsilonproteobacteria in terrestrial chemosynthetic ecosystems where 22
reduced sulfur species contribute the bulk of the available chemical energy (37, 51), it is 23
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likely that Sulfurovumales clones retrieved from Acquasanta biofilms will prove to be 1
sulfur-oxidizing lithoautotrophs, sulfur-reducing lithoautotrophs, or possibly mixotrophs 2
that can reduce sulfur using organic compounds depending on environmental conditions. 3
Gammaproteobacteria clones from Acquasanta clone libraries belong to a single 4
phylotype represented by clones AS053-B12 and AS077-B32 (Figure 4). Related isolates 5
include the sulfur-oxidizing lithoautotrophs 'Candidatus Thb. baregensis' (25, 26), 6
Thiofaba tepidiphila (42), and Thiovirga sulfuroxydans (29, 30). Representatives of the 7
clade defined by these isolates (Figure 4) are important constituents of stream biofilms in 8
other sulfidic caves. For example, (37) found close relatives of Thb. baregensis in six out 9
of six Frasassi stream biofilm clone libraries. Clones in this clade have also been 10
retrieved from Parker Cave in Kentucky (4) and Movile Cave in Romania (7). Thus, both 11
metabolisms of cultivated relatives and the distribution of related clones in the 12
environment suggest that Acquasanta Thb. baregensis relatives are sulfur-oxidizing 13
lithoautotrophs. 14
Deltaproteobacterial 16S rRNA clones are very distantly related (<90%) to 15
publicly available sequences, including environmental clones (Figure 5). With the 16
exception of the AS053-B137 phylotype (3 clones), the clones belong to a large 17
environmental clone group with no cultivated representatives. The nearest relatives of 18
these phylotypes are from marine sediment and methane seep environments including Eel 19
River sediments ("Eel-2" clade) (48), where sulfate reduction is an important process. 20
Acquasanta Deltaproteobacteria may thus represent novel sulfate-reducing bacteria. 21
However, because of their dissimilarity to characterized isolates, inferences about their 22
metabolism remain somewhat speculative. It is interesting to note that none of the diverse 23
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Deltaproteobacteria clones from the Frasassi cave system fall within the Acquasanta 1
environmental clone groups. Conversely, Acquasanta clones are not represented in the 2
Desulfocapsa clade, which contains the vast majority of Frasassi Deltaproteobacteria 3
clones (37, 39). 4
Acquasanta caves and the cooler Frasassi caves located ~80 km away provide an 5
interesting geochemical and physical context for comparing microbial communities. 6
Hydrogen sulfide in Acquasanta and Frassasi cave waters is thought to have a similar 7
source, namely partial reduction of sulfate in gypsum-bearing evaporite rocks in the 8
underlying Triassic Burano Formation (18, 20). Conductivity of sulfidic water at 9
Acquasanta is somewhat higher than at Frasassi (~2000 vs. ~9000 µS/cm), although 10
dissolved ions are present in similar ratios (18, 19). The temperature of sulfidic water in 11
the Frasassi cave system is 13-14oC, compared to 35-50°C at Acquasanta. 12
The most abundant Acquasanta clones belong to clades also containing Frasassi 13
phylotypes (i.e. Sulfurovumales in the Epsilonproteobacteria, Thb. baregensis relatives in 14
the Gammaproteobacteria) (37). However, Acquasanta phylotypes are distinct (Figures 3 15
and 4), and Frasassi clones are often more closely related to phylotypes from 16
geographically distant sulfidic caves such as Movile Cave (Romania), Parker Cave 17
(Kentucky, USA), and Lower Kane Caves (Wyoming, USA) than to clones from 18
geographically nearby Acquasanta caves. Phylogeographical patterns can arise via 19
several unrelated mechanisms, including selective pressures imposed by the environment, 20
dispersal limitations, and chance historical occurrences such as colonization events (41). 21
Because stream waters in Acquasanta caves have a significantly higher temperature than 22
previously studied sulfidic cave waters, it is worth considering whether environmental 23
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selection based on temperature could explain the phylogeographical pattern we observe. 1
However, clades within the Sulfurovumales containing Frasassi and other non-thermal 2
cave clones also contain sequences from hot sulfidic springs, suggesting that temperature 3
alone cannot account for the large genetic distances between Frasassi and Acquasanta 4
phylotypes. 5
6
Sulfur oxidizer niches 7
8
We previously proposed a simple niche model for sulfur-oxidizing bacteria in cave 9
streams of the non-thermal Frasassi cave system (37). In the model, two niche dimensions 10
(hydrodynamic shear and aqueous sulfide/oxygen ratio) controlled biofilm population 11
structures. The model described the distribution of three major biofilm types named after 12
their dominant populations: (1) Beggiatoa spp. forming sediment-water interface mats at 13
low shear and variable sulfide/oxygen ratio, (2) Thiothrix spp. forming rock-attached 14
streamers at high shear and low sulfide/oxygen ratio, and (3) filamentous 15
Epsilonproteobacteria forming streamers at high shear and high sulfide/oxygen ratio. 16
These relationships are depicted in Figure S3, with the addition of Acquasanta samples 17
from Table 1. The niche model correctly predicts that Epsilonproteobacteria are the 18
dominant biofilm populations in Acquasanta waters (high shear, high sulfide/oxygen 19
ratio). 20
Sulfidic caves have important similarities with non-thermal and moderately 21
thermal zones around hydrothermal vents, including complete darkness, high sulfide 22
concentrations, and food chains based on bacterial chemosynthesis. We could not identify 23
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any directly comparable quantitative phylogenetic studies of moderately thermal vent 1
microbial community composition in the literature. However, the niche model emerging 2
from study of sulfidic caves is at least consistent with what is known about niches of 3
cultivable mesophilic and moderately thermophilic vent chemoautotrophs (6, 43, 55). 4
Based on the physiology and genome content of hydrothermal vent isolates from mixing 5
zone habitats, Gammaproteobacteria are facultatively aerobic to strictly aerobic whereas 6
Epsilonproteobacteria are strictly anaerobic to facultatively aerobic. These trends parallel 7
the high sulfide/oxygen niche of filamentous Epsilonproteobacteria and low 8
sulfide/oxygen niche of Thiothrix spp. (Gammaproteobacteria) in turbulently mixed 9
sulfidic cave streams. 10
11
Implications and future work 12
13
Niche separation has been documented for microbial clades at a variety of 14
taxonomic levels. For example, environmental specialization correlates with marine 15
bacterioplankton clades within the Vibrionaceae based on hsp60 sequencing (27), with 16
Bacillus simplex strain groups typed by RAPD-PCR (56), with subgroups of 17
actinobacterial clade acI based on 16S rRNA sequences (45), with bacterial phyla and 18
classes in pasture soils (50), and more speculatively, with phylum or sub-phylum level 19
clades present in metagenomic data sets (60). Members of sulfur-oxidizing clades such as 20
the Sulfurovumales appear to show fidelity to a relatively narrow geochemical niche, 21
outcompeting co-existing representatives of other major sulfur oxidizing clades when 22
conditions are correct. Further effort is warranted to establish the evolutionary history of 23
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environmental specialization and niche differentiation among sulfur oxidizers and other 1
microbial functional guilds. 2
3
Acknowledgements 4
5
This work was supported by grants to J. L. M. from the National Science Foundation 6
(EAR-0527046) and NASA NAI (NNA04CC06A). D. J. T. was supported by a 7
Worldwide University Network (WUN) research mobility grant. We thank A. Montanari 8
for logistical support and the use of facilities and laboratory space at the Osservatorio 9
Geologico di Coldigioco (Italy), S. Galdenzi, S. Mariani, G. Filipponi, and the cavers of 10
the Associazione Speleologica Acquasantana C.A.I. for their expert assistance, and L. 11
Albertson, L. Hose, F. Baldoni, and S. Dattagupta for their participation in field 12
campaigns. We thank J. Patel and K. Dawson for laboratory assistance. 13
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Table and Figure Captions 1
2
Figure 1. (A) Plan view map of the Rio Garrafo caves, modified with permission from 3
(21). Contour lines indicate surface elevation in meters. (B) Cross section of Grotta 4
Nuova del Rio Garrafo, modified with permission from (18). Black circles indicate 5
sampling locations for biofilms depicted in Figure S1. Arrows indicate flow directions 6
for water (solid) and gas (dashed). Samples analyzed in the current study were collected 7
exclusively at site AS1. 8
9
Figure 2. (A) Taxonomic composition of two 16S rRNA clone libraries from Acquasanta 10
stream biofilms. Both biofilms were collected at site AS1 (Figure 1b). (B) Rarefaction of 11
16S rRNA clone libraries constructed from Acquasanta and Frasassi cave stream 12
biofilms. OTUs are defined at 98% sequence identity. Frasassi clone libraries are from 13
(37), except for library PC05-LKA (Macalady, unpublished data). 14
15
Figure 3. Maximum likelihood phylogram of 16S rRNA gene sequences from the 16
Sulfurovumales clade (Epsilonproteobacteria). The number of clones represented by each 17
Acquasanta phylotype (98% nucleotide similarity) are indicated in parentheses. Neighbor 18
joining (left) and maximum parsimony (right) bootstrap values greater than 50 are shown 19
for each node. 20
21
Figure 4. Maximum likelihood phylogram of 16S rRNA gene sequences from the 22
Thiofaba/Thiovirga clade in the Gammaproteobacteria. Numbers of clones represented 23
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by each Acquasanta phylotype are indicated in parentheses. Neighbor joining (left) and 1
maximum parsimony (right) bootstrap values greater than 50 are shown for each node. 2
3
Figure 5. Maximum likelihood phylogram of 16S rRNA gene sequences from the 4
Deltaproteobacteria. Numbers of clones represented by each Acquasanta phylotype are 5
indicated in parentheses. Neighbor joining (left) and maximum parsimony (right) 6
bootstrap values greater than 50 are shown for each node. The tree includes the 5 nearest 7
neighbors in public sequence databases for each Acquasanta phylotype. 8
9
Table 1. FISH cell area abundances and geochemical data for Acquasanta cave stream 10
biofilm samples. 11
12
Table 2. FISH probes used in this study. (2, 3, 11, 35, 39, 40, 58) 13
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Table 2. FISH probes used in this study
Probe name Sequence (5'-3') % formamide Label Target specificity Reference
EUB338a GCT GCC TCC CGT AGG AGT 0-50% FITC most Bacteria
(2)
EUB338-IIa GCA GCC ACC CGT AGG TGT 0-50% FITC Planctomycetales
(11)
EUB338-IIIa GCT GCC ACC CGT AGG TGT 0-50% FITC Verrucomicrobiales
(11)
ARCH915 GTG CTC CCC CGC CAA TTC CT 20% Cy3 most Archaea(58)
EP404 AAA KGY GTC ATC CTC CA 30% Cy3 most Epsilonproteobacteria(39)
GAM42ab GCC TTC CCA CAT CGT TT 35% Cy3 most Gammaproteobacteria
(40)
DELTA495ac AGT TAG CCG GTG CTT CCT 45% Cy3 most Deltaproteobacteria,
some Gemmatimonas group
(35)
SRB385 CGG CGT CGC TGC GTC AGG 35% Cy3 some Deltaproteobacteria,
some Actinobacteria and
Gemmatimonas group (3)aEUBMIX is a mixture of EUB338, EUB338-II, and EUB338-III
brequires competitor oligo cGam42a: GCCTTCCCACTTCGTTT
crequires competitor oligo cDELTA495a: AGTTAGCCGGTGCTTCTT
Table 1. FISH cell area abundances and geochemical parameters for Acquasanta biofilm samples.
date temp sp. cond. SO42-
O2(aq) H2S(aq) H2S(g)b
collected EUBMIX EP404 GAM42a DELTA495a SRB385oC (mS/cm) (mM) (uM) (uM) (ppm)
AS05-3 8/19/05 +++++ ++++ ++++ ++ ++ 40.4 6.37 8.4 NM NM NM NM
AS05-2 8/19/05 +++++ ++++ ++++ ++ NM 40.4 6.37 8.4 NM NM NM NM
AS07-6 6/1/07 +++++ ++++ ++++ ++ NM 44.1 6.38 10.6 9.92 3.06 801 100
AS07-7 6/1/07 +++++ ++++ ++++ ++ ++ 44.1 6.38 10.6 9.92 3.06 801 100
AS08-2 6/14/08 +++++ +++++ ++ ++ ++ 42.7 6.31 10.5 12.71 6.69 415 15
AS08-3 6/14/08 +++++ ++++ +++ ++ NM 42.7 6.31 10.5 12.71 6.69 415 15
bMeasured 1.5 m above cave stream
pHsamplePercent hybridization by each FISH probe
a
aEstimated proportion total cells, ND = none detected, + = <3%, ++ = 3-15%, +++ = 15-35%, ++++ = 35-75%, ++++ = 75-100%,
NM = not measured. No ARCH915-positive cells were detected.
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AS1
H2S(g)
AS2
AS3
A B
stream
Jones et al. Figure 1
10 m
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Gammaproteo-bacteria 10.6%
MVP-152.1%
Elusimicrobia5.3%
Epsilonproteobacteria68.1%
Spirochaetales 1.1%
Deltaproteo-bacteria 5.3%
Bacteroidetes 4.3%OP5 1.1%
Chloroflexi 1.1%
OP11 1.1%
Gammaproteo-bacteria 26.3%
MVP-157.5%
Elusimicrobia3.8%
Epsilonproteobacteria46.3%
Spirochaetales 1.3%
Deltaproteo-
bacteria 6.3%
Bacteroidetes 7.5%
OP3/Marine Group A 1.3%
Sample AS07-7(94 clones)
Sample AS05-3(80 clones)
Jones et al., Figure 2
0 20 40 60 80 100
00
10
20
30
40
50
60
7
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● Rio Garrafo biofilms
Frasassi biofilms
●
AS05-3
AS07-7
A
B
GS02-WM
RS06-101
GS02-zEL
PC05-LKA
PC05-11PC06-110
FS06-12
on April 3, 2019 by guest
http://aem.asm
.org/D
ownloaded from
Jones et al. Figure 3
Frasassi clone GS02-WM3 (DQ133917)
Frasassi (Grotta Sulfurea) clone FC1_c124 (DQ295656)
Frasassi clone GS02-WM35 (DQ133918)
Acquasanta clone AS077-B8 GU390726 (1)
Movile Cave clone MC1_b16_cl2 (DQ295688)
Frasassi clone PC06110-B99 (EU101202)
Frasassi clone PC0511-SILK24 (EF467461)
Frasassi clone PC02-LKA76 (EF467572)
Cesspool Cave clone CC1_cl43 (DQ295575)
Big Sulfur Cave clone BSC2_c21 (DQ295546)Sulfidic spring clone B3−AlvEE (AB425208)
Frasassi clone RS06101-B61 (EU101255)
hydrothermal vent clone L63−WB1 (DQ071278)
Sulfurovum lithotrophicum str. 42BKT (AB091292)
Frasassi (Grotta Sulfurea) clone FC1_c102 (DQ295645)
Frasassi clone RS06101-B1 (EU101289)
Frasassi (Grotta Sulfurea) clone FC1_c112 (DQ295653)
Acquasanta clone AS053_B79 GU390843 (5)
Movile Cave clone MC1_bact_cl30 (DQ295689)
Frasassi clone LKA78 (EF467574)
Acquasanta clone AS053_B2 GU390837 (32)
Acquasanta clone AS077_B27 GU390766 (63)
Wastewater clone A54 (EU234097)
Arcobacter cibariusCampylobacter jejuni
Helicobacter pylori
Acidithiobacillus ferrooxidans
0.1 substitutions/site
Su
lfuro
vu
ma
les
Sulfurovum sp. str. NBC37-1 (NC_009663)
Lower Kane Cave clone LKC3_270.57 (AY510215)
Lower Kane Cave clone LKC4_187.1 (DQ295682)
Cesspool Cave clone CC1_cl28 (DQ295572)
White sulfur spring clone SS_EPS2_PT1.1 (DQ295623)
Fat Man’s Misery spring clone FMM.WH2_E2.cl2 (DQ295670)
Pah Tempe hot spring clone EPS2_PT1.1 (DQ295623)
100/100
-/61
-/80
99/100
-/54
-/79
76/-100/-
99/100
94/100
75/80
74/-
99/-
62/64
100/100
89/-
100/100
100/100
98/100
97/98
66/54
56/-
on April 3, 2019 by guest
http://aem.asm
.org/D
ownloaded from
“Candidatus Thb. baregensis" (Y09280)
Frasassi biofilm clone FS0612-B7 (EU101049)
Frasassi biofilm clone GS02-zEL16 (DQ415810)
Frasassi biofilm clone PC0511-SILK8 (EF467463)
Parker Cave clone SRang1.28 (AF047617)
Frasassi biofilm clone PC0511-SILK22 (EF467476)
Frasassi biofilm clone PC06110-B13 (EU101161)
Frasassi biofilm clone FS0612-U1 (EU101140)
Subsurface water clone MS149BH1062003_5 (DQ354745)
Hydrocarbon seep clone BPC028 (AF154088)
Thiofaba tepidiphila (AB304258)
Thiovirga sulfuroxydans (AB118236)
Frasassi biofilm clone PC06110-B37 (EU101175)
Magnesite mine clone cMM319−25 (AJ536782)
Parker Cave clone SRang2.5 (AF047623)
Acidithiobacillus ferrooxidans
Acquasanta clone AS053-B125 GU390864 (21)
Acquasanta clone AS077-B32 GU390741 (10)
0.1 substitutions/site
Jones et al. Figure 4
100/100
100/100
100/100
100/100
-/94
-/100
100/100
89/61
83/98
86/50
99/-
on April 3, 2019 by guest
http://aem.asm
.org/D
ownloaded from
Jones et al. Figure 5
Marine sediment clone Hyd89−19 (AJ535233)
Marine methane seep clone 1513 (AF354149)
Gulf of Mexico sediment clone GoM_GC232_4463_Bac90 (AM745217)
Hydrothermal vent biofilm clone MS12−1−B09 (AM712339)
Yonaguni Knoll IV hydrothermal marine sediment clone OT−B08.16 (AB252432)
Hydrothermal sediment clone (AF420338)
Hydrothermal vent chimney clone PICO pp37 (AJ969442)
Acquasanta clone AS053-B25 GU390749 (2)
Acquasanta clone AS077-B51 GU390717 (5)
Iron−rich travertine clone FD03 (AB354612)
Marine sediment clone Hyd89−29 (AJ535253)
Acquasanta clone AS053-B133 GU390830 (2)
Khir Ganga India hot spring clone MO75 (EU037213)
Desulforhopalus singaporensis str. S'pore T1 (AF118453)
Frasassi biofilm clone GS02-zEL4 (DQ415869)
Frasassi biofilm clone PC0511-SILK96 (EF467470)
Desulfobulbus rhabdoformis str. M16 (U12253)
Desulfacinum subterraneum str. 101 (AF385080)
Desulfococcus multivorans str. DSM 2059 (AF418173)
Desulfobacula toluolica str. DSM 7467 (AJ441316)
Acid mine drainage clone BA71 (AF225447)
Termite gut clone M1PL1−84 (AB192071)
Desulfomonile limimaris (AF230531)
Geobacter metallireducens str. GS−15 (L07834)
Pelobacter acidigallici str. MaGal12T (X77216)
Anaeromyxobacter dehalogenans 2CP−C str. 2CP−C (AF382399)
Myxococcus fulvus str. NBRC 100072 (AB218211)
Acquasanta clone AS053-B137 GU390803 (3)
Lactobacillus acidophilus (NC_006814)
0.1 substitutions/site
Frasassi biofilm clone GS02-WM65 (DQ133927)
Frasassi biofilm clone RS06101-B26 (EU101227)
100/100
100/100
100/100
80/94
98/98
50/-
89/87
-/69
-/99
100/100
-/81-/7198/92
99/99
52/-
68/71
86/94
on April 3, 2019 by guest
http://aem.asm
.org/D
ownloaded from