1 community structure of subsurface biofilms in the thermal sulfidic caves of 1 acquasanta terme

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


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


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

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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


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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 clone RS06101-B1 (EU101289)


Acquasanta clone AS053_B79 GU390843 (5)

Movile Cave clone MC1_bact_cl30 (DQ295689)

Frasassi clone LKA78 (EF467574)



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/-


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“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 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





100/100

100/100

100/100

100/100

-/94

-/100

100/100

89/61

83/98

86/50

99/-


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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)



Iron−rich travertine clone FD03 (AB354612)

Marine sediment clone Hyd89−29 (AJ535253)


Khir Ganga India hot spring clone MO75 (EU037213)

Desulforhopalus singaporensis str. S'pore T1 (AF118453)

Frasassi biofilm clone GS02-zEL4 (DQ415869)


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)


Lactobacillus acidophilus (NC_006814)


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


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1 community structure of subsurface biofilms in the thermal sulfidic caves of 1 acquasanta terme

Documents