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Applied and Environmental Microbiology, August 2008, p. 4973-4977, Vol. 74, No. 15
0099-2240/08/$08.00+0     doi:10.1128/AEM.02915-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Diversity of Uncultured Epsilonproteobacteria from Terrestrial Sulfidic Caves and Springs{triangledown}

Megan L. Porter1 and Annette Summers Engel2*

University of Maryland Baltimore County, Department of Biological Sciences, Baltimore, Maryland 21250,1 Louisiana State University, Department of Geology and Geophysics, Baton Rouge, Louisiana 708032

Received 24 December 2007/ Accepted 28 May 2008


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ABSTRACT
 
This study expands the phylogenetic diversity of Epsilonproteobacteria using the 16S rRNA gene framework. Of the 73 lineages defined by sequence similarities at or greater than 99%, most were found at only one site. In contrast, eight lineages were retrieved from sites spanning geographic distances from 1,000 to >10,000 km.


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INTRODUCTION
 
A recent review of the Epsilonproteobacteria taxonomic class revealed that 16S rRNA gene sequences retrieved from extreme environments comprised a large clade of mostly uncultivated microbes, provisionally designated the family "Thiovulgaceae" (3). While the marine representatives of the "Thiovulgaceae" represent a diverse assemblage (see, e.g., references 11, 13, 15, 18, 22, 23, and 30), nearly 80% of the terrestrial sequences in this family originated from just one site: Lower Kane Cave (LKC), Wyoming (3, 7, 8). LKC represented the first nonmarine, natural system demonstrably influenced by the activity of Epsilonproteobacteria (7). The Epsilonproteobacteria have been shown to contribute to ecosystem function via chemolithoautotrophic metabolisms (8) and play a significant role in rock dissolution and porosity formation (9). Although six Epsilonproteobacteria operational taxonomic units (OTUs) from LKC have been described (7, 8), this diversity was dominated by the two lineages: LKC group I (LKC-GI) and LKC group II (LKC-GII). Here, we extend the diversity of terrestrial Epsilonproteobacteria by investigating the 16S rRNA sequence diversity in 15 additional sulfidic cave and spring sites (Table 1) and demonstrate that these lineages are found in novel geographic locations.


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  		TABLE 1. Epsilonproteobacterial diversity associated with sampling sites described in this and previous studiesa

16S rRNA gene sequences were amplified from DNA extracted from microbial mats collected at each site. Universal bacterial primers were used for cave sites (7) and newly developed lineage-specific primers for each LKC group (7) for the spring site DNA. Primer eps59f (forward, 5'-AGTCGAACGATGAGAGGA-3') was used for LKC-GI and eps174f (forward, 5'-CCCCATACTCCTTCTCAT-3') for LKC-GII; both of these primers were paired with a universal bacterial reverse primer. From these amplification products, 15 clone libraries were constructed and screened, and 2 to 57 clones were sequenced from each library using previously described methods (7, 8). Sequences were submitted to the RDP CHECK- CHIMERA program (20).

For phylogenetic analyses, 207 new epsilonproteobacterial sequences were combined with 139 previously published epsilonproteobacterial sequences (62 sulfidic cave and spring sequences; 77 from other habitats) and 16 outgroup taxa. Following sequence alignment (29) and refinement (6), GBlocks v0.91b (4) was used to eliminate alignment regions where there were sequence data for only a small proportion of the taxa. The full alignment file can be downloaded at http://geol.lsu.edu/Faculty/Engel/epsilon.htm. A maximum likelihood phylogeny with branch support from 100 bootstrap replicates was constructed (14) using the best-fit model (25, 26). Delineation of sequence clusters at the 99% sequence similarity predicted for species level relationships (see, e.g., references 16, 17, and 28) was done in DOTUR using the furthest-neighbor clustering algorithm (27). Species level OTUs were designated if DOTUR similarity groups were concordant with a monophyletic clade.

Campbell et al. (3) systematically evaluated 16S rRNA gene sequences derived from environmental sources and found that marine and terrestrial evolutionary lineages were phylogenetically distinct from each other, although there were far fewer terrestrially derived sequences than sequences from marine settings. The sequences recovered from the cave and spring locations represent extensive diversity within the Epsilonproteobacteria, with representatives from all of the major families except the Hydrogenimonaceae (order Campylobacterales) and Nautiliaceae (order Nautiliales) (Fig. 1). We recovered the same major clades designated by Campbell et al. (3), with the majority of new sequences belonging to the provisional family "Thiovulgaceae" (Fig. 1). These results expand the diversity and geographic ranges of the clades and add terrestrially derived sequences to marine clades I and II (3) (Fig. 1). From the current data set, there were no apparent relationships among phylogeny, habitat type (e.g., light versus dark), and geochemistry (data not shown) (Fig. 1; Table 1).


Figure 1
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  		FIG. 1. Maximum likelihood phylogeny of the newly characterized Epsilonproteobacteria terrestrial sequences, rooted to basal epsilonproteobacterial sequences (e.g., Caminibacter, AY691429 and AY691430; Hydrogenimonas, AB105048; Lebetimonas, AB167820; Nautilia, AF357197 and AJ575809; Nitratiruptor, AB175499; Thioreductor, AB175498), other proteobacterial sequences (e.g., Alphaproteobacteria, AY180103 and D84529; Deltaproteobacteria, Y11560 and X95181; Gammaproteobacteria, AJ543327 and AY776275), and Aquifex aeolicus (AJ309733) and Thermotoga maritima (AJ401021) sequences (outgroups not shown). Accession numbers for previously acquired sequences are included in brackets. Nodal supports from 100 bootstrap replicates are indicated on branches; bootstrap proportions less than 70% are not shown. Species level OTUs are based on monophyletic clades identified by DOTUR clustering at the 99% similarity level and are indicated as follows: circles represent OTUs consisting of sequences from a single location, squares represent OTUs consisting of sequences from two locations found within the same region, and stars represent OTUs consisting of sequences from two or more sites located on different continents. For clarity, monophyletic clades (i.e., a group of sequences containing an ancestor and all of its descendants) consisting of sequences from a single location are collapsed to a single branch; some of these sequence groups consist of more than one OTU (as defined by sequences with ≥99% similarity), which is indicated by more than one symbol per branch. For collapsed clades, the number of sequences is indicated in parentheses behind each location name. Abbreviations correspond to sampling sites listed in Table 1. The four main "Thiovulgaceae" clades from Campbell et al. (3) are indicated.

Based on OTU designations, ~50% of the lineages recovered were novel and 70% of the OTUs were represented by singleton and doubleton sequence groups. Based on this high number of novel OTUs, it is highly likely that additional terrestrial epsilonproteobacterial diversity remains to be characterized. Future research should continue to explore these sites, as the goal of this initial study was not to exhaustively sample all sites using all possible primer sets. Nevertheless, by using the lineage-specific approach, we may have uncovered epsilonproteobacterial diversity that would have otherwise gone undetected by using domain-specific primers (see, e.g., reference 24).

It was expected that greater geographic separation between the sampled sites would correspond to increased genetic distance among the sequences from each site (see, e.g., references 12, 16, 17, and 24). However, this hypothesis was not supported. Only eight OTUs comprised sequences retrieved from more than one site (Fig. 1). One of these OTUs comprised sequences from more than two sites, specifically, nine locations spanning >10,000 km. This large OTU was also phylogenetically affiliated with LKC-GII and was represented by ≤1% sequence divergence (Fig. 2). A recent study of the Frasassi Caves in Italy also identified sequences belonging to this OTU (e.g., clone WM35) (19), further supporting the cosmopolitan distribution of this group (21). Interestingly, sequences affiliated with LKC-GI were not found in any of the newly investigated habitats in this study.


Figure 2
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  		FIG. 2. Average pairwise sequence distances for each of the eight OTUs containing representatives from more than one site plotted against the average geographic distance among all of the sites within the OTU. The phylogenetic distances were calculated using the PHYLIP v3.6 DNADIST module (10), which was used as DOTUR input to determine OTUs. Error bars represent ± 1 standard deviation.

It is clear that the Epsilonproteobacteria are key players in biogeochemical cycling in marine habitats (see, e.g., references 3 and 23). Although it is possible that these organisms may also be significant players in terrestrial systems, to our knowledge, this is the first study to evaluate whether or not the Epsilonproteobacteria are widely distributed in terrestrial habitats. Our findings revealed that at least the two LKC lineages have contrasting patterns of geographic distribution, even within a limited 16S rRNA framework. Because we found no correlation between phylogeny and geochemical conditions or habitat type (data not shown), the disparate spatial distributions may be due to differing biogeographic, geologic, and/or hydrostratigraphic mechanisms (see, e.g., reference 3). At this time, however, any further discussion would be highly speculative because we currently do not understand the mechanisms for microbial dispersal and colonization in the terrestrial subsurface. Moreover, because the current range of sample locations is restricted to the Northern Hemisphere, future work from both noncave or karst systems and Southern Hemisphere sites will undoubtedly provide new evidence to test hypotheses related to diversity driven by geographic isolation (see, e.g., references 5, 12, and 24).


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Nucleotide sequence accession numbers.
 
The 207 new epsilonproteobacterial sequences obtained in this study have been assigned GenBank accession numbers DQ295539 to DQ295541, DQ295543 to DQ295720, and DQ295722 to DQ295747.


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ACKNOWLEDGMENTS
 
We thank the many field assistants who helped to sample the caves and springs over the years. B. J. Campbell, D. E. Northup, C. J. Schulz, and P. C. Bennett contributed to discussions regarding the implications of the results.

This research was initiated with support from the National Science Foundation LExEn grant (EAR-0085576) to P. C. Bennett at the University of Texas at Austin. Continuing work was partly supported by the College of Basic Sciences at Louisiana State University, the Louisiana Board of Regents (NSF/LEQSF 2005-Pfund-04) to sample in the Frasassi Caves, Italy, and the National Science Foundation (DEB-0640835).


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FOOTNOTES
 
* Corresponding author. Mailing address: Louisiana State University, Department of Geology and Geophysics, E235 Howe-Russell Geoscience Complex, Baton Rouge, LA 70803. Phone: (225) 578-2469. Fax: (225) 578-2302. E-mail: aengel{at}lsu.edu Back

{triangledown} Published ahead of print on 6 June 2008. Back


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Applied and Environmental Microbiology, August 2008, p. 4973-4977, Vol. 74, No. 15
0099-2240/08/$08.00+0     doi:10.1128/AEM.02915-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.




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