Halomonas and Marinobacter ecotypes from hydrothermal vent, subseafloor and deep-sea environments

Authors


  • Editor: Gary King

Correspondence: Jonathan Z. Kaye, Gordon and Betty Moore Foundation, 1661 Page Mill Road, Palo Alto, CA 94304, USA. Tel.: +1 650 213 3122; fax: +1 650 213 3003; e-mail: jon.kaye@moore.org

Abstract

Moderately halophilic and euryhaline bacteria are routinely found in cool to warm hydrothermal vent and nearby cold, deep-sea environments. To elucidate the diversity of these microorganisms – with the goal of determining which among them constitute ecotypes specifically associated with hydrothermal vent and subseafloor habitats – PCR primers were designed to detect natural populations of euryhaline Gammaproteobacteria belonging to the cosmopolitan genera Halomonas and Marinobacter. The distribution patterns of 16S rRNA gene sequence data revealed that Halomonas group 2A comprised a subseafloor population at Axial Seamount on the Juan de Fuca Ridge. Complementary biogeographic and physiological data suggested that other Halomonas clades include members that are cold adapted (Halomonas group 2B) or associated with massive sulfide deposits (Halomonas group 2C). Similarly, a monophyletic Marinobacter clade may represent Fe2+-oxidizing facultative chemoautotrophs based on the phylogenetic data presented here and previously reported phenotypic characterizations. The biogeographic distributions of Halomonas and Marinobacter isolates and clones reveal that these are cosmopolitan genera, commonly found in the deep sea and in hydrothermal vent settings. As such, they are good candidates for further laboratory investigations into the biogeochemical processes in these environments.

Introduction

Easily cultivated microorganisms from marine environments, such as Pseudomonas and Vibrio of the Gammaproteobacteria, are infrequently observed in bacterial phylogenies of community DNA based on the 16S rRNA gene (Rappé & Giovannoni, 2003). Less easily cultured microorganisms, including representatives of the ubiquitous SAR clades, comprise a significant proportion of 16S rRNA gene sequences in many pelagic marine habitats (Morris et al., 2002; Rappé & Giovannoni, 2003), and, along with other numerically dominant bacterial species, have been enriched and characterized physiologically using novel cultivation techniques (Rappéet al., 2002; Giovannoni & Stingl, 2007; Alain & Querellou, 2009).

The cultivation of ecologically and biogeochemically important taxa and culture-independent analyses of marine microbial community structure augment the importance and utility of each other. The combination of laboratory-determined physiologies and a map of the distribution of bacterial clades, such as the SAR groups in pelagic environments (Morris et al., 2002; Rappéet al., 2002) and the Epsilonproteobacteria in hydrothermal vent habitats (e.g. Campbell et al., 2006), reveals where and how microbial metabolisms may be impacting the transformations of organic compounds and inorganic nutrients. There are still significant caveats, however, with regard to making inferences of specific physiological attributes from 16S rRNA gene sequences and when distinguishing the genotypic potential of a microorganism from the phenotype expressed in situ.

In between the rare, historically cultivated heterotrophic microorganisms and numerically dominant bacterial taxa in both pelagic and hydrothermal vent environments lie groups such as Halomonas and Marinobacter (Kaye & Baross, 2000). These genera have been isolated frequently from enrichments on media with elevated Fe2+ and Mn2+ at 2–20 °C (Kaye & Baross, 2000; Edwards et al., 2003; Tempelton et al., 2005) and were the sole representatives obtained with quantitative enrichments using the same medium augmented with elevated salt (16% NaCl) and incubated at 20 °C (Kaye & Baross, 2000). Using quantitative enrichments, salt-tolerant microorganisms comprised 0.01 to>10% of the total microbial community in 43 hydrothermal vent and water column samples from the North and South Pacific Oceans (Kaye & Baross, 2000). Six Halomonas strains isolated from hydrothermal vent habitats were characterized as psychrotolerant, heterotrophic, euryhaline, slightly to moderately halophilic, cadmium resistant and facultatively anaerobic, consistent with a phenotype suitable for growth in hypothesized low-temperature hydrothermal fluids (diffuse flow) and subseafloor environments (Butterfield et al., 1997; Huber et al., 2003; Mehta et al., 2003; Kaye et al., 2004). Partial characterization of novel Marinobacter strains from hydrothermal vent habitats (Edwards et al., 2003) highlighted the ability of Marinobacter to oxidize Fe2+ from metal sulfide minerals and basalt at a low temperature and emphasized their role in the weathering of metal sulfide deposits. Complementary mineralogical and phylogenetic analyses revealed their numerical significance in clone libraries among extinct sulfides and metalliferous deposits at the seafloor (Rogers et al., 2003). Some of these Marinobacter strains are also closely related to the numerically significant euryhaline strains isolated at 20 °C from seawater and diffuse flow environments (Kaye & Baross, 2000; Edwards et al., 2003). These studies suggest a potentially important biogeochemical role for Marinobacter and Halomonas, and indicate that they may be quantitatively significant in certain habitats.

Our goal in the present study is to understand the biogeography and diversity of moderately halophilic and euryhaline Halomonas and Marinobacter in cool to warm hydrothermal vent and plume environments and the nearby cold deep sea in selected Juan de Fuca Ridge and associated ridge flank habitats to explore their significance, occurrence and possible contribution to transformations of organic carbon and iron. Herein, we designed taxonomically focused PCR primers to determine the strain and species diversity of these two genera at several Juan de Fuca Ridge environments and to ascertain whether certain subpopulations of these genera live in particular hydrothermal vent and deep-sea habitats. We propose that Halomonas and Marinobacter are ecologically important participants in hydrothermal vent settings, where they contribute to transformations of iron and carbon. Their ease of culturing makes them excellent candidates as model bacteria to use in laboratory experiments to further understand their ecological function in hydrothermal vent ecosystems.

Materials and methods

Sample collection

Low-temperature hydrothermal fluids and deep seawater were collected in the northeast Pacific Ocean (Fig. 1a; Table 1). Diffuse flow fluids were obtained at Axial Seamount and the Main Endeavour Field (MEF) on the Juan de Fuca Ridge using a suction sampler and either filtered on deck or in situ using a hydrothermal fluid and particle sampler mounted on the remote-operated vehicle ROPOS or the deep submergence vehicle Alvin. In some instances, fluids were filtered first through cellulose membranes (3.0-μm pore size; Millipore) and then Sterivex-GP filters (0.2-μm pore size; Millipore) and frozen in liquid nitrogen (Huber et al., 2002). The former fraction was defined as ‘particle-attached’ and subsequently analyzed. Other diffuse flow samples were filtered only using Sterivex-GP filters. Background deep seawater (without a detectable hydrothermal vent signal) was collected near Axial Seamount, in the axial valley near the MEF and near Baby Bare with Niskin bottles jointly mounted on a rosette with a conductivity–temperature–depth–transmissometry package. These deep seawater samples were filtered only through Sterivex-GP filters on deck. Finally, sections of sulfide structures measuring several meters in length were retrieved hot and fresh from the Mothra vent field on the Endeavour Segment during the Edifice Rex sulfide recovery program, subsampled by the mineralogical zone and frozen in liquid nitrogen (Schrenk et al., 2003).

Figure 1.

 (a) Map of the Juan de Fuca Ridge, associated ridge flanks, and sampling sites. (b) Based on data obtained from GenBank, locations of samples from which Halomonas or Marinobacter species were retrieved in clone libraries or isolated in culture. Species names, accession numbers and sample setting are listed in Tables S1 and S2.

Table 1.   Source of DNA extracts amplified with primers designed for the moderately halophilic, euryhaline and mesophilic Halomonadaceae and Marinobacter
RegionSampleSiteSample typeT (°C)Depth
(m)
Cell concentration
(mL−1)*
Size
fraction
(μm)
Volume
filtered
(L)
DateDive
or cast
PCR
  • *

    ± 95% confidence limit. mL−1 unless noted as g−1.

  • Letters correspond to clone library constructed (strong amplification); +, preliminary or inconsistent amplification; −, no amplification.

  • 55°C measured on a later dive during the cruise.

  • §

    CTD, Conductivity–temperature–depth package that was attached to a rosette of Niskin bottles for water sampling.

  • Zone FZ2b, 2–7 cm within a sulfide structure (Schrenk et al., 2003).

  • Hydrothermal plume not detected. Cell concentration assumed to be ∼5.5 × 104 (Mehta et al., 2003).

  • ND, not determined; NA, not applicable; MEF, Main Endeavour Field, Endeavour Segment, Juan de Fuca Ridge.

Axial SeamountSS001Marker 33 ventDiffuse flow3715249.7 ± 2.9 × 104>3.01.00August 1998ROPOS 462G
SS002Milky ventDiffuse flow515311.7 ± 0.5 × 104>3.01.25September 1998ROPOS 463+
FS004Marshmallow ventDiffuse flow100∼15205.9 ± 1.8 × 104>3.00.60September 1998ROPOS 469+
FS009Easy ventDiffuse flow10∼15201.2 ± 0.3 × 105>3.01.00September 1998ROPOS 473H
FS024Magnesia ventDiffuse flow5.0∼15205.0 ± 0.3 × 105>3.01.02July 1999ROPOS 488
FS045Cloud pit, marker N6Diffuse flow15.9∼15201.2 ± 0.2 × 105>3.01.00July 2000ROPOS 547
FS047Marker 33 ventDiffuse flow31.315241.2 ± 0.2 × 105>3.01.00July 2000ROPOS 551
Near Axial SeamountCTD003§ Deep seawater212954.9 ± 0.8 × 104>0.21.00July 1999CTDF
Mothra vent fieldG3Fe/SiFinnIron and zinc sulfide, silicaND22701.7 ± 0.2 × 108 g–1Total cellsNAJune 1998NA+
MEFMEF166Near Puffer ventDiffuse flow39∼22001.7 ± 0.3 × 105>0.21.00September 2000Alvin 3618+
Near MEFMEF131 On-axis deep seawater22350ND>0.23.00September 2000CTDE
Near Baby BareCTD005 Deep seawater225404.0 ± 0.6 × 104>0.23.00September 2002CTDD

Molecular analyses

Community DNA was extracted from filters (Huber et al., 2002) and rocks (Schrenk et al., 2003) as described previously. Direct cell counts were reported previously (Kaye & Baross, 2000; Huber et al., 2002; Mehta et al., 2003; Schrenk et al., 2003) or performed for this study according to a standard technique (Porter & Feig, 1980). PCR primers were designed to amplify Halomonadaceae and Marinobacter by aligning 46 full-length 16S rRNA gene sequences from members of these groups: 115F (5′-GAG TAA TGC ATA GGA ATC TGC C) and 1446R (5′-TAG GCT AAC CAC TTC TG) (see Appendix S1 for details). The annealing temperature for each primer was optimized using temperature-gradient PCR with genomic Halomonas DNA from three species as a template using the following combinations of primers: 115F and the universal bacterial primer 1492R (5′-GGT TAC CTT GTT ACG ACT T) (Lane, 1991); 1446R and the universal bacterial primer 8F (5′-AGA GTT TGA TCC TGG CTC AG); and 115F and 1446R. The PCR cocktail for each 20 μL reaction contained 1.5 mM MgCl2, 0.8 mM dNTPs, 0.25 μM of each primer, 1 × PCR buffer (Promega), 1 U Taq DNA polymerase (Promega) and 1 μL template. The profile had an initial denaturation at 94 °C for 5 min, followed by 28 cycles of denaturation at 94 °C for 30 s, annealing at 54–61 °C for 45 s and extension at 72 °C for 2 min, with a final 10-min extension at 72 °C. Optimal amplification occurred using an annealing temperature of 56 °C. Environmental DNA was amplified using the primers 115F and 1446R over a temperature gradient to confirm that optimal performance was still at 56 °C. Diffuse flow, sulfide rock and deep seawater samples were optimized separately for cycle number if a band was obtained (Table 1). Multiple PCR reactions were run with each sample using a cycle number that indicated that product saturation had not yet been reached (Suzuki & Giovannoni, 1996; Polz & Cavanaugh, 1998). Each product was reconditioned by running a three-cycle PCR on a 1 : 10 dilution of the initial product using the same cocktail and profile (Thompson et al., 2002). The reconditioned products were pooled and cleaned using Qiaquick PCR purification columns (Qiagen) and then further purified by running 10 μL on a 1% w/v agarose gel and using the Qiaquick Gel Extraction Kit (Qiagen). Most libraries were assembled from multiple independent cloning procedures (see Appendix S1 for further details).

Phylogenetic analysis

Sequences were confirmed as nonchimeric using the chimera check function of the RDP-II website (Cole et al., 2003; Hugenholtz & Huber, 2003), consistent with their seamless fit into a master alignment of closely related taxa. Variable stem-loops at bases 201–216 and 1135–1139 (Escherichia coli numbering) were excised to facilitate the alignment. Unique sequences were aligned in arb (Ludwig et al., 2004) against the Greengenes curated arb database (DeSantis et al., 2006). All sequences retained in the final tree were exported from arb and aligned in mega 4 (Tamura et al., 2007), followed by tree construction using neighbor joining with 1000 bootstraps. The accession numbers for clone libraries D, E, F, G and H are, respectively, AY687565–AY687590, AY687539–AY687564, AY687520–AY687538, AY687591–AY687627 and AY687475–AY687519.

Results

Site temperatures and cell counts

Diffuse flow samples ranged between 5 and 100 °C and background seawater was 2 °C (Table 1). Cell concentrations in the diffuse flow samples ranged from 1.7 × 104 cells mL−1 at Milky vent to 1.7 × 105 cells mL−1 near Puffer vent. Background seawater cell concentrations were 4.0–4.9 × 104 cells mL−1. The sulfide rock sample collected from the Finn sulfide chimney contained 1.7 × 108 cells g−1.

Primer construction and preliminary PCR amplification

Two primers were ultimately selected for use in the PCR amplification of Halomonas and Marinobacter 16S rRNA genes after testing a combination of five different primers (details in Appendix S1). The novel primers 115F (5′-GAG TAA TGC ATA GGA ATC TGC C) and 1446R (5′-TAG GCT AAC CAC TTC TG) were selected for optimization with the notion that using two novel primers would focus amplification on the desired taxa (because fewer undesired taxa matched both primers) and because the combination of these two primers provided maximal coverage of the 16S rRNA gene. Overlapping fully characterized taxa matched by both of these primers with zero mismatches included only certain Halomonas spp. With one base mismatch, selected members of the genera Acinetobacter, Flavobacterium, Marinobacter, Methylomonas, Moraxella, Pseudomonas, Thioalkalimicrobium and Vibrio, symbiont bacteria and additional Halomonas spp. also matched. Temperature-gradient PCR using each primer in concert with a universal bacterial primer and with each other (using genomic Halomonas and Marinobacter DNA as template) revealed that the optimum annealing temperature for each primer was 56 °C. The primer pair amplified a DNA fragment of approximately 1300 base-pairs length from pure-culture genomic extracts of numerous strains of Halomonas, Marinobacter and Cobetia, but not of Marinococcus (Gram-positive), Desulfurobacterium and Thermococcus (archaeon) or other strains of Gammaproteobacteria, including E. coli, Salinisphaera and Vibrio.

Amplification of environmental DNA extracts

The primers 115F and 1446R were used to amplify 16S rRNA genes from community DNA extracts from various diffuse flow, sulfide rock and deep seawater samples from 12 sites in the northeast Pacific Ocean (Fig. 1a; Table 1). Amplification of some samples did not produce a band, while others did so inconsistently. Presumably, the samples for which amplification was not successful did not contain sufficient numbers of organisms detectable with the primer pair used. Five samples were selected for cycle optimization (31–35 cycles each) and cloning, resulting in 17–45 16S rRNA gene sequences per sample of approximately 1200–1250 base-pairs length.

Clone libraries

One hundred and fifty-one 16S rRNA gene sequences were compiled, belonging exclusively to the genera Halomonas (96% of clones) and Marinobacter (4% of clones; Table 2). The Halomonas 16S rRNA gene sequences fell among only three phylogenetic branches, all within 16S rRNA group 2 of the genus (Arahal et al., 2002; de la Haba et al., 2010) with high bootstrap values (Figs 2 and 3). Over the region of the 16S rRNA gene used to construct phylogenetic trees, a small number of clones contained sequences identical to other clones or to previously reported sequences; most sequences were unique, however. Identical matches included: F1 and Marinobacter spp. 1802c.Splume1; H21, H32, H37 and Halomonas meridiana; and H28 and H. meridiana strain Slthf1. Within each of the three Halomonas (groups 2A, 2B and 2C) and one Marinobacter clade, clone sequences shared>99% sequence similarity to each other and to the isolates included in the given group (except in the case of outliers such as Halomonas magadiensis). The similarity between a given group and its closest-neighboring group was>98%. The source of isolation of both named and incompletely characterized Halomonas and Marinobacter taxa included in the phylogenetic analysis is summarized in Table S2.

Table 2.   Composition of clone libraries based on phylogenetic clades within Halomonas and Marinobacter as measured by the number of clones per clade
 Halomonas group 2AHalomonas group 2BHalomonas group 2CMarinobacter
  1. MEF, Main Endeavour Field, Endeavour Segment, Juan de Fuca Ridge.

  2. Libraries G and H derived from the>3.0 μm filter fraction of cool to warm diffuse flow hydrothermal fluids, whereas libraries D, E and F were constructed from cold, deep seawater samples (>0.2 μm filter fraction) located near the sites mentioned.

>3.0 μm fraction
 Marker 33 vent, G25660
 Easy vent, H45000
>0.2 μm fraction
 Near Baby Bare, D42500
 MEF axial valley, E20240
 Near Axial Seamount, F7046
Figure 2.

 Neighbor-joining phylogenetic tree highlighting Halomonas groups 2B and 2C (left) and Marinobacter (right). Clone sequences from this study are denoted by a letter followed by a number (e.g. “F11”). Bootstrap values >50% are indicated. Escherichia coli and Sulfurovum lithotrophicum (epsilonproteobacterium) are outgroups.

Figure 3.

 Neighbor-joining phylogenetic tree highlighting Halomonas group 2A. Clone sequence notation and other features as in Fig. 2 The lower part of the tree was removed to save space, but remains the same as in Fig. 2.

Discussion

Taxonomically focused PCR primers enabled the detection of the slightly to moderately halophilic and euryhaline genera Halomonas and Marinobacter in the cold deep sea and in cool to warm hydrothermal fluids. Using universal bacterial PCR primers, Halomonas 16S rRNA gene sequences have been identified previously in a clone library from Baby Bare deep seawater (from which library D was also created), where five of 84 clones belonged to Halomonas group 2A (Huber et al., 2006). In addition, Halomonas and Marinobacter were detected using 454 pyrosequencing of the V6 hypervariable region of the 16S rRNA gene in certain deep-sea hydrothermal vent samples (Huber et al., 2007). In that study, Halomonas comprised<1% of all sequence tags in two samples from Axial Seamount, and Marinobacter comprised 12% of the total bacterial community in one of those samples, which was taken in diffuse flow in a community of tubeworms. However, the detection of these groups across hydrothermal vent habitats has been spotty; for example, other clone libraries generated with universal bacterial PCR primers from hydrothermal vent and deep-sea samples collected atop and near Axial Seamount by Huber et al. (2003) did not produce any Halomonas or Marinobacter signals. Hence, the taxonomically focused primers applied here for Halomonas and Marinobacter appear to have increased our ability to detect these groups in hydrothermal fluids.

Although the ability to recover Marinobacter and Halomonas sequences in clone libraries that are generated from hydrothermal fluids using universal bacterial PCR primers is less than predicted by most probable number assays, a number of recent studies have recovered them without such a targeted approach from other hydrothermal vent sample types, most notably from solid mineral surfaces. For example, in one study of the East Pacific Rise (EPR), Halomonas and Marinobacter were detected on basalt and in the overlying seawater (Santelli et al., 2008), as indicated by the presence of sequences in Halomonas groups 2A and 2B and Marinobacter that begin with ‘EPR’ in Figs 2 and 3. Rogers et al. (2003) also recovered a high proportion of Marinobacter from extinct sulfide and metalliferous sediments from Juan de Fuca Ridge hydrothermal samples; Marinobacter overwhelmingly dominated some samples in that study. These observations suggest that Marinobacter– and possibly Halomonas– proliferate in biofilms associated with mineral particles and as such they may be missed in samples and studies that do not consider the particle-associated fraction of the community. This inference is supported by some of the sampling carried out as part of this study. For example, Halomonas group 2A dominated in the ‘particle-attached’ fraction of diffuse fluids (>3.0 μm size fraction; Fig. 3). Variations between particle-attached and free-living taxa from hydrothermal habitats have been observed similarly in other studies (Huber et al., 2002, 2003; Cowen, 2004).

We infer that the 16S rRNA gene sequences from Halomonas and Marinobacter detected in this study reflect an indigenous diversity and indicate clear population delineations among taxa. The topology of the phylogenetic trees (Figs 2 and 3) reveals distinct, analytically robust branches with broad, bushy tips that contain 16S rRNA gene sequences of>99% sequence identity. The tips, including Halomonas groups 2A, 2B and 2C, appear to be similar to the ‘microdiverse clusters’ that have been observed in other settings (Acinas et al., 2004). The clusters defined by Acinas et al. (2004) grouped at the level of>99% 16S rRNA gene sequence identity and were hypothesized to function as ecotypes that resulted from selective sweeps during their evolutionary history (Acinas et al., 2004; Koeppel et al., 2008). We propose that marine representatives of the genus Halomonas evolved in a similar fashion, resulting in the ecotypes described here.

Members of group 2A were detected in all the samples analyzed, but their enrichment in low-temperature vent fluids suggests growth in this niche. The maximum recorded temperature for growth within the genus Halomonas is 40–50 °C (Mata et al., 2002), although the maximum temperature for the growth for most members of the Halomonas 16S rRNA group 2 is 30–40 °C, including H. meridiana, Halomonas axialensis, Halomonas aquamarina, Halomonas variabilis, Halomonas neptunia, Halomonas glaciei and Halomonas sulfidaeris (Mata et al., 2002; Reddy et al., 2003; Kaye et al., 2004). However, the maximum temperature for growth may increase by several degrees under hydrostatic pressures equivalent to mid-ocean-ridge depths (Yayanos & DeLong, 1987; Takai et al., 2008). While the marker 33 hydrothermal vent may be too warm (37–55 °C) to allow the growth of certain Halomonas spp., members of groups 2A, 2B and 2C may be growing in cooler nearby subseafloor habitats and flushed into the water column during the vigorous hydrothermal flow associated with the vent (Huber et al., 2002). At 10 °C, Easy vent is a more hospitable environment for the growth of Halomonas, and indeed, each of the 45 clones from library H collected there belonged to group 2A.

The cultured members of group 2A, H. aquamarina, H. axialensis and H. meridiana, were, respectively, isolated from an unreported location and depth off the coast of California, a 27 °C diffuse flow vent on Axial Seamount in 1998 and from an Antarctic hypersaline lake (ZoBell & Upham, 1944; James et al., 1990; Kaye et al., 2004). Characterized H. meridiana strains Slthf1 and Eplume2 were, respectively, cultured from a 9 °C diffuse flow site on the Southern EPR and from a 2 °C hydrothermal plume above the MEF on the Endeavour Segment of the Juan de Fuca Ridge (Kaye et al., 2004). Hydrostatic-pressure–temperature growth experiments suggest that H. axialensis and H. meridiana strain Slthf1 can thrive in cool to warm pressurized habitats (13–30 °C,>25 MPa), but grow only very slowly or not at all under cold (2 °C) deep-sea conditions (Kaye & Baross, 2004). This observation serves as another indicator of the hypothesized endemism of this group in cool to warm subseafloor habitats.

Ninety-six percent of the clones from library D, a deep seawater sample from the eastern flank of the Juan de Fuca Ridge located 4.6 km from the basaltic outcrop at Baby Bare, fell into the H. glacieiH. neptuniaH. variabilis group 2B. This clade was also moderately well represented in the marker 33 library, but not detected in any other sample. Halomonas neptunia and H. glaciei were, respectively, isolated from a 2 °C hydrothermal plume above the MEF and from Antarctic fast ice (Reddy et al., 2003; Kaye et al., 2004). Halomonas variabilis, more distantly related to the other members of this group, was isolated from the Great Salt Lake (Fendrich, 1988). The remaining isolates in group 2B were obtained from a variety of cold environments: a 2 °C deep-sea hydrothermal plume, and Arctic and Antarctic seawater. One strain, isolated from 194 °C hydrothermal fluid from a vent on the Trans-Atlantic Geotraverse (TAG) hydrothermal mound on the Mid-Atlantic Ridge, very likely did not derive from that high-temperature environment (Okamoto et al., 2001). The clones in group 2B may represent a cold-adapted, deep-sea Halomonas lineage, given the predominance of this group in a cold deep seawater sample and given that many cultured strains were isolated from permanently cold environments.

Library E, which derived from a deep seawater sample procured in 2000 from within the axial valley of the MEF without a detectable hydrothermal plume signal, was comprised almost exclusively of clones belonging to group 2C with H. sulfidaeris as its only characterized member. Halomonas sulfidaeris was isolated from a sample of sulfide rock collected from the MEF in 1995 (Kaye et al., 2004). An incompletely characterized isolate in this group was isolated from sediment in the Sea of Okhotsk. As with libraries D and H, the low clone diversity implicates a resident population that is locally enriched. In this case, the enriched population may be linked directly to the widespread massive metal sulfide deposits for which the MEF and the encompassing 15 km stretch of the Endeavour Segment are known (Kelley et al., 2001).

The deep seawater sample taken near Axial Seamount was the only sample from which Marinobacter 16S rRNA gene sequences were amplified (library F; Fig. 2). Like Halomonas, the genus Marinobacter is nearly ubiquitous, contains moderately halophilic and euryhaline species and includes strains that have been isolated frequently from hydrothermal vent environments (Kaye & Baross, 2000; Edwards et al., 2003). Partial characterization of Marinobacter spp. from samples of oxidized sulfide rock and metalliferous sediments collected from the Endeavour Segment and Middle Valley revealed that some species are psychrophilic Fe2+ oxidizers, a phenotype consistent with success in cold, oxic hydrothermal vent environments (Edwards et al., 2003). Moreover, these and other Fe2+-oxidizing bacteria likely play a significant role in seafloor weathering of hydrothermal deposits (Edwards, 2004). The Axial Seamount samples without detectable Marinobacter sequences contained higher concentrations of hydrogen sulfide, suggesting a possible antagonistic effect on this group.

It can be difficult to infer specific phenotypic attributes from phylogeny, especially for the genus Halomonas, within which 16S rRNA gene sequences of different species are typically>97% identical (Arahal et al., 2002; de la Haba et al., 2010). Although it is not yet possible to demonstrate the precise phenotype of the bacteria represented in the clone libraries, the results suggest that certain subpopulations of Halomonas dominate over other Halomonas groups in different hydrothermal vent, subseafloor and deep-sea habitats. In addition, only members of Halomonas 16S rRNA group 2 were shown to be present in deep-sea and hydrothermal vent ecosystems, bolstering a broad ecotype division within the genus between marine isolates and strains collected from hypersaline habitats, saline soils and food-curing brines, although this distinction belies several exceptions. The groups analyzed here appear to comprise functional intraspecific ecotypes with>99% sequence identity in the 16S rRNA gene among members of a given clade. Further physiological experiments are required to decipher the phenotypic diversity among the phylogenetically closely related Halomonas and Marinobacter groups. We assume that all of the clones represent euryhaline species, but the exact temperature ranges for growth (including possible psychrophily), especially under in situ hydrostatic pressures, and possible mixotrophic or autotrophic growth on Fe2+, have yet to be determined. It would be instructive to attempt to isolate psychrophilic Halomonas and Marinobacter spp. using cold temperatures, elevated salinity and in situ hydrostatic pressure during enrichment.

Recent work has revealed that multiple species of Halomonas and Marinobacter produce novel, highly efficient Fe3+ siderophores (Martinez et al., 2000; Hickford et al., 2004; Homann et al., 2009a, b). The presence of mechanisms to specifically obtain metals emanating from vents may help explain the success of these two genera in iron-rich environments. Interestingly, at least one of the siderophore-producing Halomonas species is also a Mn2+ oxidizer, indicating that this species may be involved in both the iron and the manganese cycles in hydrothermal vent environments (Homann et al., 2009b). While these are not the only microorganisms important in iron cycling, their presence in hydrothermal fields where iron concentrations are high suggests an active role in the iron cycle for Halomonas and Marinobacter.

To determine the global distribution of Halomonas and Marinobacter species, we mapped the origins of sequences deposited in GenBank (Fig. 1b). Clones and isolates have been retrieved from every ocean, and many of the sampling sites were either influenced by hydrothermal processes or highly saline (Table S2). The occurrence of these lineages is extremely widespread in hydrothermal settings, including the Juan de Fuca Ridge, EPR, Mid-Atlantic Ridge, Kumano Nada mud volcano, Nankai Trough, Murray Seamount, Loihi Seamount, Suiyo Seamount and North Fiji Basin.

Conclusions

The application of taxonomically focused PCR primers designed to detect natural populations of Halomonas and Marinobacter revealed niche partitioning among Halomonas groups 2A, 2B and 2C. Complementary phylogenetic and physiological data suggest that Halomonas group 2A represents a subseafloor clade, whereas Halomonas groups 2B and 2C represent cold-adapted lineages, the latter possibly associated with metal sulfide deposits. A monophyletic Marinobacter clade detected may represent Fe2+-oxidizing facultative chemoautotrophs. Both Halomonas and Marinobacter are detected in cultures and clone libraries globally, revealing that they are cosmopolitan genera with localized enrichments in deep-sea, hydrothermal vent and subseafloor settings. Their widespread distribution and ease of culturing suggest that Halomonas and Marinobacter are excellent candidates as model hydrothermal vent microorganisms for laboratory-based studies of deep-sea hydrothermal vent metal cycling.

Acknowledgements

We thank Joe Hoover, Sheryl Bolton, Robin Froisland, Julie Huber, Mausmi Mehta and Matt Schrenk for laboratory assistance, samples and data, and are grateful to chief scientists John Delaney, Bob Embley, Paul Johnson, Debbie Kelley and Meg Tivey for cruise opportunities. We also thank Jody Deming, Julie Huber and Byron Crump for discussions and manuscript suggestions. Support provided by Washington Sea Grant (NA 76RG0119) and the NASA Astrobiology Institute at the Carnegie Geophysical Laboratory, Washington, DC to J.A.B. The authors declare that they have no competing interests.

Authors' contributions

J.Z.K. helped to conceive the study, participated in the sequencing of the 16S rRNA genes, performed the molecular alignments and analyses and drafted the manuscript. J.B.S. helped with phylogenetic analyses and biogeography and helped draft the discussion. K.J.E. helped with editing and manuscript conception. J.A.B. helped to conceive the study and edited the manuscript. All authors read and approved the final manuscript.

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