RESEARCH ARTICLE Hidden in plain sight: discovery of sheath-forming, iron-oxidizing Zetaproteobacteria at Loihi Seamount, Hawaii, USA Emily J. Fleming1, Richard E. Davis2, Sean M. McAllister3,, Clara S. Chan4, Craig L. Moyer3, Bradley M. Tebo2 & David Emerson1 1Bigelow Laboratory for Ocean Sciences, East Boothbay, ME, USA; 2Department of Environmental and Biomolecular Systems, Oregon Health and Science University, Portland, OR, USA; 3Department of Biology, Western Washington University, Bellingham, WA, USA and 4Department of Geological Sciences, University of Delaware, Newark, DE, USA Correspondence: David Emerson, Bigelow Abstract Laboratory for Ocean Sciences, PO Box 380, East Boothbay, ME 04544, USA. Tel.: +1 207 Lithotrophic iron-oxidizing bacteria (FeOB) form microbial mats at focused 315 2567; fax: +1 207 315 2329; flow or diffuse flow vents in deep-sea hydrothermal systems where Fe(II) is a e-mail: [email protected] dominant electron donor. These mats composed of biogenically formed Fe(III)-oxyhydroxides include twisted stalks and tubular sheaths, with sheaths Present address: Sean M. McAllister, typically composing a minor component of bulk mats. The micron diameter Department of Geological Sciences, Fe(III)-oxyhydroxide-containing tubular sheaths bear a strong resemblance to University of Delaware, Newark, DE, USA sheaths formed by the freshwater FeOB, Leptothrix ochracea. We discovered that Received 20 December 2012; revised 28 veil-like surface layers present in iron-mats at the Loihi Seamount were domi- – February 2013; accepted 28 February 2013. nated by sheaths (40 60% of total morphotypes present) compared with deeper Final version published online 15 April 2013. (> 1 cm) mat samples (0–16% sheath). By light microscopy, these sheaths appeared nearly identical to those of L. ochracea. Clone libraries of the SSU DOI: 10.1111/1574-6941.12104 rRNA gene from this top layer were dominated by Zetaproteobacteria, and lacked phylotypes related to L. ochracea. In mats with similar morphologies, ter- € Editor: Max Haggblom minal-restriction fragment length polymorphism (T-RFLP) data along with quantitative PCR (Q-PCR) analyses using a Zetaproteobacteria-specific primer Keywords Leptothrix ochracea; iron-oxidation; confirmed the presence and abundance of Zetaproteobacteria.AZetaproteobacte- Zetaproteobacteria FISH probes; Loihi ria fluorescence in situ hybridization (FISH) probe hybridized to ensheathed Seamount; convergent evolution; marine cells (4% of total cells), while a L. ochracea-specific probe and a Betaproteobacte- iron-mats; deep-sea sampling. ria probe did not. Together, these results constitute the discovery of a novel group of marine sheath-forming FeOB bearing a striking morphological similar- ity to L. ochracea, but belonging to an entirely different class of Proteobacteria. [Fe(II)] compounds often give rise to prolific and diverse Introduction microbial mat communities of sulfur-oxidizing or iron- Microbial mats are commonly found in habitats charac- oxidizing bacteria (FeOB), respectively (Ruby et al., 1981; terized by steep gradients of redox-active substrates. They Emerson & Moyer, 2010; Wankel et al., 2011). are highly productive and tend to be stratified into differ- Loihi Seamount is an active undersea volcano located ent populations of microorganisms adapted to the spe- at the eastern edge of the Hawaiian hotspot (Garcia et al., cific, yet dynamic physicochemical conditions that exist 2006). Near the Loihi summit (960 meters below sea within the mat (Teske & Stahl, 2002). Deep-sea hydro- level) are a variety of hydrothermal vent sites of low to thermal vents, both at spreading ridge axes and sea- intermediate temperature (4–60 °C) with both focused mounts, offer a case in point by supporting microbial and diffuse venting. The vent fluids at Loihi Seamount MICROBIOLOGY ECOLOGY MICROBIOLOGY mat communities that take advantage of gradients of are highly enriched in Fe(II) as well as CO2 and are lar- inorganic, redox-active compounds in the vent fluids and gely devoid of sulfide (Glazer & Rouxel, 2009). The pH oxygen in the surrounding ocean. Hydrothermal vent of the hydrothermal source fluids can be around 5.5, and fluids highly enriched in reduced sulfur- or ferrous iron plume waters can as low as 7.2 (Garcia et al., 2006). ª 2013 Federation of European Microbiological Societies FEMS Microbiol Ecol 85 (2013) 116–127 Published by John Wiley & Sons Ltd. All rights reserved Sheath-forming Zetaproteobacteria 117 Microbial mats at these vents derive their primary energy associate them with any specific vent site or condition. Fur- source from the oxidation of Fe(II), making Loihi a thermore, there are few reported observations of such model system for studying communities of marine FeOB. structures in other marine iron-mats (Hodges & Olson, The first convincing evidence for deep-sea communities 2009; Li et al., 2012). of FeOB was found at Loihi Seamount (Karl et al., 1989), In freshwater iron-mat communities, L. ochracea is eas- and it was subsequently shown that FeOB were abundant ily recognizable due to its copious production of hollow, and formed the mat-like communities responsible for tubular Fe(III)-oxyhydroxide-encrusted sheaths mostly most of the deposition of biogenic Fe(III)-oxyhydroxides devoid of cells; even in the most actively growing mats, (Emerson & Moyer, 2002). The Zetaproteobacteria,a only 10% of the sheaths contain cells (Emerson & Revsb- novel class of Proteobacteria, were discovered at Loihi ech, 1994; Fleming et al., 2011). Only recently, cultivation- Seamount and are represented by the novel obligate independent analysis has shown that L. ochracea is a mem- chemolithotroph Mariprofundus ferrooxydans (Emerson ber of the Betaproteobacteria, related to other sheath-form- et al., 2007). The Zetaproteobacteria have subsequently ing heterotrophic Leptothrix spp., and Sphaerotilus spp. been shown to be an abundant group at other deep-sea (Fleming et al., 2011). Despite L. ochracea’s relationship to and shallow marine hydrothermal systems (Emerson & these heterotrophic sheath-formers, its physiology remains Moyer, 2010). unclear; it cannot be enriched on media that support One remarkable trait of many FeOB that grow at growth of Sphaerotilus spp. or other Leptothrix spp., and, circumneutral pH is their ability to produce biomineral- unlike these organisms, it has an absolute requirement for ized extracellular structures such as stalks and sheaths Fe(II). This has led to speculation that it may be a litho- (Emerson et al., 2010). Mariprofundus ferrooxydans and troph that utilizes Fe(II) as an energy source (Emerson related marine strains produce a twisted stalk containing et al., 2010). an organic component that controls the deposition of the The role of these Leptothrix-like sheath-forming bacteria Fe(III) metabolic byproducts of cell growth (Ghiorse, in marine systems is even less understood. Cultivation- 1984; Chan et al., 2011). These stalks bear a striking simi- independent studies of marine iron-mat communities have larity to the structures produced by the freshwater FeOB not reported the presence of SSU rRNA gene sequences Gallionella ferruginea (Comolli et al., 2011). Until related to L. ochracea, or other iron-oxidizing Betaproteo- recently, this led to the assumption that the stalk-like bacteria. However, because the sheaths are infrequently structures that are commonly associated with flocculent observed, and the cells are even rarer, it is possible they Fe(III)-oxyhydroxide deposits at iron-rich deep-sea vents have simply gone undetected. In a recent expedition to the were formed by Gallionella. However, phylogenetic analy- iron-mats at Loihi Seamount, we were able to collect a thin sis has shown that Mariprofundus and Gallionella are dis- veil-like layer on the surface of active iron-mats, which was tantly related (Emerson et al., 2007) and share few highly highly enriched in sheath structures. Our goal was to deter- homologous functional genes (Singer et al., 2011). Fur- mine whether the sheath producing bacteria found at Loihi thermore, a number of cultivation-independent studies of were closely related to sheath producing L. ochracea found marine iron-mats (Davis & Moyer, 2008; Hodges & in freshwater habitats. Olson, 2009; Kato et al., 2009; Rassa et al., 2009; Forget et al., 2010; Handley et al., 2010; McAllister et al., 2011; Materials and Methods Meyer-Dombard et al., 2012) found evidence for Zetapro- teobacteria related to M. ferrooxydans, but not for bacteria Site/sample collection related to Gallionella species. Thus, it appears that the morphological, ecological, and physiological commonali- The description of microbial mat ecosystems at Loihi ties between Mariprofundus and Gallionella are not due to Seamount, including ecological and biomineralogical anal- a close genetic relatedness but may be the result of yses, has been previously published (Glazer & Rouxel, convergent evolution. 2009; Rassa et al., 2009; Emerson & Moyer, 2010; Edwards Another FeOB morphotype observed in Loihi Seamount et al., 2011; Toner et al., 2012). For this study, we focused iron-mats is filamentous cells encased in a robust sheath on collecting thin, cream-colored veil-like mats (see Fig. 1) composed primarily of Fe(III)-oxyhydroxides (Emerson & using a custom-designed biomat syringe (BS) sampler that Moyer, 2002, 2010). Morphologically, these structures bear could be operated with the manipulator arms of the remo- striking resemblance to a commonly observed freshwater tely operated vehicle (ROV),