Hidden in plain sight: Discovery of sheath-forming, iron-oxidizing Zetaproteobacteria at Loihi Seamount, Hawaii, USA

Lithotrophic iron-oxidizing bacteria (FeOB) form microbial mats at focused flow or diffuse flow vents in deep-sea hydrothermal systems where Fe(II) is a dominant electron donor. These mats composed of biogenically formed Fe(III)-oxyhydroxides include twisted stalks and tubular sheaths, with sheaths typically composing a minor component of bulk mats. The micron diameter Fe(III)-oxyhydroxide-containing tubular sheaths bear a strong resemblance to sheaths formed by the freshwater FeOB, Leptothrix ochracea. We discovered that veil-like surface layers present in iron-mats at the Loihi Seamount were dominated by sheaths (40-60% of total morphotypes present) compared with deeper (> 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 rRNA gene from this top layer were dominated by Zetaproteobacteria, and lacked phylotypes related to L. ochracea. In mats with similar morphologies, terminal-restriction fragment length polymorphism (T-RFLP) data along with quantitative PCR (Q-PCR) analyses using a Zetaproteobacteria-specific primer confirmed the presence and abundance of Zetaproteobacteria. A Zetaproteobacteria fluorescence in situ hybridization (FISH) probe hybridized to ensheathed cells (4% of total cells), while a L. ochracea-specific probe and a Betaproteobacteria probe did not. Together, these results constitute the discovery of a novel group of marine sheath-forming FeOB bearing a striking morphological similarity to L. ochracea, but belonging to an entirely different class of Proteobacteria.