title: What's new is old: resolving the identity of Leptothrix ochracea using single cell genomics, pyrosequencing and FISH.
abstract: Leptothrix ochracea is a common inhabitant of freshwater iron seeps and iron-rich wetlands. Its defining characteristic is copious production of extracellular sheaths encrusted with iron oxyhydroxides. Surprisingly, over 90% of these sheaths are empty, hence, what appears to be an abundant population of iron-oxidizing bacteria, consists of relatively few cells. Because L. ochracea has proven difficult to cultivate, its identification is based solely on habitat preference and morphology. We utilized cultivation-independent techniques to resolve this long-standing enigma. By selecting the actively growing edge of a Leptothrix-containing iron mat, a conventional SSU rRNA gene clone library was obtained that had 29 clones (42% of the total library) related to the Leptothrix/Sphaerotilus group (≤96% identical to cultured representatives). A pyrotagged library of the V4 hypervariable region constructed from the bulk mat showed that 7.2% of the total sequences also belonged to the Leptothrix/Sphaerotilus group. Sorting of individual L. ochracea sheaths, followed by whole genome amplification (WGA) and PCR identified a SSU rRNA sequence that clustered closely with the putative Leptothrix clones and pyrotags. Using these data, a fluorescence in-situ hybridization (FISH) probe, Lepto175, was designed that bound to ensheathed cells. Quantitative use of this probe demonstrated that up to 35% of microbial cells in an actively accreting iron mat were L. ochracea. The SSU rRNA gene of L. ochracea shares 96% homology with its closet cultivated relative, L. cholodnii, This establishes that L. ochracea is indeed related to this group of morphologically similar, filamentous, sheathed microorganisms.
authors: Bigelow Laboratory for Ocean Sciences, West Boothbay Harbor, Maine, United States of America. efleming@bigelow.org
authors: Emily J
authors: EJ
authors: Fleming
authors: Amy E
authors: AE
authors: Langdon
authors: Manuel
authors: M
authors: Martinez-Garcia
authors: Ramunas
authors: R
authors: Stepanauskas
authors: Nicole J
authors: NJ
authors: Poulton
authors: E Dashiell P
authors: ED
authors: Masland
authors: David
authors: D
authors: Emerson
mesh_terms: D044822:Biodiversity; D002999:Clone Cells; D023281:Genomics / Q000379:methods*; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D007501:Iron / Q000378:metabolism; D042743:Leptothrix / Q000166:cytology* / Q000235:genetics*; D008969:Molecular Sequence Data; D010802:Phylogeny; D012335:RNA, Ribosomal / Q000235:genetics; D054679:Ribosome Subunits, Small / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000379:methods*; D013696:Temperature
chemical_list: D012335:RNA, Ribosomal; D007501:Iron