title: Genome-directed isolation of the key nitrogen fixer Leptospirillum ferrodiazotrophum sp. nov. from an acidophilic microbial community.
abstract: Analysis of assembled random shotgun sequence data from a low-diversity, subsurface acid mine drainage (AMD) biofilm revealed a single nif operon. This was found on a genome fragment belonging to a member of Leptospirillum group III, a lineage in the Nitrospirae phylum with no cultivated representatives. Based on the prediction that this organism is solely responsible for nitrogen fixation in the community, we pursued a selective isolation strategy to obtain the organism in pure culture. An AMD biofilm sample naturally abundant in Leptospirillum group III cells was homogenized, filtered, and serially diluted into a nitrogen-free liquid medium. The resulting culture in the terminal dilution grew autotrophically to a maximum cell density of approximately 10(6) cells/ml, oxidizing ferrous iron as the sole energy source. 16S rRNA-internal transcribed spacer region clone library analysis confirmed that the isolate is a member of Leptospirillum group III and that the culture is axenic. We propose the name Leptospirillum ferrodiazotrophum sp. nov. for this iron-oxidizing, free-living diazotroph. This study highlights how environmental sequence data can provide insights for culturing previously uncultured microorganisms.
authors.affiliation: Department of Environmental Science, Policy, and Management, 151 Hilgard Hall, University of California, Berkeley, CA 94720-3111, USA. gtyson@nature.berkeley.edu
authors.forename: Gene W
authors.initials: GW
authors.lastname: Tyson
authors.forename: Ian
authors.initials: I
authors.lastname: Lo
authors.forename: Brett J
authors.initials: BJ
authors.lastname: Baker
authors.forename: Eric E
authors.initials: EE
authors.lastname: Allen
authors.forename: Philip
authors.initials: P
authors.lastname: Hugenholtz
authors.forename: Jillian F
authors.initials: JF
authors.lastname: Banfield
mesh_terms: D001419:Bacteria / Q000145:classification* / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D001426:Bacterial Proteins / Q000235:genetics; D018441:Biofilms / Q000254:growth & development*; D003470:Culture Media; D004269:DNA, Bacterial / Q000032:analysis; D021903:DNA, Ribosomal Spacer / Q000032:analysis; D017753:Ecosystem; D016680:Genome, Bacterial; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000378:metabolism*; D008906:Mining; D009586:Nitrogen Fixation / Q000235:genetics*; D009876:Operon; D010802:Phylogeny; D012336:RNA, Ribosomal, 16S / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D003470:Culture Media; D004269:DNA, Bacterial; D021903:DNA, Ribosomal Spacer; D012336:RNA, Ribosomal, 16S; D007501:Iron