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.

FIG. 1.

Comparison of the nif operon identified in the AMD biofilm community genomic data and the nif operon of L. ferrooxidans L3.2 (22). Dashed lines indicate missing sequence in L. ferrooxidans. CHP, conserved hypothetical protein.

FIG. 2.

Phylogenetic tree inferred from NifH protein identified in the AMD biofilm community (boldface) and reference isolate microbial genomes. Branch points supported (bootstrap values, >74%) by all inference methods used are indicated by solid circles, and those supported by most inference methods are indicated by open circles. Branch points without circles were not resolved (bootstrap values, <75%) as specific groups in different analyses. The bar represents 10% estimated sequence divergence. Outgroups are Chlorobium tepidum TLS (group II) and Methanothermobacter thermoautotrophicum (group III).

FIG. 3.

FISH images of the isolate. (A) Cells hybridized with Cy5-labeled EUB338mix; (B) FITC-labeled LF655 (Leptospirillum group I, II, and III); (C) Cy3-labeled LF1252 (only Leptospirillum group III); (D) combination of all three probes.

FIG. 4.

Maximum-likelihood dendrogram of Leptospirillum genera based on comparative analyses of 16S rRNA gene data. Group designations are bracketed on the right. Branch points supported (bootstrap values, >74%) by all inference methods used are indicated by solid circles, and those supported by most inference methods are indicated by open circles. Branch points without circles were not resolved (bootstrap values, <75%) as specific groups in different analyses. Shown in blue are the 16S rRNA gene sequence for each of the genomes recovered in the community genomics analysis (34) and in red is the 16S rRNA sequence of the Leptospirillum group III isolate. The outgroup used was Nitrospira marina. The bar represents 10% estimated sequence divergence.