New group in the Leptospirillum clade: cultivation-independent community genomics, proteomics, and transcriptomics of the new species "Leptospirillum group IV UBA BS"

Abstract

Leptospirillum spp. are widespread members of acidophilic microbial communities that catalyze ferrous iron oxidation, thereby increasing sulfide mineral dissolution rates. These bacteria play important roles in environmental acidification and are harnessed for bioleaching-based metal recovery. Known members of the Leptospirillum clade of the Nitrospira phylum are Leptospirillum ferrooxidans (group I), Leptospirillum ferriphilum and "Leptospirillum rubarum" (group II), and Leptospirillum ferrodiazotrophum (group III). In the Richmond Mine acid mine drainage (AMD) system, biofilm formation is initiated by L. rubarum; L. ferrodiazotrophum appears in later developmental stages. Here we used community metagenomic data from unusual, thick floating biofilms to identify distinguishing metabolic traits in a rare and uncultivated community member, the new species "Leptospirillum group IV UBA BS." These biofilms typically also contain a variety of Archaea, Actinobacteria, and a few other Leptospirillum spp. The Leptospirillum group IV UBA BS species shares 98% 16S rRNA sequence identity and 70% average amino acid identity between orthologs with its closest relative, L. ferrodiazotrophum. The presence of nitrogen fixation and reverse tricarboxylic acid (TCA) cycle proteins suggest an autotrophic metabolism similar to that of L. ferrodiazotrophum, while hydrogenase proteins suggest anaerobic metabolism. Community transcriptomic and proteomic analyses demonstrate expression of a multicopper oxidase unique to this species, as well as hydrogenases and core metabolic genes. Results suggest that the Leptospirillum group IV UBA BS species might play important roles in carbon fixation, nitrogen fixation, hydrogen metabolism, and iron oxidation in some acidic environments.

Fig 1

Fluorescence in situ hybridization images of UBA BS biofilms. (A) Sample Aug07, showing DNA (blue; most small dots are Archaea) and general bacteria (green). (B) Sample Aug07, showing general bacteria (green) and Leptospirillum group IV UBA BS (yellow). (C) Sample Jun08, showing general bacteria (green) and L. ferrodiazotrophum (red). (D) Sample Aug07, showing general bacteria (green), L. ferrodiazotrophum (red), and Leptospirillum group II 5wayCG type (blue). (E) Sample Island 2, showing general bacteria (blue), L. ferrodiazotrophum (green/light blue). (F) Sample Island 2, showing general bacteria (blue) and Leptospirillum group IV UBA BS (purple/pink). The images in panels A and B were taken from the same field of view, as were the images in panels E and F. The white arrows indicate Leptospirillum group III cells. Bars, 1 μm.

Fig 2

(A) Percentages of uniquely mapping community genomics reads from the Nov05 sample to assembled genomes from AMD organisms. LeptoIV, Leptospirillum group IV UBA BS; LeptoIII, Leptospirillum ferrodiazotrophum; LeptoII_CG, Leptospirillum group II 5way CG type; Actino1 and Actino2, actinobacterial bins 1 and 2 (from our lab), respectively; Sulfo, Sulfobacillus bin; Fer1, Ferroplasma type I; Fer2_env, Ferroplasma type II. (B) Percent NSAF values in three community proteomics data sets (III and IV refer to L. ferrodiazotrophum [group III] and Leptospirillum group IV UBA BS protein abundances, respectively).

Fig 3

Neighbor-joining 16S rRNA phylogenetic tree of Leptospirillum spp. (1,000 bootstraps). Percent bootstrap values are shown at the nodes. The scale bar indicates 10% sequence divergence. The sequence of a Deltaproteobacterium was used as an outgroup. str., strain.

Fig 4

Multiple-sequence alignment of MCOs, showing the conserved copper center residues, T1 (∗) and T2/T3 (⧫). The alignment includes sequences from Leptospirillum group IV UBA BS (box outlined by a broken line) (lines 1), Nitrosomonas europaea (lines 2), Nitrosococcus halophilus (lines 3), Herminiimonas arsenicoxydans (lines 4), Shewanella woodyi ATCC 51908 (lines 5), Deinococcus maricopensis DMS 21211 (lines 6) and Flavobacterium frigoris PS1 (lines 7). Gaps introduced to maximize alignment are indicated by dashes.

Fig 5

Diagram of the nitrogen fixation operon in Leptospirillum group IV UBA BS, L. ferrodiazotrophum, and L. ferrooxidans. NifX-assoc, NifX-associated protein; Fdx, ferredoxin; [FeS], FeS cluster assembly protein. Gaps (brackets) in the Leptospirillum group IV UBA BS operon correspond to genes: (i) genes encoding two hypothetical (Hypo) proteins, nifS, aminotransferase gene; and (ii) nifU, nifT, genes encoding two hypothetical proteins, ferredoxin gene, nifW, and genes encoding two final nitrogen regulatory proteins P-II. aa, amino acids.

Fig 6

(A and B) Three-dimensional (3D) structure representations of the hydrogenase HydB in Leptospirillum group IV modeled on the Escherichia coli protein. (A) HydB full model; (B) higher-magnification image of the active site highlighting catalytic residues (NiFe-binding residues [red] and Fe₂-binding residues [brown]). Conserved structural regions are shown in blue-green, while less-conserved regions are shown in magenta. (C) Multiple-alignment screenshot showing catalytic residues (Cys75 and Cys78 [rose] and Glu56 [peach]) and other perfectly conserved residues around the active site (green). Alignment includes hydrogenase sequences from Leptospirillum group IV UBA BS (line 1), E. coli (lines 2 and 3), Allocromatium vinosum (line 4), Bradyrhizobium japonicum (line 5), Desulfovibrio vulgaris (line 6), Wolinella succinogenes (line 7), and Desulfovibrio baculatus (line 8). The shades of yellow and brown indicate degrees of conservation.

Fig 7

NSAF clustering in Leptospirillum group IV UBA BS compared to L. ferrodiazotrophum in three UBA BS biofilms. Cluster 1 was overrepresented by L. ferrodiazotrophum, and cluster 2 was overrepresented by Leptospirillum group IV UBA BS. Clusters 1 and 2 contained genes involved in the following functional categories (number of genes in cluster 1; number in cluster 2): energy-related genes (8; 17), carbohydrate metabolism (10; 20), nucleotide metabolism (2; 5), amino acid metabolism (10; 16), lipid and fatty acid metabolism (4; 0), vitamin and cofactor biosynthesis (5; 9), ribosomal proteins (0; 20), peptide processing (7; 4), chemotaxis and flagella (1; 9) and phosphate uptake (0; 3).