Community genomic and proteomic analyses of chemoautotrophic iron-oxidizing "Leptospirillum rubarum" (Group II) and "Leptospirillum ferrodiazotrophum" (Group III) bacteria in acid mine drainage biofilms

Abstract

We analyzed near-complete population (composite) genomic sequences for coexisting acidophilic iron-oxidizing Leptospirillum group II and III bacteria (phylum Nitrospirae) and an extrachromosomal plasmid from a Richmond Mine, Iron Mountain, CA, acid mine drainage biofilm. Community proteomic analysis of the genomically characterized sample and two other biofilms identified 64.6% and 44.9% of the predicted proteins of Leptospirillum groups II and III, respectively, and 20% of the predicted plasmid proteins. The bacteria share 92% 16S rRNA gene sequence identity and >60% of their genes, including integrated plasmid-like regions. The extrachromosomal plasmid carries conjugation genes with detectable sequence similarity to genes in the integrated conjugative plasmid, but only those on the extrachromosomal element were identified by proteomics. Both bacterial groups have genes for community-essential functions, including carbon fixation and biosynthesis of vitamins, fatty acids, and biopolymers (including cellulose); proteomic analyses reveal these activities. Both Leptospirillum types have multiple pathways for osmotic protection. Although both are motile, signal transduction and methyl-accepting chemotaxis proteins are more abundant in Leptospirillum group III, consistent with its distribution in gradients within biofilms. Interestingly, Leptospirillum group II uses a methyl-dependent and Leptospirillum group III a methyl-independent response pathway. Although only Leptospirillum group III can fix nitrogen, these proteins were not identified by proteomics. The abundances of core proteins are similar in all communities, but the abundance levels of unique and shared proteins of unknown function vary. Some proteins unique to one organism were highly expressed and may be key to the functional and ecological differentiation of Leptospirillum groups II and III.

FIG. 1.

Map showing sampling locations.

FIG. 2.

Phylogenetic tree based on 16S rRNA genes of Leptospirillum spp. (maximum-likelihood method). Statistically supported bootstrap values are labeled at the nodes. The scale bar represents 0.10 changes per site, or 10%. Filled squares indicate isolates, while filled circles indicate composite genomes.

FIG. 3.

Diagram of the genome of Leptospirillum group II (outer circle) and orthologs in Leptospirillum group III (inner circle, color coded by scaffold). A histogram of percent identity between orthologs is shown by black bars on the inner ring. Heat maps of protein identification values (NSAF) are given by six middle rings (gray, no identification).

FIG. 4.

Protein abundance values (NSAF) for Leptospirillum group II and III orthologs in UBA, ABfront (AB-F), and ABend (AB-E) samples. Red, overrepresented; green, underrepresented; black, median; gray, no identification. The functional categories for the most abundant proteins are in three clusters. Cluster A (only part shown) represents transcription, translation, ribosomal structure, and biogenesis (7 proteins); coenzyme transport and metabolism (6 proteins); transport and secretion (5 proteins); energy production and conversion (3 proteins); and other functions (22 proteins). Cluster B represents transport and secretion (10 proteins); translation, ribosomal structure, and biogenesis (5 proteins); posttranslational modification, protein turnover, and chaperones (4 proteins); lipid transport and metabolism (2 proteins); and other functions (25 proteins). Cluster C represents energy production and conversion (10 proteins); cell motility (10 proteins); amino acid transport and metabolism (6 proteins); transcription, translation, ribosomal structure, and biogenesis (6 proteins); and other functions (24 proteins).

FIG. 5.

Inferred abundances (NSAF) of proteins of unknown function in Leptospirillum groups II and III. (A) Orthologs; (B) proteins unique to Leptospirillum group II; (C) proteins unique to Leptospirillum group III. Red, overrepresented; green, underrepresented; black, median; gray, no identification.

FIG. 6.

Proposed CO₂ fixation pathway (rTCA) for Leptospirillum groups II and III. 1, PFOR; 2, PEP synthase; 3, PEP carboxylase (PEPC); 4, malate dehydrogenase; 5, fumarate hydratase; 6, fumarate reductase; 7, succinyl-CoA synthetase; 8. PFOR (second copy); 9, isocitrate dehydrogenase; 10, aconitate hydratase; 11, succinyl-CoA synthetase (second copy) and citrate synthase.

FIG. 7.

(A) Alignment of PEP carboxylase regions containing conserved active site residues (in red) from Maize and E. coli (47) and from Leptospirillum group II. Other, identical residues are shown in purple. (B) Predicted protein structure of PEP carboxylase in Leptospirillum group II, with active site residues shown in red.

FIG. 8.

(A) Diagram of the chemotaxis gene cluster in Leptospirillum groups II and III. Orthologs are shown in gray, and unique proteins are shown in a black pattern. MCP, methyl-accepting chemotaxis sensory transducer. Cartoons show predicted methyl-dependent (B) and methyl-independent (C) chemotaxis systems in Leptospirillum groups II and III, respectively. Adaptation chemotaxis proteins: R, CheR; B, CheB; and V, CheV.

FIG. 9.

Diagram of CRISPR/CAS loci associated with the extrachromosomal plasmid. Black bars show repeat sequences, while bars between the repeats represent spacer sequences. (While cas genes are displayed accurately, the sets of spacers are shown schematically.) A spacer at CRISPR locus 13 (extrachromosomal plasmid) targets a cas3 gene at CRISPR locus 19 (plasmid-like contig 15511). The inset displays a portion of the cas3 gene targeted by the spacer shown bold.