Genomic and physiological characterization of the chromate-reducing, aquifer-derived Firmicute Pelosinus sp. strain HCF1

Abstract

Pelosinus spp. are fermentative firmicutes that were recently reported to be prominent members of microbial communities at contaminated subsurface sites in multiple locations. Here we report metabolic characteristics and their putative genetic basis in Pelosinus sp. strain HCF1, an isolate that predominated anaerobic, Cr(VI)-reducing columns constructed with aquifer sediment. Strain HCF1 ferments lactate to propionate and acetate (the methylmalonyl-coenzyme A [CoA] pathway was identified in the genome), and its genome encodes two [NiFe]- and four [FeFe]-hydrogenases for H(2) cycling. The reduction of Cr(VI) and Fe(III) may be catalyzed by a flavoprotein with 42 to 51% sequence identity to both ChrR and FerB. This bacterium has unexpected capabilities and gene content associated with reduction of nitrogen oxides, including dissimilatory reduction of nitrate to ammonium (two copies of NrfH and NrfA were identified along with NarGHI) and a nitric oxide reductase (NorCB). In this strain, either H(2) or lactate can act as a sole electron donor for nitrate, Cr(VI), and Fe(III) reduction. Transcriptional studies demonstrated differential expression of hydrogenases and nitrate and nitrite reductases. Overall, the unexpected metabolic capabilities and gene content reported here broaden our perspective on what biogeochemical and ecological roles this species might play as a prominent member of microbial communities in subsurface environments.

Fig 1

Results of cell suspension assays to determine reduction of Cr(VI) by strain HCF1 under fermentative conditions with lactate (20 mM) as the electron donor. (A) Dissolved Cr(VI) concentrations as determined by the DPC assay. (B) Dissolved total Cr concentrations as determined by ICP-MS in aliquots of the same samples represented in panel A. Cell densities were approximately 2.2 × 10⁹ cells/ml. Note that the slightly lower time-zero concentrations for lactate-containing samples in panel A compared to panel B are likely due to a minor effect of lactate on the DPC assay (9).

Fig 2

Results of cell suspension assays to determine reduction of Fe(III)-NTA by strain HCF1 under fermentative conditions with lactate (20 mM) as the electron donor. Data points represent averages of duplicates, and error bars represent 1 standard deviation. Cell densities were approximately 1.3 × 10⁹ cells/ml.

Fig 3

Lactate fermentation under anaerobic growth conditions in the presence and absence of nitrate. (A) Concentrations of lactate, acetate, and propionate (left-hand axis) and nitrate and nitrite (right-hand axis). Nitrate and nitrite are shown only for nitrate-amended replicates. Data points represent averages of duplicates, and error bars represent 1 standard deviation. Black arrows indicate the sampling times for transcriptional analysis. (B) Transcript copy number (normalized to rpoB) determined by RT-qPCR for three genes, narG (Hcf1DRAFT_02301), nrfH copy 1 (Hcf1DRAFT_00451), and nrfH copy 2 (Hcf1DRAFT_02324), in biological duplicates with and without nitrate (represented in panel A). Bar heights represent averages (n = 5 or 6, including technical replicates). Relative standard deviations averaged 24% and ranged from 13 to 46% (see Table S4 in the supplemental material).

Fig 4

Maximum likelihood phylogenetic tree of Pelosinus sp. strain HCF1 and representative Firmicutes. The bar represents distances calculated as described in Materials and Methods. Numbers at internal nodes are the percentages of 100 bootstrap samples in which the organisms to the right of the node were monophyletic.

Fig 5

(A) Proposed pathway for lactate fermentation to acetate and propionate in Pelosinus sp. strain HCF1 based on genomic analysis (see the text). Abbreviations: Pta, phosphotransacetylase; Ack, acetate kinase; PFL, pyruvate formate-lyase; PFOR, pyruvate:ferredoxin/flavodoxin oxidoreductase; DH, dehydrogenase; CoA, coenzyme A. (B) Genomic organization of selected genes putatively involved in the methylmalonyl-CoA pathway in strain HCF1 and Veillonella parvula. Note that PCR finishing was used to establish that Hcf1DRAFT_02210 and Hcf1DRAFT_03695 are contiguous in the genome. LAO, lysine-arginine-ornithine.

Fig 6

Phylogenetic composition of the flow-through column microbial community from which strain HCF1 was isolated, based upon SSU rRNA gene pyrotag sequence analysis. OTU, operational taxonomic unit.