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Genome-enabled studies of anaerobic, nitrate-dependent iron oxidation in the chemolithoautotrophic bacterium Thiobacillus denitrificans.

ABSTRACT: Thiobacillus denitrificans is a chemolithoautotrophic bacterium capable of anaerobic, nitrate-dependent U(IV) and Fe(II) oxidation, both of which can strongly influence the long-term efficacy of in situ reductive immobilization of uranium in contaminated aquifers. We previously identified two c-type cytochromes involved in nitrate-dependent U(IV) oxidation in T. denitrificans and hypothesized that c-type cytochromes would also catalyze Fe(II) oxidation, as they have been found to play this role in anaerobic phototrophic Fe(II)-oxidizing bacteria. Here we report on efforts to identify genes associated with nitrate-dependent Fe(II) oxidation, namely (a) whole-genome transcriptional studies [using FeCO3, Fe(2) (+), and U(IV) oxides as electron donors under denitrifying conditions], (b) Fe(II) oxidation assays performed with knockout mutants targeting primarily highly expressed or upregulated c-type cytochromes, and (c) random transposon-mutagenesis studies with screening for Fe(II) oxidation. Assays of mutants for 26 target genes, most of which were c-type cytochromes, indicated that none of the mutants tested were significantly defective in nitrate-dependent Fe(II) oxidation. The non-defective mutants included the c 1-cytochrome subunit of the cytochrome bc 1 complex (complex III), which has relevance to a previously proposed role for this complex in nitrate-dependent Fe(II) oxidation and to current concepts of reverse electron transfer. A transposon mutant with a disrupted gene associated with NADH:ubiquinone oxidoreductase (complex I) was ~35% defective relative to the wild-type strain; this strain was similarly defective in nitrate reduction with thiosulfate as the electron donor. Overall, our results indicate that nitrate-dependent Fe(II) oxidation in T. denitrificans is not catalyzed by the same c-type cytochromes involved in U(IV) oxidation, nor have other c-type cytochromes yet been implicated in the process.

SUBMITTER: Beller HR

PROVIDER: S-EPMC3753534 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Genome-enabled studies of anaerobic, nitrate-dependent iron oxidation in the chemolithoautotrophic bacterium Thiobacillus denitrificans.

Beller Harry R HR Zhou Peng P Legler Tina C TC Chakicherla Anu A Kane Staci S Letain Tracy E TE A O'Day Peggy P

Frontiers in microbiology 20130827

Thiobacillus denitrificans is a chemolithoautotrophic bacterium capable of anaerobic, nitrate-dependent U(IV) and Fe(II) oxidation, both of which can strongly influence the long-term efficacy of in situ reductive immobilization of uranium in contaminated aquifers. We previously identified two c-type cytochromes involved in nitrate-dependent U(IV) oxidation in T. denitrificans and hypothesized that c-type cytochromes would also catalyze Fe(II) oxidation, as they have been found to play this role ...[more]

PMID: 24065960

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Project description:Beller, H. R., T. E. Letain, A. Chakicherla, S. R. Kane, T. C. Legler, and M. A. Coleman. 2006. Whole-genome transcriptional analysis of chemolithoautotrophic thiosulfate oxidation by Thiobacillus denitrificans under aerobic vs. denitrifying conditions. Journal of Bacteriology 188:7005-7015. Thiobacillus denitrificans is one of the few known obligate chemolithoautotrophic bacteria capable of energetically coupling thiosulfate oxidation to denitrification as well as aerobic respiration. As very little is known about the differential expression of genes associated with key chemolithoautotrophic functions (such as sulfur-compound oxidation and CO2 fixation) under aerobic versus denitrifying conditions, we conducted whole-genome, cDNA microarray studies to explore this topic systematically. The microarrays identified 277 genes (approximately ten percent of the genome) as differentially expressed using Robust Multi-array Average statistical analysis and a 2-fold cutoff. Genes upregulated (ca. 6- to 150-fold) under aerobic conditions included a cluster of genes associated with iron acquisition (e.g., siderophore-related genes), a cluster of cytochrome cbb3 oxidase genes, cbbL and cbbS (encoding the large and small subunits of form I ribulose 1,5-bisphosphate carboxylase/oxygenase, or RubisCO), and multiple molecular chaperone genes. Genes upregulated (ca. 4- to 95-fold) under denitrifying conditions included nar, nir, and nor genes (associated respectively with nitrate reductase, nitrite reductase, and nitric oxide reductase, which catalyze successive steps of denitrification), cbbM (encoding form II RubisCO), and genes involved with sulfur-compound oxidation (including two physically separated but highly similar copies of sulfide:quinone oxidoreductase and of dsrC, associated with dissimilatory sulfite reductase). Among genes associated with denitrification, relative expression levels (i.e., degree of upregulation with nitrate) tended to decrease in the order nar > nir > nor > nos. Reverse transcription, quantitative PCR analysis was used to validate these trends. Keywords: bacterial metabolism

Project description:Microbial nitrate-dependent Fe(II) oxidation is known to contribute to iron biogeochemical cycling; however, the microorganisms responsible are virtually unknown. In an effort to elucidate this microbial metabolic process in the context of an environmental system, a 14-cm sediment core was collected from a freshwater lake and geochemically characterized concurrently with the enumeration of the nitrate-dependent Fe(II)-oxidizing microbial community and subsequent isolation of a nitrate-dependent Fe(II)-oxidizing microorganism. Throughout the sediment core, ambient concentrations of Fe(II) and nitrate were observed to coexist. Concomitant most probable number enumeration revealed the presence of an abundant nitrate-dependent Fe(II)-oxidizing microbial community (2.4 x 10(3) to 1.5 x 10(4) cells g(-1) wet sediment) from which a novel anaerobic, lithoautotrophic, Fe(II)-oxidizing bacterium, strain 2002, was isolated. Analysis of the complete 16S rRNA gene sequence revealed that strain 2002 was a member of the beta subclass of the proteobacteria with 94.8% similarity to Chromobacterium violaceum, a bacterium not previously recognized for the ability to oxidize nitrate-dependent Fe(II). Under nongrowth conditions, both strain 2002 and C. violaceum incompletely reduced nitrate to nitrite with Fe(II) as the electron donor, while under growth conditions nitrate was reduced to gaseous end products (N2 and N2O). Lithoautotrophic metabolism under nitrate-dependent Fe(II)-oxidizing conditions was verified by the requirement of CO2 for growth as well as the assimilation of 14C-labeled CO2 into biomass. The isolation of strain 2002 represents the first example of an anaerobic, mesophilic, neutrophilic Fe(II)-oxidizing lithoautotroph isolated from freshwater samples. Our studies further demonstrate the abundance of nitrate-dependent Fe(II) oxidizers in freshwater lake sediments and provide further evidence for the potential of microbially mediated Fe(II) oxidation in anoxic environments.

Dataset Information

Genome-enabled studies of anaerobic, nitrate-dependent iron oxidation in the chemolithoautotrophic bacterium Thiobacillus denitrificans.

Publications

Genome-enabled studies of anaerobic, nitrate-dependent iron oxidation in the chemolithoautotrophic bacterium Thiobacillus denitrificans.

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