Characterization and genomic analysis of chromate resistant and reducing Bacillus cereus strain SJ1

<p>Abstract</p> <p>Background</p> <p>Chromium is a toxic heavy metal, which primarily exists in two inorganic forms, Cr(VI) and Cr(III). Chromate [Cr(VI)] is carcinogenic, mutational, and teratogenic due to its strong oxidizing nature. Biotransformation of Cr(VI) to less-toxic Cr(III) by chromate-resistant and reducing bacteria has offered an ecological and economical option for chromate detoxification and bioremediation. However, knowledge of the genetic determinants for chromate resistance and reduction has been limited so far. Our main aim was to investigate chromate resistance and reduction by <it>Bacillus cereus </it>SJ1, and to further study the underlying mechanisms at the molecular level using the obtained genome sequence.</p> <p>Results</p> <p><it>Bacillus cereus </it>SJ1 isolated from chromium-contaminated wastewater of a metal electroplating factory displayed high Cr(VI) resistance with a minimal inhibitory concentration (MIC) of 30 mM when induced with Cr(VI). A complete bacterial reduction of 1 mM Cr(VI) was achieved within 57 h. By genome sequence analysis, a putative chromate transport operon, <it>chrIA</it>1, and two additional <it>chrA </it>genes encoding putative chromate transporters that likely confer chromate resistance were identified. Furthermore, we also found an azoreductase gene <it>azoR </it>and four nitroreductase genes <it>nitR </it>possibly involved in chromate reduction. Using reverse transcription PCR (RT-PCR) technology, it was shown that expression of adjacent genes <it>chrA</it>1 and <it>chrI </it>was induced in response to Cr(VI) but expression of the other two chromate transporter genes <it>chrA</it>2 and <it>chrA</it>3 was constitutive. In contrast, chromate reduction was constitutive in both phenotypic and gene expression analyses. The presence of a resolvase gene upstream of <it>chrIA</it>1, an arsenic resistance operon and a gene encoding Tn7-like transposition proteins ABBCCCD downstream of <it>chrIA</it>1 in <it>B. cereus </it>SJ1 implied the possibility of recent horizontal gene transfer.</p> <p>Conclusion</p> <p>Our results indicate that expression of the chromate transporter gene <it>chrA</it>1 was inducible by Cr(VI) and most likely regulated by the putative transcriptional regulator ChrI. The bacterial Cr(VI)-resistant level was also inducible. The presence of an adjacent arsenic resistance gene cluster nearby the <it>chrIA</it>1 suggested that strong selective pressure by chromium and arsenic could cause bacterial horizontal gene transfer. Such events may favor the survival and increase the resistance level of <it>B. cereus </it>SJ1.</p

Transcriptional and functional characterization of a putative azoreductase gene in Shewanella oneidensis MR-1

Shewanella oneidensis MR-1 is a facultatively anaerobic gamma-proteobacterium that can respire anaerobically by utilizing a wide variety of alternative electron acceptors including different soluble and insoluble metals. The MR-1 gene SO3585 is annotated as a putative azoreductase and was shown in previous functional genomics studies to be significantly upregulated at both the mRNA and protein levels in response to acute chromate exposure. Azoreductases are enzymes that catalyze the reductive cleavage of azo dyes to aromatic amines. Azo dyes pose a serious concern as xenobiotic pollutants that are released into the environment as industrial effluents from textile and dye manufacturing. SO3585 shares 32% amino acid sequence identity to the azoreductase from Bacillus cereus, and 28% sequence identity to Pseudomonas putida ChrR and Escherichia coli YieF, two soluble flavoproteins shown to exhibit chromate reductase activity. However, a Hidden Markov Model-based search of the TIGRFAM & Pfam database indicates that SO3585 most likely belongs to the NADPH-dependent FMN reductase family of proteins. Reverse transcription-PCR was used to show that so3585 is co-transcribed with two tightly clustered downstream genes, so3586 (encoding a predicted glyoxalase family protein) and so3587 (a hypothetical membrane-associated protein). Using 5\u27 Rapid Amplification of cDNA Ends analysis, the transcriptional start site of the so3585-86-87 operon was localized to an adenine residue positioned 26 bp upstream of the annotated so3585 translational start codon. Phenotype characterization demonstrated that an MR-1 strain harboring a nonpolar in-frame deletion of the so3585 gene was similar to the wild type MR-1 strain in its ability to decolorize the azo dyes Orange II and Direct Blue 15 under aerobic conditions. However, growth studies showed that the mutant was hypersensitive to different transition metals, most notably Cd(II) and Cu(II). Reduction experiments showed the mutant to be initially impaired in its chromate reduction ability, although both strains eventually showed complete reduction of chromate within a similar time period. Biochemical analysis of a purified recombinant SO3585 indicated no detectable chromate or azo dye reductase activity associated with this S. oneidensis protein. In conclusion, the data suggest that the annotation of azoreductase is not correct and that the protein, in combination with SO3586 and SO3587, is likely involved in detoxification mechanisms under metal stress