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

Abstract

Background: 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 Bacillus cereus SJ1, and to further study the underlying mechanisms at the molecular level using the obtained genome sequence.

Results: Bacillus cereus 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, chrIA1, and two additional chrA genes encoding putative chromate transporters that likely confer chromate resistance were identified. Furthermore, we also found an azoreductase gene azoR and four nitroreductase genes nitR possibly involved in chromate reduction. Using reverse transcription PCR (RT-PCR) technology, it was shown that expression of adjacent genes chrA1 and chrI was induced in response to Cr(VI) but expression of the other two chromate transporter genes chrA2 and chrA3 was constitutive. In contrast, chromate reduction was constitutive in both phenotypic and gene expression analyses. The presence of a resolvase gene upstream of chrIA1, an arsenic resistance operon and a gene encoding Tn7-like transposition proteins ABBCCCD downstream of chrIA1 in B. cereus SJ1 implied the possibility of recent horizontal gene transfer.

Conclusion: Our results indicate that expression of the chromate transporter gene chrA1 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 chrIA1 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 B. cereus SJ1.

Figure 1

Chromate reduction and growth curves of B. cereus SJ1. B. cereus SJ1 growth curves in LB medium with (■) and without (○) 1 mM K₂CrO₄. (♦), Cr(VI) reduction of B. cereus SJ1 in LB medium (pH 7.0) with 1 mM K₂CrO₄. (▲), LB medium (pH 7.0) amended with 1 mM K₂CrO₄ without bacterial inoculation as a control. Error bars represent standard deviation of triplicate samples.

Figure 2

SEM micrographs of B. cereus SJ1 cells. (a), B. cereus SJ1 cells grown in LB medium for 24 h without K₂CrO₄; (b), B. cereus SJ1 cells grown in LB medium amended with 1 mM K₂CrO₄ for 24 h. Scale bars: 1 μm.

Figure 3

Comparison of genetic determinants of chromate resistance in other bacterial strains versus B. cereus SJ1. (a) Genetic context of the chromate operon chrIA and arsenic resistance operon arsRBCDA in B. cereus SJ1. (b) Genetic context of the chromate operon chrIA1 in B. thuringiensis serovar konkukian str. 97-27. B. thuringiensis str. 97-27 [GenBank: AE017355]; B. anthracis str. Ames Ancestor [GenBank: AE017334]; B. anthracis str. Ames [GenBank: NC003997]; B. anthracis str. Sterne [GenBank: AE017225]; B. cereus E33L [GenBank: CP000001]; B. thuringiensis str. Al Hakam [GenBank: NC008600] and B. cereus ATCC 10987 [GenBank: AE017194].

Figure 4

Chromate resistance and reduction of B. cereus SJ1. Chromate reduction (A) and resistance (B) analysis of B. cereus SJ1 uninduced (◊) and induced with (■) 1 mM K₂CrO₄ for 8 h before bacterial inoculation in LB medium (pH 7.0). B. cereus SJ1 was incubated for 48 h before growth was measured for Cr resistance determination. (▲), amended with 1 mM K₂CrO₄ without bacterial inoculation as a control. Error bars represent standard deviation of triplicate samples.

Figure 5

RT-PCR analysis of putative chromate reduction genes nitR and azoR. M, 1 kb DNA ladder. r, negative control for RT, obtained using total RNA (after DNase I treatment) as the template for PCR amplification, to verify that no genomic contamination was present in the RNA extract; c, RT-PCR product using the first strand cDNA as the template; g, PCR positive control obtained using genomic DNA from B. cereus SJ1 as the template. 0, 1 and 3 after r and c represent samples uninduced and induced by 0.3 mM K₂CrO₄ for 1 h and 3 h, respectively. Lanes 1-7, nitR1 (locus_tag: BCSJ1_00500, 592 bp); Lanes 8-14, azoR (locus_tag: BCSJ1_06081, 413 bp); Lanes 15-21, nitR2 (locus_tag: BCSJ1_14230, 480 bp); Lanes 22-28, nitR3 (locus_tag: BCSJ1_17540, 546 bp); Lanes 29-35, nitR4 (locus_tag: BCSJ1_02410, 477 bp); Lanes 36-38, RT-PCR of 16 S rRNA genes. The arrow indicates a non-specific band.

Figure 6

RT-PCR analysis of chrA, chrI induction and chrI-chrA1 co-transcription. The M, r, c, g were identical to these of Figure 5. (A), RT-PCR analysis of expression of chrA's. Lanes 1-7, chromate resistance gene chrA1 (locus_tag: BCSJ1_04594, 946 bp); Lanes 8-14, chrA2 (locus_tag: BCSJ1_18833, 491 bp); Lanes 15-21, chrA3 (locus_tag: BCSJ1_18828, 354 bp). (B), RT-PCR analysis of chrI induction and chrI-chrA1 co-transcription. 5 and 15 after r and c represent samples induced by 0.3 mM K₂CrO₄ for 5 min and 15 min, respectively. Lanes 1-7, transcriptional regulator gene chrI (locus_tag: BCSJ1_04599, 604 bp); Lanes 8-14, chrI-chrA1 (1,130 bp). Lanes 15-17, RT-PCR of 16 S rRNA genes. The arrow indicates a non-specific band.