| The wide use of chromium (Cr) and its compound in electroplating, leather tanning, textile and preservation industries with subsequent sewage disposal causes severe contamination of global soil-water systems. 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, just a few highly efficient chromate-reducing bacteria has been found, knowledge of the genetic determinants for chromate resistance and reduction has been limited so far.Two chromate resistant and reducing strains, Bacillus cereus SJ1 and Lysinibacillus fusiformis ZC1, were isolated from chromium-contaminated wastewater of a metal electroplating factory, the characterization of chromate reduction was evaluated. The minimal inhibitory concentration (MIC) of K2CrO4 for SJ1 and ZC1 were 30 mM and 60 mM, respectively. The chromate resistance of SJ1 was inducible while for ZC1 it was constitutive. A complete bacterial reduction of 1 mM Cr(VI) was achieved within 57 h for SJl and 12h for ZC1.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 chrl was induced by 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 SJ1 implied the possibility of recent horizontal gene transfer, which made SJ1 adapte for the chromate contaminated environment. The expression of the chromate transporter gene chrA1 was most likely regulated by chrl. By an alignment of most PadR-like regulators which form an operon with the chromate transporter gene chrA, two highly conserved basic amino acids (lysine and arginine) were identified in Chrl and the homologs that might be involved in chromate binding and recognition because they would carry a positive charge under physiological conditions. Even though the physiological function of ChrI has not been verified due to the absence of a genetic system for this Gram positive strain, ChrI is most likely the first identified chromate responsive regulator.The chromate reduction efficience of L. fusiformis ZC1 is the highest that ever been reported. The Cr(VI) reduction ability of L. fusiformis ZC1 was enhanced by sodium acetate and NADH. L. fusiformis ZC1 showed multiple metal(loid) resistances. By whole genome sequence analysis, strain ZC1 was found to contain a large number of putative chromate resistance, chromate reducing and metal(loid) resistance genes. Chromate resistance of ZC1 was constitutive in both phenotypic and gene expression analyses. Furthermore, expression of adjacent putative chromate reduction related genes nitR and yieF was also found to be constitutive. The large numbers of NADH-dependent and constitutively expressed chromate reductase genes may be responsible for the rapid chromate reduction in order to detoxify Cr(Ⅵ) and to metabolize and survive in the harsh wastewater environment.The results of this dissertation have important theory meaning and innovation on exploitation of chromate-resistant and reducing microorganism resource, the characterization of microbial chromate reduction, the molecular mechanisms of bacterial chromate resistant regulation and environmental adaption. |
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