| Arsenic,a toxic metalloid element existing in the form of either organic or inorganic salts,is widely spread in the environment.Over exposure to arsenic can lead to a series of human diseases including diabetes and cancers.The pressure of survival in arsenic-containing environments has prompted the evolution of detoxification systems in almost all microorganisms.The most comprehensively studied mechanisms,and therefore the best understood mechanisms,are those of bacteria.A Bacillus strain designated CDB3 isolated from an arsenic contaminated cattle dip site,has been found to possess a novel ars cluster with eight genes in the order of arsRYCDATorf7orf8,one of the longest ars operons identified to date.Furthermore,it is the first time that the two novel genes,orf7 and orf8,were found to be associated with known ars genes and located on the same operon.In this study,we investigated the functions of arsA,arsD,arsT,orf7 and orf8 in arsenic resistance using a plasmid-based gene knockout approach in the ars gene deficient Escherichia coli strain AW3110.In addition,a bioinformatics study was carried out to screen and inverstigate novel arsenic resistance genes on putative ars operons from genome-available prokaryotes.The results of arsenic resistance assays in arsenic-sensitive strain E.coli AW3110 suggested that arsA was involved in arsenic resistance pathway.The mutation of arsA showed a significant decrease in resistance to arsenite,suggesting that CDB3 ArsA is likely to interact with ArsY,forming ArsAY complex that can enhance the tolerance of the cells to arsenic.An ars gene mutant plasmid,pWZ6(arsD?A?)was transformed into E.coli AW3110 competent cells,followed by arsenic resistance assays.The results showed that the last two pairs of cycteines(Cys112,113 and Cys119,120)located at the end of C-terminal of ArsD are essential for maintaining its regulatory function.According to the genomic sequencing,another three putative thioredoxin reductase genes,apart from the arsT located on arsl,were possessed by Bacillus sp.CDB3.However,the integral function domains were only conserved by ArsT based on bioinformatics analysis.The results of RT-PCR demonstrated that CDB3 arsl was capable of producing two transcripts,an 8-gene long transcript and a 3-gene short transcript,which also evidenced that the two novel genes,orf7 and orf8,could express properly in heterologous E.coli AW3110 host.Cells harboring orf7 mutant showed the identical resistance curves to that of positive control under both arsenate and arsenite treatments,implying that orf7 might be irrelevant to arsenic resistance.However,the contribution by orf7 to arsenic resistance can not be left out,which warrants further investigation.Orf8 is composed of 141 amino acids.Cells containing orf8 mutant showed a remarkable decrease in resistance to arsenate,though not arsenite,suggesting that the function of orf8 is only associated with arsenate.In addition,the introduce of orf8 into E.coli JM109 could further enhance its arsenic resistance.In addition to the functional analysis of genes on Bacillus sp.CDB3 arsl,we were also interested in looking for novel ars genes that are located on ars operons.577 putative ars operons were identified,from which 242 putative ars genes were predicted and 90 showed sequence similarities with known genes.Among these genes,75 genes could be assigned to 13 biological processes,of which the establishment of localization and regulation of biological process accounted for 29.3%and 13.3%,respectively.Of 90 genes,55 genes were classified to 8 sorts of molecular function.The genes associated with transferase activity,hydrolase activity and nucleotide binding totally accounted for 39%.6 putative ars genes that occur more than 10 times were highlighted,which are major facilitator transporter(32),putative permease(47),redox active disulfide protein 2(42),GCN5-like N-acetyltransferase(13),glyoxalase/bleomycin resistance protein/dioxygenase(27)and glyceraldehyde-3-phosphate dehydrogenase(10). |
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