Identification And Functional Study On Bacterial Arsenic Detoxification Genes

Arsenic is known as a toxic metalloid,which primarily exists in inorganic forms of arsenite[As(â…¢)]and arsenate[As(â…¤)]in the natural environment and can be transformed by microbial redox processes.As(â…¢) is much more toxic and mobile than As(â…¤),hence microorganisms can greatly influence the human health and safety by the processes of arsenic redox transformation.This dissertation focused on the studies of As(â…¢) oxidation and cytoplasmic As(â…¤) reduction of arsenite-resistant bacteria and performed two research subjects.The main results were as follows:Four soil samples were collected in China from regions with high(TS), intermediate(SY),and low(LY and YC) arsenic contamination.A total of 58 arsenite-resistant bacteria(TS1-45,SY1-8,LY1-4,and YC1) were isolated from these four samples with three different arsenic-contaminated levels.12 high As(â…¢) resistance bacteria(MICs>20 mM) were only isolated from the highly arsenic-contaminated TS site including Acinetobacter,Agrobacterium,Arthrobacter, Comamonas,Rhodococcus,Stenotrophomonas and Pseudomonas.Using the KMnO₄ screening method,5 As(â…¢)-oxidizing bacteria(Achromobacter sp.SY8, Pseudomonas sp.TS44,Agrobacterium spp.TS43,TS45 and LY4) were identified from the 58 strains that displayed a higher average As(â…¢) resistance level than the 53 non-As(â…¢) oxidizers.Five aoxB fragments encoding the large subunit of As(â…¢) oxidase and 51 As(â…¢) transporter genes[18 arsB,12 ACR3(1) and 21 ACR3(2)]were successfully amplified from these strains using PCR with degenerate primers.The aoxB particularly existed in the As(â…¢)-oxidizers whereas arsB,A CR3(1) and A CR3(2) could occur in both As(â…¢)-oxidizers and non-As(â…¢) oxidizers.Phylogenetic trees of 16S rRNA gene,deduced AoxB,and deduced ArsB/Acr3(1)p/Arc3(2)p were constructed by the neighbor-joining method and analyzed in detail.According to the comparison of these trees,5 aoxBs had the similar phylogeny with 16S rRNA gene, but the discrepancies could be detected in 8 ACR3(2) and 1 arsB which were probably acquired by horizontal gene transfer(HGT).These results indicated that soils with long-term arsenic contamination may result in the evolution of highly diverse arsenic-resistant bacteria and elevated As(â…¢) resistance level.Further analyses revealed that HGT promoted the lateral evolution of arsenic-resistant bacteria.This study is the first report of comparative study on arsenic-resistant bacteria and their resistance genes from different arsenic contaminated soils in China,also the first report of detecting HGT of As(â…¢) transporters.Two of the above As(â…¢)-oxidizing bacteria(Achromobacter sp.SY8 and Pseudomonas sp.TS44) were used as the second research subject.A 17.5 kb DNA sequence containing the complete arsenite oxidase(aox) gene cluster (aoxX-aoxS-aoxR and aoxA-aoxB-aoxC-aoxD) was isolated from SY8 using a Fosmid library approach.Similarly,a 14.6 kb DNA sequence including the complete aox cluster(arsD-arsA-aoxA-aoxB) and the arsenic resistance(ars) gene cluster (arsC1-arsR-arsC2-ACR3-arsH-DSP-GAPDH-MFS) was obtained from TS44 by inverse PCR method.According to RT-PCR experiments,SY8 aoxXSR and aoxABCD transcribed as two different transcripts and belonged to two operons;TS44 aox and ars clusters transcribed as a single transcript in their respective cluster and each of them belonged to one operon.All of these genes were found to be up-regulated by the addition of As(â…¢),As(â…¤) or antimonite[Sb(â…¢)],except TS44 arsC1-arsR appeared to be expressed constitutively.Bioinformatic analyses revealed that the SY8 aox cluster was regulated by a two-component signal transduction system which was different from the reported work of Agrobacterium tumefaciens 5A.Hence,we predicted that a novel two-component regulatory model of bacterial As(â…¢) sensing and signal transduction could exist in SY8.The organization of TS44 aox cluster was unusual since it contained structural genes only and arsDA in its upstream.However, except TS44,no aox cluster with structural genes only was searched in GenBank database.The first five genes of TS44 ars cluster were widely reported to be associated with arsenic resistance,but the last three genes were not.The above three points indicated that these arsenic detoxification gene clusters of SY8 and TS44 were complex and had interesting and novel arrangements.The results of this dissertation have made great contributions to the exploitation of arsenic-resistant microorganisms,the microbial ecology of arsenic-contaminated environments,the bacterial arsenic metabolisms,and the molecular mechanisms of bacterial arsenic resistance.