Characterization of Arsenic-Resistant Bacteria and their ars Genotype for Metal Bioremediation

The estuarine and coastal waters of Goa situated on the West coast of India, are known to be highly contaminated with arsenic. The rivers Mandovi and Zuari flowing through the iron and manganese mining areas are daily used for the transportation of over 300 barges of iron-ore to the harbour. However, studies related to the identification of ars genotype in arsenic-resistant bacteria inhabiting these estuarine waters are not available. This has prompted us to isolate diverse arsenic- resistant bacteria from the Mandovi and Zuari estuarine systems and characterize the ars genotype among the isolates. Additionally, we report the cloning of arsenic detoxification genes (arsB and arsC) from the arsenic-resistant isolates. These genes, in particular arsC gene could be successfully utilized in designing potential arsenic remediation strategies. A total of 50 heterotrophic bacteria were isolated through serial dilutions on agar plates from marine and estuarine (Mandovi and Zuari) waters of Goa, India that were prone to heavy metal pollution. These bacterial isolates were screened to determine their tolerance to arsenate and arsenite. PCR analysis was performed using genomic DNA and plasmid DNA of arsenic resistant bacteria with primers specific for ars genes (arsA, arsB and arsC). With the primers used in the present study, no amplification of the ars genes was observed with genomic DNA as the template, but plasmid DNA resulted in the amplification. Hence, it is evident that ars genes are anchored to plasmid rather than to chromosomal DNA. Out of the 36 arsenic-resistant bacteria, only 17 harboured the ars genes on the plasmid DNA. Phylogenetic analysis based on 16S rRNA sequence analysis showed that these isolates belonged to the phyla γ-Proteobacteria, Actinobacteria and Firmicutes. The ars genotype characterization in 36 bacterial isolates (resistant to 100 mM of sodium arsenate) revealed that only 17 isolates harboured the arsA, B and C genes on the plasmid DNA. The arsA, B and C genes were individually detected using PCR in 16, 9 and 13 bacterial isolates respectively indicating the prevalence of arsA > arsC > arsB in the isolates. Molecular identification of the 17 isolates bearing the ars genotype was carried using 16S rDNA sequencing. A 409 bp fragment of arsC gene coding for arsenate reductase and a 1300 bp full length arsB gene encoding arsenite efflux pump were isolated from the genera Halomonas and Acinetobacter. While full length (1300 bp) arsB gene was isolated from the strain Halomonas species SPK23 (Acc.No.EU024298), a putative arsC comprising 409 bp was obtained from Acinetobacter species VKPM 14 (Acc.No. EF570879) and Halomonas species SPK 23 (Acc.No. EF583905). The full length arsB gene isolated from the strain Halomonas sp. SPK23, showed 98% homology with E.coli chromosomal arsB gene, 82% with plasmid R773 arsenical resistance operon genes, 81% with Acidiphilium multivorum plasmid pKW301 and Klebsiella oxytoca plasmid pMH12, and 79% with Enterobacter cloacae arsB gene. It showed 97% homology with partial cds of Pseudomonas putida strain RS-17 arsB gene, 96% with Acinetobacter sp. VKPM45 plasmid arsenite/antimonite transporter gene, and 95% with Vibrio cholerae arsenite/antimonite transporter gene. Phylogenetic analysis of arsB and C genes indicated their close genetic relationship with plasmid borne ars genes of E.coli and arsenate reductase of plant origin. The putative arsenate reductase gene isolated from Acinetobacter species complemented arsenate resistance in E.coli WC3110 and JM109 validating its function. Hence, this study dealing with isolation of native arsenic resistant bacteria and characterization of their ars genes will be useful for development of efficient arsenic bioremediation strategies. The naturally occurring arsenic-resistant isolates are more environmentally acceptable and safe for detoxification of arsenic. Hence, isolation of such arsenic-resistant species has considerable ecological advantage. However, characterization of arsenic metabolizing genes is required for their successful exploitation in in situ arsenic bioremediation. The arsenite/antimonite efflux pump and arsenate reductase encoding genes isolated in the present study could be used to engineer either bacteria or plants which can in turn, help in remediation of arsenic polluted sites. Also, arsenate reductase gene could be co-expressed along with genes encoding metallothionein proteins for developing an efficient bioremediation/phytoremediation technology. Further, we report the isolation of a 1459 bp full-length cDNA sequence encoding a phytochelatin synthase (PCS) from subabul (Leucaena leucocephala), designated as LlPCS1. The SDS PAGE analysis resulted in a recombinant protein of molecular weight 66 kDa. The deduced 485 amino acid sequence of LlPCS1contains three Cys-Cys motifs and 13 single Cys residues, but only 4 of them (Cys-56, Cys-90/91, and Cys-109) in the N-terminal half of the LlPCS1 protein are conserved unlike in other known PCS polypeptides. The relative level of heavy metal tolerance imparted by AtPCS1 (isolated from Arabidopsis thaliana) and LlPCS1 to E.coli was analyzed. When bacterial cells expressing LlPCS1 were grown in the presence of heavy metals such as arsenite, arsenate and cadmium, cellular metal