Doctor of Philosophy
School of Biological Sciences - Faculty of Science
Bhat, N. Somanath, Characterization of arsenic resistant bacteria and a novel gene cluster in bacillus sp. CDB3, PhD thesis, School of Biological Sciences, University of Wollongong, 2007. http://ro.uow.edu.au/theses/66
Arsenic is a toxic metalloid of the nitrogen family which is found in both natural environments and sites contaminated in a number of ways. Arsenate acts as a phosphate analog which interferes with phosphate uptake and utilization and arsenite disrupts enzymatic function. Micro organisms have evolved a variety of mechanisms in coping with toxicity of arsenic and the best known detoxification pathway involves the arsenic resistance (ars) cluster. To date, a number of ars clusters have been characterized at the molecular level.
Contamination of cattle dip-site soils with arsenic along with other toxicants such as DDT represents a major pollution problem in agricultural sites and has been shown to have distinct effects on the soil microbial populations. Little is known of the micro organisms at these sites. This thesis investigated the identity of some of the rhizosphere bacteria isolated from arsenic contaminated dip-site soils of north-eastern New South Wales and functionally characterized some of the ars genes cloned from one strain.
Five bacterial strains isolated from cattle dip-sites (referred to as CDB) were identified based on morhological, biochemical and 16S rDNA sequence characters. These bacterial strains belonged to four different genera, CDB1- Arthrobacter sp., CDB2- Ochrobactrum sp., CDB3 & CDB4- Bacillus spp and CDB5- Serratia sp. The arsenic resistance profiles of these bacteria were quite different. The highest resistance to arsenite was by CDB5 followed by CDB4, CDB3, CDB2 and CDB1. CDB2 was exceptionally tolerant to arsenate, exhibiting normal growth on agar containing 200 mM arsenate, an almost saturated concentration, while all the other strains showed a minimum inhibitory concentration to arsenate of 75 mM.
From Bacillus sp. CDB3, a novel ars gene cluster was cloned and sequenced (Luo, 2006), which revealed eight intact open reading frames organised in a unique gene order, arsRYCDATIP when compared to other ars gene clusters. This gene cluster was found to exist on the chromosome, unlike the arsRDABC clusters which exist on plasmids of Gram negative bacteria.
Bioinformatic analysis was undertaken to examine any novel sequence characters. Alignment of the CDB3 ArsD with known ArsDs showed that the CDB3 ArsD protein lacks the four C-terminal cysteine residues (Cys112-Cys113 and Cys119- Cys120), which have been demonstrated to be required for induction in E. coli pR773 ArsD, suggesting the regulatory mechanism by CDB3 ArsD may be different from that of E. coli pR773. This was the case, when the ArsD of CDB3 was mutated and compared to the intact ArsD as presented in this thesis, the E. coli cells bearing mutant ArsD showed sensitivity to arsenic. The membrane bound AsIII pump encoded by the second ORF is a YqcL type protein but may still associate with an ATPase encoded by the fifth ORF. Phylogenetic analysis of arsenite efflux pumps also revealed the existence of a sub-group of the ArsB group proteins, adding to the diversity of the arsenite pump protein family. Further investigation was done to determine whether the CDB3 ArsA, (which is known to couple to ArsB) can couple to YqcL and vice versa. Results indicated that CDB 3 ArsA can couple to both YqcL and E. coli ArsB resulting in elevated resistance to the host, confirming the functionality of ArsA. No data on ArsA coupling with YqcL is available and this thesis was the first instance to show the interaction of YqcL with ArsA in extruding arsenite out of the cell.
Transcriptional regulation of CDB3 ars gene cluster was performed by reporter gene and northern analysis. Northern analysis showed that the mRNA transcription could read through the whole cluster, indicating that the CDB3 ars gene cluster can be transcribed under the control of a single promoter. However, some short transcripts were also detected. These results showed that the ars gene cluster can be transcribed as whole cluster, but under certain conditions it may be transcribed as two or more sub-operons. This was further studied by the reporter gene analysis which indicated active promoters in front of arsR, arsD and arsT. Gel mobility shift assays also displayed the existence of a promoter in front of arsD, where the purified ArsD protein was shown to bind to its own promoter (between arsC and arsD) in addition to the arsR promoter. It appeared that the regulation of expression of the CDB3 ars gene cluster 1 is a very interesting but complicated mechanism.