Year

2004

Degree Name

Master of Science (Research)

Department

School of Biological Sciences - Faculty of Science

Abstract

Arsenic is a naturally occurring toxic metalloid that has been widely used in agriculture, industry and medicine throughout the world. As it occurs quite commonly throughout the environment, many living organisms have established a tolerance or resistance to its biologically toxic effects. The most common form of resistance in bacteria is centred on the efflux mechanism that is controlled by the arsenic resistance (ars) genes. Diversely arranged ars genes have been widely found on both chromosomes and plasmids of various bacteria. A Sinorhizobium strain (As4) isolated from the soil of arsenic-contaminated cattle dip-sites in Northern NSW, Australia, is able to actively grow in the presence of high concentrations of arsenic. Further examination led to the identification of a novel ars gene cluster (As4 ars gene cluster) that contains six or more genes, including a trxB gene that co-functions with the arsC gene. The first five genes are organised in a unique order � arsRBCDA when compared to the common order of arsRDABC found in other ars operons such as pR773. Computer analysis predicted that more than one transcription unit might exist in this gene cluster with arsRBC and the downstream genes (arsDAtrxB) transcribed separately. A previously conducted Northern blot analysis seemed to support this view, but no definitive conclusion could be reached without further experiments. This thesis describes the design, construction and use of a series of reporter gene plasmids, in which the promoter P(subscript arsR) and arsR gene or/and putative promoter P(subscript arsD) and arsD gene were fused upstream to the promoterless lacZ gene of plasmid pUJ8. Activity of the different constructs was monitored by a ?-galactosidase assay. The results indicated that the putative promoter P(subscript arsD) may not exist, and that the expression of the ars genes were regulated by ArsR and ArsD, which can be induced by arsenite. The transcript identification by RT-PCR analysis also demonstrated that the mRNA transcription could read through the predicted putative transcriptional terminator between arsC and arsD, indicating that the As4 ars gene cluster was transcribed under the control of a single promoter, which is in line with the results from the reporter gene analysis. Bioinformatic analysis of the ArsR, ArsD and ArsA proteins of the As4 ars gene cluster was undertaken to examine specific consensus sequences. Alignment of the As4 ArsD with seven other ArsDs shows that the As4 ArsD protein lacks a particular pair of cysteine residues, which have been demonstrated to be required for induction in pR773 ArsD, suggesting the regulatory mechanism of As4 ArsD may be different from that of pR773. Bioinformatic analysis of the non-coding region in front of arsD in the As4 ars gene cluster revealed some specific sequences, including two inverted repeats, and that a putative regulatory protein-binding site exists within this region. Therefore, some suggestions as to the function of the region and the regulatory mechanism of the As4 ars gene cluster are made. It is possible that the cluster can be transcribed to both a short mRNA (arsRBC) and a long mRNA (arsRBCDAtrxB) depending on the concentrations of inducers, with ArsD binding to the putative regulatory elements in front of arsD to interfere with the transcription of the downstream genes. Alternatively, it may be that the inverted repeats in front of arsD can form a stem-loop to regulate the downstream genes at a post-transcriptional level. The phylogenetic analysis and comparison of As4 Ars proteins with their counterparts encoded by other ars operons also indicated that unlike the arsRDABC operons which exist on the plasmids of Gram-negative bacteria, the As4 ars operon possibly originated from an arsRBC operon of a Gram-positive organism by acquisition of an arsDA operon at the end of arsC.

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