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Characterization of Arsenic Resistant Bacteria and a Novel Gene Cluster in 'Bacillus' Sp University of Wollongong Research Online University of Wollongong Thesis Collection University of Wollongong Thesis Collections 2007 Characterization of arsenic resistant bacteria and a novel gene cluster in 'Bacillus' sp. CDB3 N. Somanath Bhat University of Wollongong Recommended Citation 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 Research Online is the open access institutional repository for the University of Wollongong. For further information contact Manager Repository Services: [email protected]. Characterization of Arsenic Resistant Bacteria and A Novel Gene Cluster in Bacillus sp. CDB3 A thesis submitted in (partial) fulfillment of the requirements for the award of the degree of DOCTOR OF PHILOSOPHY (PhD) From THE UNIVERSITY OF WOLLONGONG by N. SOMANATH BHAT Master of Science {Microbiology), University of Bangalore, India Master of Science {Biotechnology), University of Wollongong, Australia. SCHOOL OF BIOLOGICAL SCIENCES -2007- I CERTIFICATION I, N. Somanath Bhat, declare that this thesis, submitted in fulfillment of the requirements for the award of Doctor of Philosophy, in the School of Biological Sciences, University of Wollongong, is wholly my own work unless otherwise referenced or acknowledged. The document has not been submitted for qualifications at any other academic institution. N. Somanath Bhat July 2007 II TABLE OF CONTENTS TITLE PAGE I CERTIFICATION II TABLE OF CONTENTS Ill LIST OF PUBLICATIONS VIII LIST OF FIGURES IX LIST OF TABLES XIV ABBREVIATIONS XV ABSTRACT XVIII ACKNOWLEDGEMENTS XXI Chapter 1: Introduction (Literature review) 1.1 History and Chemical Forms of Arsenic 2 1.2 Ecology of Arsenic 4 1.3 Microbial Transformations of Arsenic 6 1.4 Uses of Arsenic 7 1.5 Toxicity of Arsenic 9 1.6 Arsenic Resistance in Micro organisms 11 1.6.1 Mechanism of Arsenic resistance 11 1.6.1.1 Oxidation 11 1.6.1.2 Uptake Minimization 11 1.6.1.3 Extrusion 12 1.6.2 Arsenic Resistant Species Characterized at Molecular Level 15 1.7 Proteins Encoded by the ars Operons 22 1.7.1 ArsR: Trans-acting repressor protein 22 1.7.2 ArsD: Secondary regulator protein and Chaperone 25 1.7.3 ArsB: Integral membrane protein 28 1.7.3.1 Efflux mechanisms of arsenic resistance in micro organisms... 31 1.7.4 ArsA: Oxyanion-stimulated ATPase 33 1.7.5 ArsC: Arsenate Reductase 36 1.8 ArsR-ArsD Metalloregulatory Circuit Model 40 1.9 Background and Aims of This Thesis 41 III Chapter 2: Characterization and Identification of Arsenic Resistant Bacterial Strains Isolated from Cattle Dip Sites 2.1 Introduction 48 2.2 Material and Methods 50 2.2.1 Bacterial strains 50 2.2.2 Identification of bacteria 50 2.2.3 16S rDNA sequencing 51 2.2.3.1 Nucleic acid preparation 51 2.2.3.2 PCR amplification and purification of the product 51 2.2.3.3 Sequencing methods 53 2.2.3.4 Sequence analysis , 53 2.2.4 Phylogenetic analysis 54 2.2.5 Taxonomy tree 54 2.2.6 Measurement of minimum inhibitory concentration 54 2.2.7 Growth inhibition 55 2.3 Results 56 2.3.1 Morhology and Gram stain of the CDB strains 56 2.3.2 Extraction of genomic DNA and PCR amplification products of 16SrDNA 57 2.3.3 Sequences of 16S rDNA 58 2.3.4 Homologous identity and multiple sequence alignments 60 2.3.5 Phylogenetic analysis and identification of the strains 75 2.3.6 Taxonomy tree 76 2.3.7 Antibiotic resistance profile of the isolated bacterial strains 77 2.3.8 Arsenic and antimony resistance profiles of isolated bacterial strains 78 2.4 Discussion 81 Chapter 3: Genetic Characterization and Sequence Analysis of CDB3 ars Gene Cluster 1 3.1 Introduction 86 3.2 Materials and Methods 90 3.2.1 Bacterial strains and plasmids 90 3.2.2 Preparation of DIG labeled DNA probe by PCR 90 3.2.3 DNA extraction and restriction digestion 91 3.2.4 Localization ofCDB3ars cluster 1 91 3.2.5 Southern blot hybridization 92 3.2.6 Computational sequence analysis 93 3.3 Results 94 3.3.1 Probe preparation 94 3.3.2 Southern blot analysis 94 TV 3.3.3 Cluster localization 95 3.3.4 Homology analysis of CDB3 Ars proteins 97 3.3.4.1 ArsR 97 3.3.4.2 ArsD 98 3.3.4.3 ArsA 103 3.3.4.4 YqcL 108 3.3.4.5 ArsC 110 3.3.5 Phylogenetic analysis of CDB3 ArsR, ArsD, YqcL, ArsC and ArsA protiens 112 3.4 Discussion 117 Chapter 4: Phylogenetic and Protein Property Studies of Arsenite Efflux Pumps in Bacteria 4.1 Introduction 122 4.2 Materials and Methods 125 4.2.1 Sequence search and alignments 125 4.2.2 Sequence homology values 125 4.2.3 Phylogenetic analysis 125 4.2.4 Prediction of transmembrane domains 126 4.2.5 Membrane topology 126 4.3 Results 127 4.3.1 Multiple sequence alignments of arsenite membrane transporters and their sequence homology 127 4.3.2 Phylogenetic analysis 136 4.3.3 Other protein properties 138 4.3.4 Membrane topology of arsenic pump proteins 141 4.4 Discussion 145 Chapter 5: Functionality of the CDB3 ars A Gene 5.1 Introduction 149 5.2 Materials and Methods 151 5.2.1 Bacterial strains and plasmids 151 5.2.2 Plasmid mini-prep and restriction digestion 152 5.2.3 Ligation and electro transformation.......... 153 5.2.4 Arsenic resistance assays 154 5.3 Results 155 5.3.1 Construction of plasmid pSB 14 155 5.3.2 Effects of CDB3 ArsA on arsenite resistance of arsRBC bearing and ars knock-out E. coli cells 157 5.4 Discussion 159 V Chapter 6: Functionality of the CDB3 arsD Gene 6.1 Introduction 162 6.2 Materials and Methods 164 6.2.1 Bacterial strains and plasmids 164 6.2.2 Deletion mutagenesis of arsD 165 6.2.3 Arsenic resistance assays 165 6.2.4 PCR amplification of arsD gene 166 6.2.5 Plasmid construction and transformation of E. coli 167 6.2.6 DNA sequencing 167 6.2.7 Expression and purification of ArsD 168 6.2.8 Preparation of DNA fragments and DIG labelling 169 6.2.9 Gel mobility shift assay 169 6.3 Results 171 6.3.1 Functional involvement of the CDB3 arsD gene in arsenic resistance 171 6.3.2 Construction of N and C terminal ArsD constructs 180 6.3.3 ArsD expression 189 6.3.4 Purification of His tagged ArsD proteins 191 6.3.5 DNA-Protein interaction studies 194 6.4 Discussion 196 Chapter 7: Transcriptional Regulation of CDB3 ars Gene Cluster 1 7.1 Introduction 200 7.2 Materials and Methods 202 7.2.1 Bacterial strains and plasmids 202 7.2.2 RNA extraction 203 7.2.3 Preparation of DIG labeled DNA probes by PCR 203 7.2.4 Northern-blot analysis 204 7.2.5 P-Galactosidase assay 206 7.3 Results 208 7.3.1 RNA extraction 208 7.3.2 Probe preparation 209 7.3.3 mRNA analysis 213 7.3.4 Reporter gene assay 214 7.4 Discussion 217 7.4.1 CDB3 ars cluster 1 can be transcribed as a single mRNA 217 7.4.2 Sub operons may exist 219 Chapter 8: Concluding Remarks 223 VI BIBLIOGRAPHY 229 APPENDICES 250 APPENDIX 1: BACTERIAL MEDIA 251 APPENDIX 2: BUFFERS AND SOLUTIONS 252 APPENDIX 3: Multiple sequence alignments of the 16S rDNA sequence of CDB1, CDB2, CDB3, CDB4 and CDB5 with those of related bacteria 256 APPENDIX 4: Nucleotide and protein sequences of the ars gene cluster 1 arsRYCDATIP from CDB3 266 APPENDIX 5: Multiple sequence alignment of the arsenite membrane transporter proteins belonging to E. coli type ArsB and a sub-group 268 Appendix 5.1: Multiple sequence alignments of ArsB- like proteins from different strains compared with related species acquired from Genbank through blastx 268 Appendix 5.2: Kyte Doolittle hydropathy plots generated by Pep Window showing the three groups of arsenite membrane transporter proteins 271 APPENDIX 6: Multiple sequence alignments of CDB3 ArsT, ArsI and ArsP with those of related bacteria 274 Appendix 6.1: Multiple alignment of the amino acid sequences of bacterial TrxB proteins 274 Appendix 6.2a: Structural multiple alignment of the amino acid sequence of HesB-like domain proteins 275 Appendix 6.2b: Structural multiple alignment of the amino acid sequences of IscA 275 Appendix 6.3: Structural multiple alignment of the amino acid sequences of CDB3 Orf8 and homologous protein phosphatases 276 APPENDIX 7: Flow chart showing the steps followed in assessing the p- galactosidase activity of lacZ fusion constructs 277 VII LIST OF PUBLICATIONS • Baldeo. K. Chopra*, Somanath Bhat*, Irina P. Mikheenko, Zhiqiang Xu, Ying Yang, Xi Luo, Hancai Chen, Lukas van Zwieten, Ross McC. Lilley and Ren Zhang (2007). "The Characteristics of Rhizosphere Microbes and their Interaction with Plants in Arsenic contaminated Soils from Cattle dip-sites" Science of the Total Environment. 378:331-342. • Somanath Bhat et. al., "A novel ars operon of Bacillus sp. (CDB3) isolated from cattle dip-sites confers increased resistance to arsenate and arsenite in Escherichia coli" (in preparation) • Somanath Bhat et. al., "Metalloregulation of repressor protein ArsD of Bacillus sp. CDB3 isolated from cattle dip sites" (in preparation) • Somanath Bhat et. al., "Phylogenetic study of arsenite efflux pumps among prokaryotes and eukaryotes" (in preparation) * Equal first authors VIII LIST OF FIGURES Figure 1.1: Periodic table showing the Arsenic metalloid as As 2 Figure 1.2: The chemical structure of some common arsenic compounds arsenate, arsenite, MMA and DMA 4 Figure 1.3: Schematic illustration of the genes of the ars operon from E.
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