Molecular analysis and characterisation of plasmid pTFS from Thiobacillus ferrooxidans ATCC33020 Clifford Noel Dominy In fulfilment of the requirements for the degree of UniversityDoctor of Philosophy of in theCape Faculty ofTown Science, University of Cape Town. CAPETOWN FEBRUARY 1997 -· ·- --·~----··~~, { . ')_ '. The copyright of this thesis vests in the author. No quotation from it or information derived from it is to be published without full acknowledgement of the source. The thesis is to be used for private study or non- commercial research purposes only. Published by the University of Cape Town (UCT) in terms of the non-exclusive license granted to UCT by the author. University of Cape Town ii To my wife Patricia, for her patience, love and understanding over the years. and to my parents, Ashton and Denise Dominy for their encouragement and support. lll CERTIFICATION OF SUPERVISOR In terms of paragraph 9 of "General regulations for the degree of Ph.D." I as supervisor of the candidate Clifford Noel Dominy, certify that I approve of the incorporation in this thesis of material that has already been published or submitted for publication. Professor D.E. Rawlings Department of Microbiology University of Cape Town IV Acknowledgements I am greatly indebted to my supervisor, Professor Doug Rawlings for his enthusiastic guidance and support over the duration of this project. His confidence and optimism have served as a source of inspiration over the years. Many thanks go to members of the "Thiobacillus Research Unit", both past and present, who freely gave advice (and reagents) in times of crises. Specifically I would like to thank Dave Berger for his initial supervision of the project and Shelly Deane for invaluable help with the pulsed field gel electrophoresis. Thanks also go to Ann Jaffray, Di de Villiers, Anne-Marie Clennel, Stan Botman, Charlie Hendrikse, Nikki Campbell and Genevieve Wilson whose administrative and technical assistance eased my life considerably within the department. A special thanks must go to Di James for advice and assistance with, what must have been, every commercial sequencing kit in existence between 1991 and 1996. I am indebted to Nicolette Coram who finished off the "last few kb" of pTF5 sequencing as part of her B.Sc (Hons) studies. I also feel obliged to thank "The Louts", in particular Will Bourn, Jono Lane, Joe Santangelo, Gordon D. Brown and Ant Smith, for many an evening spent in "The Laboratory" critically evaluating various aspects of this work. V TABLE OF CONTENTS . ABSTRACT ............................................................................... VI ABBREVIATIONS .................................................................... VIII CHAPTER 1 Literature Review .......................................... 1 1.1 Plasmid studies in the genus Thiobacillus ....... 3 1.2 Insertion sequences from T ferrooxidans ....... 15 1.3 Development of genetic tools for the genus Thiobacillus .................................................... 17 1.4 The molecular biology of redox active proteins and their genes in the thiobacilli........................ 20 1.5 The role of heavy metals in the bioleaching envtronment ..................................................... 35 1.6 Aims of this project ......................................... 38 CHAPTER2 Activation of metronidazole by plasmid pTF5 ............................................................... 40 2.0 Sutnmary ........................................................ 41 2.1 Introduction .................................................... 41 2.2 Materials and methods .................................... 48 2.3 Results ............................................................ 52 2.4 Discussion ....................................................... 76 vi CHAPTER3 Characterisation of genes from plasmid pTF5 which encode putative iron-sulphur proteins .... 82 3.0 Summary- ......................................................... 83 3.1 Introduction ..................................................... 83 3.2 Materials and methods ..................................... 93 3.3 Results ............................................................. 96 3.4 Discussion ........................................................ 108 CHAPTER4 Distribution analysis and characterisation of pTF5 and related plasmids in T. ferrooxidans isolates .................................. 113 4.0 Summary-......................................................... 114 4.1 Introduction ..................................................... 114 4.2 Materials and methods ..................................... 118 4.3 Results ............................................................. 121 4.4 Discussion ........................................................ 125 CHAPTER 5 General conclusions ....................................... 128 APPENDIX A pTF5 complete nucleic acid sequence........... 139 APPENDIX B Media, buffers and solutions ......................... 151 APPENDIX C General techniques ......................................... 155 APPENDIX D Amino acid codes ........................................... 158 APPENDIX E Physical maps of vectors and transposons ....... 159 REFERENCES ............................................................................ 162 Vl ABSTRACT Plasmid pTF5, an endogenous plasmid of Thiobacillus ferrooxidans was isolated and cloned from a T. ferrooxidans ATCC33020 pHC79 cosmid library. The plasmid was isolated due to its ability to confer metronidazole sensitivity to an Escherichia coli metronidazole resistant mutant. The region responsible for this phenotype was narrowed down to a 319 hp fragment which did not appear to contain an open reading frame. A nearly identical fragment had been previously reported as being part of an origin of vegetative replication (oriV) of the narrow host-range plasmid, pTFI91 from T. ferrooxidans TFI91 (Chakravarty et al. 1995). Plasmid pTF5 differed from pTFI91 in at least two aspects; its size, (pTF5 was 19.8 kbp versus 9.8 kbp for pTFI91), and in the number of iterons present in its oriV. Plasmid pTF5 was completely sequenced and found to contain four open reading frames (ORFs) which encoded putative iron-sulphur containing proteins. Two of the ORFs with amino acid homology to ferredoxin-like (fdxA) and fnr-like regulatory genes respectively, appeared to be divergently transcribed from the other two ORFs, one of which had amino acid sequence similarities to a prismane-like protein (psmA) and the other to a NADH oxidoreductase protein (redA). In vitro transcription­ translation analysis showed that only the fdxA gene was recognised by a heterologous E. coli system, with the psmA and redA genes requiring E. coli-derived promoters in order to be expressed. Evidence of a "transpositional scar" was detected between the fnr-like and fdxA genes, due to the presence of two incomplete ORFs with sequence identity to a transketolase gene and transposase gene from E. coli and T. ferrooxidans respectively. Plasmid pTF5 was present in T. ferrooxidans at a copy number of 2 to 4 per genome. As was the case with other T. ferrooxidans pTFI91-like plasmids, pTF5 could not replicate in E. coli (Rawlings and Woods 1985, Valenti et al. 1990, Chakravarty et al. 1995) and was not detected in the Thiobacillus thiooxidans or Leptospirillum ferrooxidans strains tested. Pulsed field gel electrophoresis showed that the approximately 6 kbp of sequence of pTF5, which Vll included the iron-sulphur protein-encoding ORFs, was also present on the chromosome of strain ATCC33020. These ORFs were not essential to the survival of the host as a number of T. ferrooxidans strains did not contain either the plasmid or the chromosomally located ORFs. Some of the T. ferrooxidans strains tested appeared not to contain plasmids, although pTFS-like plasmid sequence was detected and had apparently become integrated into the hosts chromosome. The iron-sulphur protein-encoding ORFs of pTFS had organisational similarity to simple electron transport pathways which in other bacteria are responsible for the degradation of aromatic compounds. Due to the possibility of a redox active role by products of the pTFS ORFs, resistance to a number of heavy metal compounds was tested in E. coli strains which contained constructs which expressed the proteins in vitro. The presence of these constructs in the heterologous host did not alter the tolerance of the E. coli strain to either uranium or silver. viii ABBREVIATIONS A adenosine aa amino acids Amp ampicillin ATCC American Type Culture Collection hp base pairs BSA bovine serum albumin C cytosine C­ carboxy terminal ( of protein) cccDNA covalently closed circular DNA CoA coenzyme A Cm chloramphenicol oc degree's Centigrade d day(s) Da daltons DMSO dimethyl sulfoxide DNA deoxyribonucleic acid Dnase deoxyribonuclease dNTP deoxynucleotide triphosphate DTT 1,4-dithio-L-threitol EDTA ethylene-diaminetetra-acetic acid Em erythromycin EtBr ethidium bromide g gram_s G guanme h hour(s) IHF integration host factor IPTG isopropyl-B-D-thiogalactopyranoside IS insertion sequence element kbp kilobase pairs kDa kilodaltons LB Luria broth M molar min minutes Mr relative molecular mass N­ amino terminal ( of protein) NADH nicotinamide adenine dinucleotide (reduced) nt nucleotide IX OD6oo optical density at 600 nanometres ORF open reading frame oriV origin of vegetative replication PAGE polyacrylamide gel electrophoresis r resistant (superscript) RBS ribosome binding site RNA ribonucleic acid sensitive (superscript) s seconds