University of Wollongong Research Online University of Wollongong Thesis Collection University of Wollongong Thesis Collections 1999 Investigation of the molecular biology and contribution to virulence of Bordetella bronchiseptica urease David McMillan University of Wollongong Recommended Citation McMillan, David, Investigation of the molecular biology and contribution to virulence of Bordetella bronchiseptica urease, Doctor of Philosophy thesis, Department of Biological Sciences, University of Wollongong, 1999. http://ro.uow.edu.au/theses/1049 Research Online is the open access institutional repository for the University of Wollongong. For further information contact the UOW Library: [email protected] Investigation of the molecular biology and contribution to virulence of Bordetella bronchiseptica urease A thesis submitted in fulfilment of the requirements for the award of the degree Doctor of Philosophy from University of Wollongong by David McMillan, BSc (Hons) Department of Biological Sciences 1999 n Declaration This thesis is submitted in accordance with the regulations of the University of Wollongong in fulfilment of the degree of Doctor of Philosophy. It does not include any material previously published by another person except where due reference is made in the text. The experimental work described in this thesis is original work and has not been submitted for a degree in any University or Institution. ffl Acknowledgments This thesis could not have been completed with the support of many friends and family. Firstly I would like to thank my supervisor, Dr Mark Walker, for his invaluable advice on the research and for also giving me the inspiration to continue when progress was slow. I also extend my gratitude to the people of Labi 11. Thanks then to Peter, Adam, Tania, Jason, Nick, Darren, Yan, Jody and Tamantha. To my friends outside of the lab, who provided me with recreation and relaxation when it was most needed, I also thank you. Some of you may have moved on, but I thank you no less. Thanks also go to Dr C. A. Guzman and Dr G. S. Chhatwal for hosting me at the GBF in Germany. Dr Guzman also undertook the mouse colonisation and intracellular invasion assays. Finally, I thank my parents. They have provided me with love, a home and unswerving support over the years taken to complete this thesis. rv Abstract Bordetella bronchiseptica is a common pathogen of the mammalian respiratory tract. Infection by this organism results in a number of respiratory conditions which may be fatal to the host. Urease is a potential virulence determinant of B. bronchiseptica that has been implicated in the pathogenesis of several other microorganisms. The urease operon of B. bronchiseptica is encoded within an 8.9 kb DNA fragment which contains the structural genes {ureA, ureB and ureC) and accessory genes (ureD and ureG) homologous to urease genes from other bacteria. Uniquely, the ureE and ureF genes are fused to form a hybrid protein, UreEF, which may result in tighter coordination of the putative functions of the individual accessory genes. The operon contains an additional open reading frame, UreJ, found only also in the Alcaligenes eutrophus urease operon. UreJ is also 37% homologous with HupE from Rhizobium leguminosarum bv. viciae, and may be involved in nickel transport across the bacterial membrane. A transcriptional activator, designated Bordetella bronchiseptica urease regulator (bbuR), is located directly upstream and in the opposite orientation to the urease operon. BbuR shares homology with members of the LysR regulatory protein family. Other members of this family have been shown to regulate the expression of urease in Klebsiella aerogenes (NAC), and to induce the expression of a set of genes in Escherichia coli (OxyR) which protects the bacteria from phagolysosomal attack after intracellular invasion. A putative BbuR binding site (5'-ATA-N9-TAT-3'), identical to the NAC-binding consensus sequence, was also found 27 bp upstream of the urease promoter in B. bronchiseptica. The results of regulation studies show that urease is repressed by the Bordetella virulence gene locus (bvg). Urease was not inducible by 10 mM urea nor up-regulated in nitrogen limiting conditions. To investigate the role of BbuR in the regulation of urease, BbuR mutants of the BB7865 and the avirulent strain BB7866 were constructed by homologous recombination. The BbuR deficient BB7865 Bl lost the ability to regulate the expression of urease, suggesting that BbuR may be an intermediary in the bvg regulation of urease. As BbuR is homologous to OxyR, and urease has been suggested to protect bacteria intracellularly, an evaluation of the intracellular survival of urease-negative mutants of BB7865 and BB7866 in comparison to their urease-positive parental strains was also undertaken. This experiment demonstrated that increasing the concentration of urea in the assay increased survival of the wild-type but not urease-negative strains after 24 h, suggesting that urease may have a role in intracellular survival. To further address the role of urease in respiratory infection, we compared the ability of BB7865, the bbuR mutant strain BB7865 Bl which constitutively expresses urease, and the urease negative mutant BB7865 U5 to colonise and persist on the murine respiratory tract. The results showed that the constitutive expression, or abolishment of urease activity, had no significant effect on respiratory infection in this model. A DNA probe containing the gene encoding UreA of B. bronchiseptica hybridised to chromosomal DNA from Bordetella pertussis Tohama I indicating the presence of cryptic urease genes in this urease negative species. PCR primers designed to amplify part of ureD and the urease promoters from B. bronchiseptica were also able to amplify identically sized DNA fragments from B. bronchiseptica, B. pertussis and B. parapertussis ATCC15311. Nucleotide sequence analysis of these regions revealed no differences in the ureD open reading frame between each species. A cluster of mutations in both B. pertussis and B. parapertussis was found upstream of the urease promoter, in a region proximal to the bbuR promoter. The inability of B. pertussis to produce urease may therefore reflect mutations in regulatory elements, and not mutations in the urease locus itself. The biochemical data together with the results from the intracellular invasion experiments suggest that urease is somehow involved in post-infection processes. The possible presence of compensatory mechanisms may explain why this hypothesis is not supported by the murine respiratory colonisation assay data. VI Table of Contents Declaration II Acknowledgments Ill Abstract IV Table of Contents VI List of Figures VIII List of Tables IX Abbreviations X INTRODUCTION 1 1.1 Overview 1 1.2 The Genus Bordetella 1 1.2.1 Phylogenetic relationship of Bordetella species 2 1.2.2 Bordetella bronchiseptica 3 1.2.3 Phenotypic Modulation 4 1.2.4 Intracellular survival of Bordetella species 21 1.3 Urease 24 1.3.1 Introduction 24 1.3.2 Genetics of Urease 26 1.3.3 Biochemistry of Urease 28 1.3.4 Regulation of urease expression 34 1.3.5 Contribution to Pathogenesis 39 1.4 Project Aims 43 METHODS 44 2.1 Bacterial strains, plasmids and media 44 2.2 Genetic techniques 44 2.3 Cosmid Cloning of DNA 47 2.4 Chromosomal DNA Extraction 48 2.5 Plasmid extraction 49 2.6 Polymerase chain reaction 51 2.7 Southern hybridisation 53 2.8 Nucleotide sequence determination and analysis 55 2.9 Conjugation of E. coli and B. bronchiseptica 57 2.11 Urease assays 57 2.12 Tissue culture methods and invasion assays 58 2.13 Construction of urease mutants of B. bronchiseptica 59 RESULTS 60 3.1 Molecular analysis of the urease locus of B. bronchiseptica 60 3.2 Characterisation of urease activity of B. bronchiseptica 83 3.3 Analysis of urease activity in other Bordetella species 89 3.4 In vitro and in vivo analysis of urease mutants of B. bronchiseptica 93 DISCUSSION 98 CONCLUSION 108 REFERENCES 109 APPENDIX I : Growth Media and Solutions 138 APPENDIX II: Primers for PCR and DNA Sequencing 143 APPENDIX III: Publications arising from this thesis 144 List of Figures 1.1 Evolutionary relationships in the Genus Bordetella 3 1.2 The bvg locus 8 1.3 Catalytic activity of urease 25 3.1 (a) Urease activity of wild-type and urease-deficient B. bronchiseptica. (b) Southern blot analysis of chromosomal DNA from wild-type and urease- deficient B. bronchiseptica probed with miniTn5/Xm2 61 3.2 Restriction map of the mutated urease locus of B. bronchiseptica 62 3.3 Southern analysis of B. bronchiseptica strains probed with pMC3 63 3.4 Subcloning the mutated urease gene from the cosmid pMCl 65 3.5 Nucleotide and deduced amino acid sequence of the urease gene cluster of B. bronchiseptica 66 3.6 Physical map of the cloned DNA fragments used to sequence the urease gene cluster 73 3.7 Alignment of the deduced amino acid sequences from B. bronchiseptica with urease subunits from other bacteria 75 3.8 Phylogenetic analysis of UreC 79 3.9 (a) Homology of UreJ with other proteins (b) Hydrophilicity plot of UreJ 81 3.10 Homology of BbuR with other LysR proteins 82 3.11 (a) Growth and expression of urease by B. bronchiseptica. (b) Urease activity of B. bronchiseptica in varying growth conditions 85 3.12 (a) Construction of the recombinant vector for homologous recombination with the bbuR gene of B. bronchiseptica. (b) Southern blot analysis of chromosomal DNA from wild-type and bbuR deficient B. bronchiseptica probed with pMC48 87 3.13 Comparison of the urease activities of wild-type B. bronchiseptica and their bbuR deficient derivatives 88 3.14 Urease activity of B. pertussis, B. parapertussis and B. avium 89 3.15 (a) Southern blot analysis of chromosomal DNA from Bordetella species probed with pMC3. (b) PCR of Bordetella spp. using primers designed after partial DNA sequencing of the urease locus 91 3.16 Comparison of the nucleotide sequences of the B.