Ca r d if f UNIVERSITY PRIFYSGOL Ca ERDY[§> Molecular Analysis of the Diversity and Pollutant Tolerance of the BurkholderiaGenus Thesis presented for the Degree of Philosophiae Doctor By George William Payne, BSc (Hons) Biotechnology In Candidature for the Degree of Philosophiae Doctor Cardiff School of Biosciences Cardiff University May 2006 UMI Number: U584883 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. Dissertation Publishing UMI U584883 Published by ProQuest LLC 2013. Copyright in the Dissertation held by the Author. Microform Edition © ProQuest LLC. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 DECLARATION This work has not previously been accepted in substance for any degree and is not being concurrently submitted in candidature for any degree. Signed ....... ..................................(candidate) 23,os.oc,...... STATEMENT 1 This thesis is a result of my own investigations, except where otherwise stated. Other sources are acknowledged by footnotes giving explicit references. A bibliography is appended. Signed (candidate) Date STATEMENT 2 I hereby give my consent for my thesis, if accepted, to be available for photocopying and for inter-library loan, and for the title and summary to be made available to outside organisations. Signed (candidate) ACKNOWLEDGEMENTS I would like to thank the following: • Dr Eshwar Mahenthiralingam, Professor Andrew Weightman and Dr Hefin Jones at the School of Biosciences, Cardiff University for their outstanding help, support and encouragement. • The UK Natural Environment Research Council (NERC) for funding this PhD studentship (grant NER/S/A/2002/10347A) and additional funding from the Environmental Genomics Thematic Program (grant NER/T/S/2001/00299). • My final year undergraduate students, Sara Morgan, Helen Rose and Daniel Brown for their assistance with the work in Chapters two, three and four respectively. • Prof J. M. Tiedje and Dr. A Ramette for welcoming me on my placement in East Lansing, Michigan and for their help and support, especially furthering my understanding of phylogenetics. • Dr Louise O’ Sullivan for provision of the STM mutant bank and her technical guidance. • Dr. K. E. Ashelford for assistance with in silico primer analysis. • Collaborators from the International Burkholderia cepacia Working Group (IBCWG) for their provision of bacterial strains and methods. SCIENTIFIC PUBLICATIONS, CONFERENCES AND PRESENTATIONS Publications: Payne, G. W., P. Vandamme, S. H. Morgan, J. J. Lipuma, T. Coenye, A. J. Weightman, T. H. Jones, and E. Mahenthiralingam. 2005. Development of a recA gene based identification approach for the entire Burkholderia genus. Applied & Environ Microbiol 71:3917-27 Payne, G. W., A. Ramette, H. L. Rose, A. J. Weightman, T. H. Jones, J. M. Tiedje, and E. Mahenthiralingam. 2006. Application of a recA gene-based identification approach to the maize rhizosphere reveals novel diversity in Burkholderia species. FEMS Microbiol Lett 259:126-32 Posters: Payne G. W., A. J. Weightman, T. H. Jones, S. H. Morgan, P. Vandamme and E. Mahenthiralingam. Expansion of the recA gene based identification approach to include the entire Burkholderia genus. 4th NERC Environmental Genomics programme science workshop. April 18 & 19, 2005. Manchester, UK Payne G. W., A. J. Weightman, T. H. Jones, S. H. Morgan, P. Vandamme and E. Mahenthiralingam. Expansion of the recA gene based identification approach to include the entire Burkholderia genus. 10th International Symposium on Microbial Ecology. August 22-27, 2004. Poster 319 Cancun, Mexico O’Sullivan L., G. W. Payne, A. Marchbank, A. Fivian, H. L. Rose, T. H. Jones, A. J. Weightman and E. Mahenthiralingam.. Environmental Burkholderia: fitness and phylogeny. 3rd NERC Environmental Genomics programme science workshop. April 5 & 6, 2004. Manchester, UK Payne G. W., A. J. Weightman, T. H. Jones and E. Mahenthiralingam. Genomic analysis of diversity and mechanisms associated with bioremediation by Burkholderia species. 2nd NERC Environmental Genomics programme science workshop. April 14 & 15, 2003. Bristol, UK Presentations: Expansion of the recA gene based identification approach to include the entire Burkholderia genus. 9th International Burkholderia cepacia Working Group meeting. April 2-4, 2004. Vancouver, Canada Burkholderia Species Diversity in an Environmental Sample. Molecular Analysis of recA. 3rd All Wales Microbiology Meeting. 14-16 March, 2005. Gregynog, Aberystwyth, Wales, UK Expansion of the recA gene based identification approach to include the entire Burkholderia genus. 2nd All Wales Microbiology Meeting. 15-17 March, 2004. Gregynog, Aberystwyth, Wales, UK SUMMARY Burkholderia is an important bacterial genus containing species of ecological, biotechnological and pathogenic interest which are widely distributed in the natural environment. This study describes the development of molecular methods to identify Burkholderia and also examines the genetic basis for their tolerance to halogenated pollutants. The recA gene is a useful target for speciation of B. cepacia complex bacteria. Novel PCR primers were evaluated by testing their specificity against a panel of Burkholderia species. Nucleotide sequencing and phylogenetic analysis of 869 bp of the recA gene differentiated between putative and known Burkholderia species, including the B. cepacia complex. A genus-specific recA PCR yielding 385 bp was also able to identify all Burkholderia examined. Phylogenetic analysis of 188 novel recA genes clarified the taxonomic position of several important Burkholderia strains, revealing the presence of four novel B. cepacia complex recA lineages. Although the recA phylogeny could not differentiate B. cepacia complex strains recovered from clinical infection versus the natural environment, it facilitated the identification of clonal strain types of from both niches. A recA-based cultivation-independent approach was used to examine Burkholderia diversity associated with the maize rhizosphere and cord forming fungi. Robust sequence datasets were created from the 869 bp recA fragments and screened to identify recA phylotypes matching B. cepacia complex species previously cultivated from the maize samples. Burkholderia related to known species as well as novel phylogenies were identified from the maize and fungal samples; in both cases the phylogenetic resolution of recA was sufficient to distinguish all individual species. Transposon mutagenesis of B. vietnamiensis G4 was used to elucidate the genetic basis of resistance to halogenated phenol derivatives. Mutations resulting in increased susceptibility to 2,4-dichlorophenol included genes encoding enolase, toluene tolerance, regulators and a lipoprotein. Gene complementation restored the resistant phenotype of two mutants, a GTP-binding protein and a methyltransferase gene. iv CONTENTS DECLARATION............................................................................................................................. ACKNOWLEDGEMENTS............................................................................................................. SCIENTIFIC PUBLICATIONS, CONFERENCES AND PRESENTATIONS............................ SUMMARY.......................................................................................................................................iv CONTENTS.......................................................................................................................................v LIST OF FIGURES.........................................................................................................................viii LIST OF TABLES............................................................................................................................ x LIST OF ABBREIVATIONS............................................................................................................xi CHAPTER 1: INTRODUCTION........................................................................................................... 1 1.1 BACTERIAL SYSTEMATICS....................................................................................................1 1.1.1 Introduction to classification...........................................................................................1 1.1.2 Classification by phenotypic characters.......................................................................3 1.1.3 Classification by genotypic characters.........................................................................5 1.1.4 Polyphasic taxonomy, a consensus approach to bacterial systematics.................7 1.2 MOLECULAR TAXONOMY......................................................................................................8 1.2.1 16S rRNA gene sequence analysis in bacterial systematics ....................................8 1.2.2 The re c A gene as a molecular chronometer in systematics ....................................8 1.2.3 Multi locus sequence typing (MLST) ............................................................................10 1.3 TAXONOMIC OVERVIEW OF THE GENUS BURKHOLDERIA AND ITS ECOLOGY.....11 1.3.1 Taxonomic history and