Copyright is owned by the Author of the thesis. Permission is given for a copy to be downloaded by an individual for the purpose of research and private study only. The thesis may not be reproduced elsewhere without the permission of the Author. Analysis of a Helicobacter pylori operon incorporating flagellar export genes A thesis presented in partial fulfllment of the requirements for the degree of Doctor of Philosophy in the Institute of Molecular BioSciences at Massey University, New Zealand Steffen Porwollik May 1999 Abstract Abstract Motility of Helicobacter species has been shown to be essential for successful colonization of the host. Previous studies indicated that the regulation of flagellar biosynthesis in the human gastric pathogen Helicobacter pylori differs from the suggested model for Gram-negative Enterobacteriaeceae. In this study, the organization of two H. pylori genes involved in export of flagellar structural proteins was investigated. A 7 kb fragment of the H. pylori 17874 genome was cloned. Sequence determination and analysis revealed a putative operon comprising an ORF of unknown function (ORF03), and genes for the isoleucyl-tRNA synthetase (ileS), an Agrobacterium tumejaciens VirBll homolog (virBl l ), an ATPase involved in flagellum-specific protein export (jli/), a presumptive flagellar export channel component (jliQ), and a homolog of an enzyme necessary for cell wall biosynthesis (murB) . The genetic organization of this region was found to be conserved in a panel of clinical H. pylori isolates, and in H. pylori 915 and SSl . The locus was also identified in the genome sequences of the H. pylori strains J99 and 26695. Cotranscription of ORF03, ileS, virBl l , flil, fliQ and murB was demonstrated by RT-PCR. Primer extension experiments identified the major transcription start site, which coincided with the A residue of the initiation codon of ORF03. A promoter element was inferred that resembled the E. coli (J70 consensus sequence. In addition, a minor transcription start site was detected upstream from ileS. Non-polar mutation of virBl l , flil and fliQ was generated by an allele replacement strategy. Engineered H. pylori flil and fliQ mutant strains were completely aflagellate and nonmotile, whereas a virBl l mutant still produced flagella and displayed slightly greater motility. The fliI and fliQ mutant strains produced severely reduced levels of flagellin and the hook protein AgE, although reduction was less stable in the flil mutant. Production of OMP4, a member of the outer membrane protein family identified in H. pylori 26695, was diminished in both the virBl l and the flil mutant. This suggested related functions of the putative virulence factor transport protein (VirB 11) and the flagellar export component (FIiI). ii Acknowledgments Acknowledgments I would like to express my sincere gratitude to my chief supervisor Dr Paul O'Toole for providing much scientific guidance and encouragement throughout every stage of this project. I am thankful for the many scientific discussions, and for immediate feedback concerning all the report and paper and thesis versions. Many thanks also for introducing me to real-life competetive science, and for giving me the opportunity to attend conferences both in and outside of New Zealand. And for modifying my American accent with this Irish gibberish. I am also grateful to my second supervisor, Dr Jan Schmid, for his contribution of ideas. Thanks for always encouraging a slightly less conventional approach to problems in molecular biology, which very often helped a lot. I am glad that someone with your microbiological background took part in the effort that lead to this thesis. Doug Hopcroft from The Horticultural and Food Research Institute of New Zealand, Palmerston North, provided the much needed expertise for the electron microscopy experiments. Thank you for your time and effort. A special thanks goes to the people from the Helipad crew, without whom I would never have pulled through. Thanks, Tash, for being the perfect lab organizer & Kiwi slang teacher, and always a good person to talk to when experiments went down the drain. It was great having you around. To Millis, cheers for some very valuable and enjoyable dialogue I had missed for too long. To Kirsty, Basil, James & Ross, thanks for making lab work a lot funnier and more pleasant than it could be. I thankfully acknowledge fmancial support from the award of a Massey University Doctoral Scholarship, and the Molecular Genetics Research Scholarship of the former Department of Microbiology and Genetics. Travel funding was gratefully received from the Massey University Graduate Research Fund, the Department of Microbiology and Genetics, and the Institute of Molecular BioSciences at Massey University. Many people have assisted me in various ways throughout the course of this work, by providing support and words of encouragment, or just by listening when I needed to talk. Thank you to all these people, especially Martin, Andrew & Cheyenne. Thanks to my friends in Palmy. I'm glad I met you. Danke, Katja, fiir all die Briefe. Und Ralf, Carola, Eike, Lutz & Betty, danke dafiir, daB ich mich in Berlin noch immer zu Hause fiihl' . Ich hab Euch vermiBt. ill Related Publications Related Publications Some of the material presented in this thesis has been published. Porwollik, S., Noonan, B. & Q'Toole, P.W. (1999). Molecular characterization of a flagellar export locus of Helicobacter pylori. Infect Immun 67:2060-2070. Porwollik, S. & Q'Toole, P.W. (1998). Molecular characterization of a flagellar export locus of Helicobacter pylori. Gut 43 (Suppl 2): A02/31 IV Abbreviations A+T content of deoxyadenylate and deoxythymidylate in DNA aa amino acid ab antibody ABI Applied Biosystems AMV avian myeloblastosis virus Ap ampicillin APS ammonium persulphate ATP adenosine triphosphate BBH basal body-hook complex BCIP 5-bromo-4-chloro3-indolyl phosphate BLAST basic local alignment search tool BSA bovine serum albumine cAMP cyclic adenosine monophosphate CAP catabolite gene activator protein CBA columbia base agar cDNA complementary DNA Cm chloramphenicol colEl colicin El CRP cyclic adenosine monophosphate receptor protein CSPD disodium 3-(4-methoxyspiro{ 1,2-dioxetane-3,2' -(5'- chloro )tricyclo[3.3.1.1. ]decan} -4-yl)phenyl phosphate CTP cytidine triphosphate dA TP 2' deoxyadenosine triphosphate DEPC diethylpyrocarbonate dGTP 2' deoxyguanosine triphosphate DIG digoxigenin DNA deoxyribonucleic acid DNase deoxyribonuclease dNTP deoxynucleoside triphosphate dTTP 2' deoxythymidine triphosphate dUTP 2' deoxyuridine triphosphate EDT A ethy lenediarninetetraacetic acid EPB electroporation buffer f1 bacteriophage f1 Fab variable sequence fragment of immunoglobulin Abbreviations v Abbreviations FSB final sample buffer FP forward primer G+C content of deoxyguanylate and deoxycytidylate in DNA GSP general secretory pathway GTP guanosine triphosphate IL interleukin IPTG isopropyl-B-D-galactoside Kan kanamycin LB Luria-Bertani broth LBA Luria-Bertani agar LPS lipopolysaccharide MALT mucosa-associated lymphoid tissue md monoclonal MCS multicloning site MOPS 3-(N-morpholino) propane sulphonic acid n/a not applicable NBT 4-nitro blue tetrazolium chloride NCBI National Center for Biotechnology Information Neo neomycin OD optical density ORF open reading frame ori origin of replication PBS phosphate-buffered saline pd polYclonal PCR polymerase chain reaction PEG polyethylene glycol pI isoelectric point PIR protein information resource r resistant RNA ribonucleic acid RNase ribonuclease RP reverse primer rpm revolutions per minute RT room temperature RT -PCR polymerase chain reaction involving an initial reverse transcriptase step SDS sodium dodecyl sulphate SM-TBS skim milk powder in Tris-buffered saline vi Abbreviations SV 40 simian virus 40 TB terrific broth TBS Tris-buffered saline TEMED NNN'N' tetramethylethylenediamine TIGR The Institute for Genomic Research T m melting temperature TNF tumor necrosis factor Tris tris(hydroxymethyl)methylamine TSB tryptic soy broth U unit UTP uridine triphosphate Vol volume w/v weight per volume X -gal 5 -bromo-4-chloro-3-indoly 1-B-D-galactoside In addition, the conventional one-letter codes for amino acids, deoxyribonuc1eosides and ribocuc1eosides were applied: amino acids: G, A, V, L, I, P, F, Y, W, S, T, C, M, N, Q, D, E, K, R, H for glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, tyrosine, tryptophan, serine, threonine, cysteine, methionine, asparagine, glutamine, aspartate, glutamate, lysine, arginine and histidine, respectively deoxyribonucleosides: A, C, G, T for deoxyadenylate, deoxycytidylate, deoxyguanylate and deoxythymidylate, respectively ribonucleosides: A, C, G, U for adenylate, cytidylate, guanylate and uridylate, respectively vii Table of Contents Table of Contents ABSTRACT ................................................................................. ii ACKNOWLEDGMENTS ........................... . . . . . . ... .. . ........................ iii RELATED PUBLICATIONS ........................................................ .iv ABBREVIATIONS ................................................ . ....................... v TABLE OF CONTENTS . . ...... . . .. ...................................... ....... .... viii LIST OF FIGURES ............................................ ... . ......... . ... .. . . . . . xii LIST OF TABLES . . ... . .. . .......... . ................................................. xiv 1 . INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1.1 A BACTERIUM THAT CAUSES GASlRIC DISEASE: HEUCOBACTER PYWRl ........................ 1 1.1.1 History and general features . ......................................................... 1 1.1.2 H. pylori pathogenesis .................................................... . . . . ... . . . . . 3 1 . 1 .2. 1 Pathogenic factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 1 . 1 .2.2 Clinical outcomes and treatment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 1.1.3 The H. pylori genome ..................................... .......................... 10 1.2 PROTEIN SECRETION SYSTEMS IN BACTERIA ................................... ........ ......... 12 1.3 FLAGELLAR BIOSYN11IESIS ................................................................... .. 19 1.3.1 Structure and assembly of a flagellu1n in Gram-negative bacteria .............. 19 1 .3 . 1 . 1 Flagellum morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . .. . . . . . . . . . . . . .. .... ...... . .. .. . . . . . ... .. 19 1 .3 . 1 .2 Assembly of the flagellum . . . . . . . . . . . . ... . . . .. . . .. .. . . . . . . . . . . .. . . . . . . . . . .. .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1 1.3. 1 .3 Regulation of flagellar gene expression . ... . .. .. .. . . . . . . ... . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. ... .. 23 1.3.2 The flagellar protein export apparatus ............................................. 26 1.3.3 Unique aspects of H. pylori flagellum biology ................................... 28 1.4 AIMs OF THIS STUDY .................................................................. .. . . . .. . . . 31 2. MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 3 2.1 BACTERIAL STRAINS, CULTURE ANDSTORAGE CONDmONS ............................... .... 33 2.2 MEDIA AND SUPPLEMENTS ....................................................... ............... 34 2.3 OLIGONUCLEOTIDE PRIMERS ............................... ............................... ..... 35 2.4 VECTORS AND RECOMBINANT PLASMIDS ... .......... . ............ .... ............. . .. ......... 36 2.5 ANTISERA ... . . . . . . . . ...... . ........................ . ..... . . . . . . . . . . .. . . ... ............ . . . . . . . . .. 38 2.6 DNA ISOLATION .................................. . .. . . . ...................... . ................ 38 2.6.1 Plasmid preparation ......................... . . . ....................... . .............. 38 2.6. 1 . 1 Easy plasmid minipreparation . . . ..... . . . . . . . . ... . . . ... . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . ..... . . . . . . . . . . . . 38 2.6.1.2 Wizard plasmid minipreparation . . . .. . . . . . . . . . . . . . . . . . . . . . . . . ...... .. . .. . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . .. 39 viii Table of Contents 2.6.1.3 High Pure plasmid preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 2.6.1.4 AB! plasrnid preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 2.6.2 Preparation of genomic DNA from Helicobacter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .40 2.7 DNA ANALYSIS METHODS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 1 2. 7 . 1 DNA agarose gel electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 1 2.7 .2 DNA restriction endonuclease treatment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 1 2 .7 .3 DNA quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 2 .7 .4 Southern blotting and hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42 2 .7 .5 DNA sequencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 2.8 DNA AMPLIFICATION BY POLYMERASE CHAIN REACTION (PCR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 2.9 CLONING PROCEDURES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 2.9.1 DNA preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 2.9.2 Ligation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 2.9.3 Transformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 2.9.3.1 Preparation of competent bacterial cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 2.9.3.2 Transformation of E. coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 2.9.3.3 Transformation of H. pylori . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 2. 10 GENERAL PRECAtJfIONS FOR RNA WORK . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 2 . 1 1 RNA ISOLATION FROM H. PYWRl CELLS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 2. 1 1 . 1 Total RNA isolation using TRIzol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 2. 1 1 .2 RNA isolation using density gradient ultracentrifugation . . . . . . . . . . . . , . . . . . . . . . 5 1 2 . 1 2 RNA ANALYSIS METHODS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 2. 1 2. 1 RNA agarose gel electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 2.12.2 RNA quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 2. 1 2.3 Northern blotting and hybridization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 2.12.3.1 Northern analysis using DIG-labelled DNA probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 2.12.3.2 Northern analysis using ECL TM-labelled DNA probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 2.12.3.3 Northern analysis using radiolabelled DNA probes ............................................... 54 2.12.3.4 Northern analysis using DIG-labelled riboprobes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54 2. 1 2.4 Transcript analysis by RT-PCR. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 2. 1 2.5 Transcript analysis by primer extension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 2. 1 3 PROTEIN SAMPLE PREPARAT ION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 2. 13 . 1 Whole cell lysates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 2. 13 .2 Cell fractionation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 2. 14 PROTEIN ANALYSES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 2. 14. 1 Protein quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 ix Table of Contents 2.14.2 Protein electrophoresis . .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . .. . . . 59 2.14.3 Western blotting and hybridization . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . 60 2.15 MICROSCOPY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 2.15.1 Phase contrast microscopy . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 2.15.2 Electron microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . 62 3. RESULTS .................................................................. 63 3.1 CLONING OFTHEHEUCOBACFER PYLORI Ful GENE ........................................... 63 3.1.1 The ",ZAP excisant pHP042 ........................................................ 63 3.1.2 Subcloning of an H. pylori DNA segment comprisingjlil. . . . . . . . . . . . . . . . . . . . . . 64 3.2 SEQUENCEANALYSI SOFTHE PSPI02 INSERT .................................................. 69 3.2.1 Sequencing strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 3.2.2 Identification of the genetic elements on the pSP 102 insert . . . . . . . . . . . . . . . . . . . . . 71 3.2.3 Further sequence features of the pSP 102 insert . . . . . . . . . . . . . . . . . .. . . .. . . . . . . . . . . . 80 3.3 CONSER VATIONOFTHEPUTATIV EOPERONINH. PYWRI . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 3.41'RANsCRIPT ANALySES ......................................................................... 86 3.4.1 Transcript detection attempts by Northern blotting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86 3.4.2 RT-PCR transcript analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90 3.4.3 Promoter mapping by primer extension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92 3.5 KNOCKOUT MUTAGENESIS OF THE GENES OF THE EXPORT LOCUS ............................... 95 3.5.1 Allele replacement strategy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95 3.5.2 Preparation of the mutagenic constructs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 3.5.2.1 The virBJ J mutagenic constructs pSPI17 and pSPl1 8 .......................................... 97 3.5.2.2 The fliI knockout plasmids pSP1 08 and pSP110 .................................................. 99 3.5.2.3 ThefliQ disrupting plasmjds pSPI07 and pSPI09 .............................................. 1 00 3.5.3 Generation of H. pylori virBll ,jlil andjliQ mutants . . . . . .. . . . . . . . . . . . . . . . . . . 100 3.6 NON-POLARITY OF THE MUTATIONS .......................................................... 102 3.7 PHENOTYPIC CHARACTERIZATlONOFTHEH. PYWRIMUfANfS . . . . . . . . . . . . . . . . . .. . . . . . . . . . . 105 3.7.1 Growth characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 106 3.7.2 Motility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106 3.7.3 Flagellum production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107 3.7.4 Expression of structural flagellar components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107 3.7.4.1 Flagellin and FlgE expression patterns in subcellular fractions of H. pylori . . ........... 1 07 3.7.4.2 Stability of the effects of the mutageneses on flagellar protein production ................ 109 3.7.5 Production of H. pylori virulence factors . . . ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111 3.7.6 Expression of outer membrane proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . 114 x Table of Contents 4. DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 6 4.1 MAJOR F INDINGS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116 4.2 CON1R1BUTION OF TH IS STUDY TO THE F IELD . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . . ........ . 116 4.3 DISCUSS ION OF THE EXPER IMENTAL DATA . . . . . . . . . . . . . ....... . . . ..... . .. . . . . . . . . . . . . ... . . . . . . 120 4.3.1 The genetic organization of the investigated operon ........................... 120 4.3.2 Conservation of the operon in H. pylori ........................................ 124 4.3.3 Transcriptional regulation of the operon ......................................... 126 4.3.4 Successful mutagenesis of H. pylori 17874 virBll, fliI andfliQ ............ 128 4.3.5 Phenotypic consequences of the virBll, flil andfliQ knockout ............. 130 4.4 FuTuRE STUDIES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133 APPENDIX 1 Physical maps of the pHP and pSP series of plasrnids ........... 137 APPENDIX 2 The complete sequence of the pSP102 insert ...................... 149 REFEREN CES ......................................................................... 162 Xl List of Figures List of Figures Figure 1.1. Electron micrograph of a mucosal biopsy with active chronic gastritis . . . . 2 Figure 1.2. Schematic diagram of currently classified bacterial protein secretion systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 Figure 1.3. Model of the Agrobacterium T-complex transport apparatus . . . . .... . . ..... 18 Figure 1.4. Structure of the flagellum of a Gram-negative bacterium like S. typhimurium and E. coli. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . 20 Figure 3.1. An internal segment of the H. pylori 1 7874 fliI gene has joined the rest of the pHP042 insert by scrambled cloning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 Figure 3.2. Cloning of the H. pylori 1 7874 fliI gene . . .. . . . . . . . .. . . . . . .. . . . ....... . . . . . . .. 67 Figure 3.3. The flil probe hybridized near the insert end of pSP 1 0 1 . . . . . . . . . . . . . . . . . . . . 68 Figure 3.4. The 0 . 7 kb Hindill fragment of the pSP102 insert is contiguous with the pSPI0 l insert in the H. pylori 1 7874 genome . . .. . . . ........... . . . . ..... . . .. . . . .. . . 70 Figure 3.5. Sequence determination of the pSPI02 insert . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72 Figure 3.6. Comparison of the pSP102 insert with the corresponding regions from the H. pylori J99 and 2 6695 genomes .... . . . . . . . .. . . . . . . . . . . . . . . . . . . . . .. . . .. . .. . . . . . ... . . 73 Figure 3.7. Similarity of H. pylori 1 7874 VirBl l to presumptive ATP binding nucleoprotein transport components . . . ............. . ..... . ... . .. . . . . . . . . . . . . . . . . . . . . . . . . . 76 Figure 3.8. Conserved sequence motifs in H. pylori 1 7874 Flil. . . . . . . . . . . .. . . . . . ..... . . 78 Figure 3.9. Similarity in hydropathicity plots of H. pylori 1 7874 FliQ and components of type ill protein export systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79 Figure 3.10. The genetic linkage of the components of the putative operon is conserved in H. pylori . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82 Figure 3.1 1. The flanking regions of the putative operon are not strictly conserved in H. pylori . . . . . ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84 X11 List of Figures Figure 3.12. The components of the putative H. pylori operon are not conserved in H. mustelae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. ... . . . .. . . . . . . . . . . . . . .... .. ... .. . . . ... . . . . . 8 5 Figure 3.13. Transcript detection by Northern analysis using gene specific probes .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 9 Figure 3.14. ORF03, ileS, virB 1 1 , flil, fliQ and murB are cotranscribed . . . . .. . . . . . . . . 91 Figure 3.15. Generation of defined cDNA fragments by primer extension ............. 93 Figure 3.16. Transcription start sites and inferred promoters of the operon . . . . . . . . .. . . 94 Figure 3.17. Schematic representation of the strategy for deletion-insertional knockout of H. pylori genes (allele replacement strategy) .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96 Figure 3.18. Schematic representation of the relevant genomic DNA regions in the H. pylori wild type and knockout mutants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . 98 Figure 3.19. PCR verification of H. pylori knockout mutants ......................... 103 Figure 3.20. The deletion-insertion mutations are non-polar. . . . . . . . . .. . . . . . . . . . . . . . . . . 1 04 Figure 3.21. Electron micrographs of negatively stained preparations of H. pylori cells . ........................................................................................ 108 Figure 3.22. Altered flagellin and FlgE production levels in H. pylori virB l l , fliI and fliQ mutants. . .................................................................... 110 Figure 3.23. Influence of passage on flagellin and FlgE production levels in the H. pylori mutants .......................................................................... 112 Figure 3.24. Expression of UreB, CagA and VacA is not altered in the H. pylori virB 1 1 , flil and fliQ mutants compared to the 17874 wild type . .................... 113 Figure 3.25. Influence of the knockout mutations on expression of two outer membrane proteins (HopB and OMP4) in H. pylori . . .. . ... .. . ..... . ................. 115 Figure 4.1. Major results of the investigations presented in this thesis . . . . . . . . . . . . . . . . 117 xiii List of Tables List of Tables Table 1.1. General features of protein secretion systems in Gram-negative bacteria . .. 14 Table 1.2. Presumed homologs in type ID secretion systems and the flagellar protein export apparatus . . ................... . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28 Table 2.1. Bacterial strains . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . .. . ... . ... . ... . . . .. . . . .. . . . . .. . ... : ... 33 Table 2.2. Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 Table 2.3. Media supplements and antibiotics ............ . . . . . . . . . . ... . . .................... 34 Table 2.4. Oligonucleotide primers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 Table 2.5. Plasmids used in this study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 Table 2.6. Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 Table 3.1 . Restriction analysis of the two plasmid variants identified by the fliI probe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66 Table 3.2. Genetic elements present in the pSP102 insert . . . .. .. .. . . . . . . . . . . . . . . . . . . . . . . . . 74 Table 3.3. Summary of transcript detection attempts by Northern analysis . . . . . . . . . . . . . 88 Table 3.4. Mutagenesis of H. pylori by electroporation or natural transformation ... 101 Table 3.5. Calculated PCR product sizes from H. pylori wild type and mutant genomic DNA . ......... . ....... . . .. . ..... . . .. . .. . . .. . . . . ........... . . . . . . . .. . . . . .. 102 xiv