Please read the abstract in the section 00front of this documentDissertation (MSc (Microbiology))--University of Pretoria, 2005.Microbiology and Plant Pathologyunrestricte

Van Schalkwyk, Antoinette

Dissertation (MSc (Microbiology))--University of Pretoria, 2005.Please read the abstract in the section 00front of this documentMicrobiology and Plant Pathologyunrestricte

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Characterization of a putative pilus assembly and secretion system in Pseudomonas aeruginosa DSM1707 by ANTOINETTE VAN SCHALKWYK Submitted in partial fulfilment of the requirements of the degree Master of Science in the Faculty of Natural and Agricultural Sciences University of Pretoria Pretoria November 2003                                                   © University of Pretoria Characterization of a putative pilus assembly and secretion system in Pseudomonas aeruginosa DSM1707 by ANTOINETTE VAN SCHALKWYK Submitted in partial fulfilment of the requirements of the degree Master of Science in the Faculty of Natural and Agricultural Sciences University of Pretoria Pretoria November 2003    ACKNOWLEDGEMENTS To my Heavenly Father, for every day and every bug. To my parents, Livio and Jacques, for being there every day. To Jacques Theron, for believing in me and the Htp system. To Prof. Brazel, R. MacDonald, B. Steyn, M. Oosthuizen , and A. Hall, for helping me with their expertise. To Dr Thierry Jouenne, and every-one at Rouen, for helping me with 2D gells and sequencing. To the NRF-CNRS, for funding the project and providing the means to travel. To the 9th floor, for being my home away from home, especially my big brothers Francois and Boet.    / ... I1 SUMMARY Characterization of a putative pilus assembly and secretion system in Pseudomonas aeruginosa DSM1707 by ANTOINETTE V AN SCHALKWYK Supervisor: 	 Prof. 1. Theron Department of Microbiology and Plant Pathology University of Pretoria Co-supervisor: 	 Prof. V.S. Brozel Department of Microbiology and Plant Pathology University of Pretoria for the degree M.Sc Pseudomonas aeruginosa, an ubiquitous environmental bacterium and an opportunistic human pathogen, forms biofilms through a series of interactions between the cells and adherence to surfaces. Adherence of P. aeruginosa to surfaces is often mediated by surface appendages such as flagella and type IV pili . In this study, a gene cluster in P. aeruginosa was identified in silico that encoded predicted protein products with homology to those encoded by two recently described novel pilus biogenesis and assembly systems of Actinobacillus actinomycetemcomitans and Caulobacter crescentus, respectively. Both these systems are involved in the production of a novel class of pili, which, in A. actinomycetemcomitans, are associated with the ability of the bacterium to bind non­specifically to inert surfaces. The homologous genes in P. aeruginosa, which have not been characterized previously, were named htp for homologous to type IV pilus biogenesis genes. To determine the functional importance of the htp gene cluster in P. aeruginosa, the htpD, htpE and htpDEF open reading frames (ORFs), which are highly conserved in the respective pilus biogenesis systems, were targeted for insertional inactivation. Whereas HtpD may function as an NTPase, the amino acid sequence of HtpE and HtpF indicate membrane localization, but no obvious functions. The respective htp ORFs were inactivated in P. aeruginosa strain DSM 1707 by homologous recombination with appropriately constmcted allelic exchange vectors to generate mutant strains DSMHtpD, DSMHtpE and DSMHtpDEF. The DSMHtpDEF mutant strain was found to be severely growth-impaired and was    consequently excluded from further analysis. Comparative analysis of the wild-type P. aeruginosa DSM1707 and mutant DSMHtpD and DSMHtpE strains revealed that whereas the DSMHtpE strain generally resembled the wild-type strain, the DSMHtpD strain was impaired in its ability to grow as a biofilm, and electron microscopic studies revealed that the cells of DSMHtpD were notably longer compared to the wild-type DSM1707 and mutant DSMHtpE cells. Furthermore, two-dimensional gel electrophoretic analysis of the extracellular proteins of the wild-type P. aeruginosa DSM1707 and mutant DSMHtpD strains revealed differences between the extracellular proteomic profiles. Based on the results obtained during the course of this investigation, it can be proposed that the newly identified hlp system of P. aeruginosa plays a role in the ability of this bacterium to successfully colonize abiotic surfaces. The more severe perturbations resulting from inactivation of the hlpD ORF furthermore suggests that the encoded putative NTPase protein plays an important role in the putati ve hlp pilus biogenesis/secretion system. Thus, it would appear that multiple factors are available to P. aeruginosa to facilitate its binding to various surfaces and possibly for interbacterial adhesion. The existence of different attachment mechanisms could reflect the complex needs of P. aeruginosa during colonization of diverse environmental niches.    iv T ABLE OF CONTENTS . ACKNOWLEDGEMENTS SUMMARY II LIST OF ABBREVIATIONS IX LIST OF FIGURES XI LIST OF TABLES XV CHAPTER 1: LITERATURE REVIEW 	 1 1.1 	 GENERAL INTRODUCTION 2 1.2 	 BIOFILM DEVELOPMENT 3 1.2.1 Steps in biofilm development 	 3 1.3 	 BACTERIAL COMPONENTS REQUIRED FOR INITIAL ATTACHMENT 6 1.3 .1 	 Importance of flagella , pili and adhesins in bacterial attachment to surfaces 6 1.4 	 FIMBRIAL EXPRESSION AND ASSEMBLY 8 1.4.1 Type 1 and P pili 	 8 1.4.1.1 	 General characteristics 9 1.4.1.2 	 Model for biogenesis of type 1 pili and P pili of E. coli 10 1.4.2 	 Curli 14 1.4.2.1 	 General characteristics 14 1.4.2.2 	 Model for biosynthesis of curJi fimbriae 15 1.4.3 	 Type IV pili 16 1.4.3.1 	 General characteristics 16 1.4.3.2 	 Model for type IV pilus biogenesis 17 1.4.3.3 	 Relationship of the type IV pilus biogenesis system with the type II secretion system 20 1.5 	 NOVEL FIMBRIAL EXPRESSION AND ASSEMBLY SYSTEMS 22 1.5.1 	 The Flp fimbriae of Actinobacillus actinomycetemcomitans 22 1.6 	 AIMS OF THIS STUDY 24    v CHAPTER 2: 	 IN SILICO IDENTIFICATION OF A NOVEL PUTATIVE PILUS BIOGENESIS SYSTEM IN Pseudomonas aerugillosa 2.1 INTRODUCTION 2.2 MATERIALS AND METHODS 2.2.1 Computer analysis 2.3 RESULTS 2.3.1 Sequence analysis 2.3.1.1 	 Identification of a gene cluster in P. aeruginosa homologous to the Jlp-rcp-tad and pilA-cpa gene clusters 2.3.1.2 	 Homology searches 2.3.2 In silico analysis of the P. aeruginosa PA4305 - PA4297 gene cluster 2.3.2.1 	 Identification of putative consensus promoter sequences 2.3.2.2 	 Transcriptional organization of the P. aeruginosa PA4305 - PA4297 gene cluster 2.3.3 Properties of the putative Htp proteins of P. aeruginosa 2.3.3.1 	 Proteins predicted to be localized to the outer membrane 2.3.3.2 	 Proteins predicted to be localized to the peri plasmic space 2.3 .3.3 	 Proteins predicted to be localized to the inner membrane 2.3.3.4 	 Proteins predicted to be localized to the cytoplasm 2.3 .3.5 	 Characterization of the putative prepilin, HtpP 2.4 DISCUSSION CHAPTER 3: 	 CONSTRUCTION OF htp MUTANT STRAINS OF P. aerugillOsa DSM1707 3.1 INTRODUCTION 3.2 MATERIALS AND METHODS 3.2.1 Bacterial strains, plasmids and culture conditions 3.2.2 Genomic DNA isolation 3.2.3 DNA amplification 3.2.3 .1 	 Oligonucleotide primers 3.2.3.2 	 Polymerase chain reaction (PCR) amplification 26 27 29 29 30 30 30 32 33 33 34 37 39 40 43 43 44 50 55 56 58 58 58 59 59 59    VI 3.2.4 Agarose gel electrophoresis 	 61 3.2.5 Purification of DNA fragments from agarose gels 	 61 3.2.6 Restriction endonuclease digestions 	 62 3.2.7 Cloning of DNA fragments into plasmid vectors 	 62 3.2.7.1 	 Ligation of DNA fragments 62 3.2.7.2 	 Preparation of competent E. coli DH5a cells 63 3.2.7.3 	 Transformation of competent E. coli DH5a cells 63 3.2.8 Extraction and purification of plasmid DNA 	 63 3.2.8.1 	 Plasmid DNA extraction 63 3.2.8.2 	 Purification of plasmid DNA 64 3.2.9 Nucleic acid sequencing 	 64 3.2.9.1 	 Sequencing reactions 64 3.2.9.2 	 Sequence analysis 65 3.2.10 Construction of allelic exchange vectors 	 65 3.2.10.1 	 Cloning of a gentamicin resistance cassette into pUC 18 67 3.2.10.2 	 Cloning of the full-length htpDEF ORFs into pUC18 67 3.2.10.3 	 Construction of allelic exchange vector pUC18-htpD-Gent 69 3.2.10.4 	 Construction of allelic exchange vector pUC 18-htpE-Gent 69 3.2.10.5 	 Construction of allelic exchange vector pUC18-htpDEF-Gent 69 3.2.11 Construction of mutant P. aeruginosa DSM1707 strains 	 71 3.2.11.1 Preparation of competent P. aeruginosa DSM1707 cells 71 3.2.1l.2 Transformation of competent P. aeruginosa cells 71 3.2.11 .3 	 Triparental conjugation 72 3.2.12 Characterization of mutant P. aentginosa DSM1707 strains 72 3.2.12.1 	 Southern blot analysis 72 3.2.12.1.1 Preparation of labeled probes 	 72 3.2.12.1 .2 Preparation of the membranes 	 73 3.2.12.1.3 Nucleic acid hybridization 	 74 3.2.12.2 	 PCR analysis 74 3.2.] 2.2.1 Oligonucleotide primers 75 3.2.12.2.2 PCR amplification 	 75 3.2.13 Complementation analysis 	 75 3.2.13.1 	 Cloning of a kanamycin resistance gene into pBluescript SKU 76 3.2.13.2 	 Construction of complementation plasmid pJB-htpDEF-Kan 76    vii 3.3 	 RESULTS 76 3.3.1 	 Construction of pUC] 8-Gent 78 3.3 .2 	 Construction of a recombinant pUC] 8 vector containing the full-length htpD, htpE and htpF ORFs 80 3.3.2.] Construction of plasmid pUC] 8-IFOR 80 3.3.2.2 Construction of plasmid pGEM-NFIR 	 82 3.3.2.3 Construction of recombinant plasmid pUC 18-DEF 85 3.3.2.4 Sequence analysis 	 87 3.3.3 Construction of different allelic exchange vectors 	 87 3.3.3. ] Construction of allelic exchange vector pUC] 8-htpD-Gent 87 3.3.3.2 Construction of allelic exchange vector pUCl S-htpE-Gent 88 3.3.3.3 Construction of allelic exchange vector pUC] 8-htpDEF-Gent 90 3.3.4 Construction of mutant strains of P. aeruginosa DSM] 707 92 3.3.5 Characterization of the putative P. aeruginosa htp mutant strains 94 3.3 .5.1 Southern blot analysis 	 94 3.3.5.2 PCR analysis 	 96 3.3.6 Construction of complementation plasmid pJB-DEF-Kan 	 97 3.4 	 DISCUSSION 100 CHAPTER 4: INVOLVEMENT OF THE htp GENE CLUSTER OF P. aeruginosa DSM1707 IN BIOFILM FORMATION AND PROTEIN SECRETION 105 4.1 	 INTRODUCTION 106 4.2 	 MATERIALS AND METHODS 108 4.2.] Bacterial strains, plasm ids and culture conditions 108 4.2.2 	 Batch assays 108 4.2.2.] Culturable counts 109 4.2.2.2 Total protein concentration 	 109 4.2.3 	 Light microscopy 110 4.2.4 	 Transmission and scanning electron microscopy 110 4.2.5 	 Characterization of mutant cultures ]11 4.2.5.1 Gram staining 	 111    viii 4.2.5.2 Fluorescent in situ hybridization III 4.2.5.2.1 Preparation of gl ass slides III 4.2.5.2.2 Oligonucleotide probe 112 4.2.5.2.3 Hybridization of whole cells 112 4.2.5.3 16S rDNA analysis 113 4.2.5.3.1 PCR amplification of the 16S rDNA gene 113 4.2.5 .3.2 Nucleic acid sequencing and analysis 113 4.2.6 Two-dimensional gel electrophoretic analyses of extracellular proteins 114 4.2.6.1 Preparation of extracellular protein samples 114 4.2.6.2 Concentration of protein samples 114 4.2.6.3 Two-dimensional gel electrophoresis 115 4.2.6.4 Staining of 2DE gels 115 4.2.6.5 Image analysis 116 4.2.6.6 N-terminal amino acid sequencing and protein identification 116 4.3 RESULTS 116 4.3 .1 Growth curves 117 4.3.2 Microscopic analysis of biofilm phenotypes 120 4.3 .3 Characterization of the mutant cultures 120 4.3.4 Biofilm development on glass wool 123 4.3.5 Electron microscopic characterization of cellular morphology 125 4.3 .5.1 Scanning electron microscopy 125 4.3.5.2 Transmission electron microscopy 128 4.3.6 Two-dimensional gel electrophoresis of extracellular proteins 133 4.4 DISCUSSION 136 CHAPTER 5: CONCLUDING REMARKS 144 REFERENCES 151    ix A amp' ATP bp ca. °C ddH20 DMSO DNA dNTP DTT EDTA e.g. EtOH Fig. gent' h IPTG kan' kb kDa lacZ LB-broth M rnA MCS mg min ml mM nm LIST OF ABBREVIATIONS Absorbance ampicillin resistance adenosine triphosphate base pair approximately degrees Celsius deionized distilled water dimethyl sulfoxide deoxyribonucleic acid deoxyribonucleoside-5 '-tri phosphate dithiothreitol ethylenediaminetetra-acetic acid for example ethanol figure gentamicin resistance hour isopropy I ~-D-thiogalactoside kanamycin resistance kilobase pairs kilodalton p-galactosidase gene Luria-Bertani broth litre molar milliampere multiple cloning site milligram minute millilitre millimolar nanometer nucleotide ammonium acetate    x OD ORF peR PEG pmol RNA rpm s SDS TE tet' TN-medium 2D 2-DE U /1g /1 1 UHQ UV V v/v w/v X-gal optical densi ty open reading frame polymerase chain reaction polyethylene glycol picomoJe ribonucleic acid revolutions per minute second sodium dodecyl sulphate Tris-EDTA · tetracycline resistance tryptone-nitratc medium two-dimensional two-dimensional electrophoresis units microgram microlitre ultra high quality ultraviolet volts volume per volume weight per volume 5-bromo-4-chloro-3-indolyl-p-D-galactopyranoside    xi LIST OF FIGURES FIGURE NUMBER Fig. 1.1 Model of biofilm development. 5 Fig. 1.2 Operons encoding type 1 and P pili in uropathogenic E. coli strains. 9 Fig. 1.3 Model for biogenesis of P pili by the chaperone-usher pathway. 13 Fig. 1.4 Models for type II secretion and type IV pilus biogenesis. 21 Fig. 2.1 Diagrammatic representation of the P. aernginosa open reading frames (PA4306 - PA4297). which display homology to the pilus biogenesis systems of A. actinomycetemcomitans and C. crescentus. respectively. 31 Fig. 2.2 Sequence of the predicted PA4306 and PA4305 promoters. 34 Fig. 2.3 Organization of the newly identified htp gene cluster in P. aeruginosa compared to theflp-rcp-tad gene cluster in A. actinomycetemcomitans. H. ducreyi and P. multocida. the pitA-cpa gene cluster in C. crescentus. and the ctp gene cluster in A. tumefaciens. 35 Fig. 2.4 Alignment of the deduced amino acid sequences of P. aernginosa PA4295 with several members of the prepilin peptidase family. 38 Fig. 2.5 Alignment of the deduced amino acid sequence of P. aernginosa HtpB with several members of the pulD/pIV family of outer membrane channel proteins. 41 Fig. 2.6 Alignment of the deduced amino acid sequence of P. aernginosa HtpD with several members of the TrbBIVirB 11 family of secretion A TPases. 45 Fig. 2.7 Alignment of the deduced amino acid sequence of P. aernginosa HtpP with several proteins of theflp subfamily of pili. 47    xii Fig. 3.1 Schematic presentation of specific primer annealing positions and direction of sequencing in the htpDEF open reading frames and adjacent P. aeruginosa nanking DNA in pUC18-DEF. 66 Fig. 3.2 Strategy for the construction of pUC18-DEF harboring the full-length htpD, htpE and htpF open reading frames of P. aeruginosa DSM1707. 68 Fig. 3.3 Strategies for the construction of the allelic exchange vectors pUC18-htpD-Gent, pUC18-htpE-Gent and pUC18-htpDEF-Gent. 70 Fig. 3.4 Strategy for the construction of the complementation plasmid pJB-DEF-Kan harboring the htpDEF open reading frames of P. aeruginosa DSM1707 under transcriptional control of the lac promoter obtained from plasmid pBluescript. 77 Fig.3.5a Plasmid map of the recombinant plasmid pUC18-Gent containing a cloned copy of a gentamicin resistance cassette. 81 Fig. 3.5b Agarose gel electrophoretic analysis of the recombinant plasmid pUC18-Gent constructed by cloning the gentamicin resistance cassette into the Sad and SmaI sites of pUC18. 81 Fig. 3.6a Agarose gel electrophoretic analysis of the recombinant plasmid pGEM-IFOR constructed by cloning the Tad-IF - Tad-OR amplicon into pGEM®-T Easy vector DNA. 83 Fig. 3.6b Agarose gel electrophoretic analysis of recombinant plasmid pUC18-IFOR. 83 Fig. 3.7 Agarose gel electrophoretic analysis of the recombinant plasmid pGEM-NFIR constructed by cloning the Tad-NF - Tad-IR ampJicon into pGEM®-T Easy vector DNA. 84 Fig. 3.8a Plasmid map of the recombinant plasmid pUC18-DEF. 86 Fig. 3.8b Agarose gel electrophoretic analysis of recombinant plasmid pUC18-DEF constructed by cloning the Tad-NF - Tad-IR amplicon from pGEM-NFIR into the NotI and KpnI sites of pUC18-IFOR. 86    XIII Fig. 3.9a Plasmid map of the allelic exchange vector pUC l8-htpD-Gent. 89 Fig. 3.9b Agarose gel electrophoretic analysis of the recombinant plasmid pUC 18-htpD-Gent. 89 Fig. 3.1 Oa Plasmid map of the allelic exchange vector pUC 1 8-htpE-Gent. 91 Fig. 3.1 Ob Agarose gel electrophoretic analysis of the recombinant plasmid pUC18-htpE-Gent constructed by cloning a gentamicin resistance cassette into the NotI site of the htpE open reading frame of plasmid pUC18-htpE. 91 Fig. 3.11 a Plasmid map of the allelic exchange vector pUC18-htpDEF-Gent. 93 Fig. 3.11 b Agarose gel electrophoretic analysis of the allelic exchange vector pUCI8-htpDEF-Gent constructed by cloning the gentamicin resistance cassette from pUCl8-Gent into the Pst! sites of htpD and htpF open reading frames contained in plasmid pUCI8-DEF. 93 Fig. 3.12a Southern blot analysis of genomic DNA extracted from P. aernginosa wild-type and mutant strains using a labeled gentamicin gene as probe. 95 Fig. 3.12b Southern blot analysis of genomic DNA extracted from P. aernginosa wild-type and mutant strains using pUC18 DNA as labeled probe. 95 Fig. 3.13 PCR analysis of the mutant strain DSMHtpE using various primer sets. 98 Fig. 3.14a Plasmid map of the complementation plasmid pJB-DEF-Kan. 101 Fig. 3.14b Agarose gel electrophoretic analysis of the intermediate plasmids used in the construction of the complementation plasmid pJB-DEF-Kan. 101 Fig. 4.1 Optical density and total cellular protein concentration of wild-type P. aeruginosa DSM1707, mutant DSMHtpE and DSMHtpE containing plasmid construct pJB-DEF-Kan. 118    xiv Fig. 4.2 Optical density and total cellular protein concentration of wild-type P. aeruginosa DSM1707, mutant DSMHtpD and DSMHtpD containing plasmid construct pJB-DEF-Kan. 119 Fig. 4.3 Photomicrographs showing the biofilm phenotypes of the wild-type P. aeruginosa DSM 1707 strain and the mutant DSMHtpE and DSMHtpD strains. 121 Fig 4.4 In situ hybridization of cells of wild-type P. aeruginosa DSM1707 and mutant strains. 124 Fig. 4.5 Propensity of DSMHtpE cells to occur as a biofilm. 126 Fig. 4.6 Propensity of DSMHtpD cells to occur as a biofilm. 127 Fig. 4.7 SEM inspection of wild-type P. aeruginosa DSM1707 and mutant DSMHtpE and DSMHtpD cells. 129 Fig.4.8a Representative electron micrographs of thin sections through resin-embedded cells observed by transmission electron microscopy (TEM). 131 Fig.4.8b Enlarged transmission electron micrographs of cells from 24 h (stationary phase) cultures of mutant DSMHtpD and DSMHtpD containing plasmid construct pJB-DEF-Kan. 132 Fig. 4.9 Two-dimensional images of extracellular proteins of wild-type P. aeruginosa DSM1707 and mutant DSMHtpD. 134    xv LIST OF TABLES T ABLE NUMBER Table 2.1 Characteristics of the P. aeruginosa hlp genes and encoded gene products, listed in conjunction with homologous genes 48 Table 3.1 Bacterial strains and plasmids used Table 4.2 Summary of the extracellular proteins identified from 2DE gels and unique 60 Table 3.2 Primers used 61 Table 4.1 Bacterial strains and plasmids used 108 to either P. aeruginosa DSM 1707 and mutant DSMHtpD 137    

Characterization of a putative pilus assembly and secretion system in Pseudomonas aeruginosa DSM 1707

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Characterization of a putative pilus assembly and secretion system in Pseudomonas aeruginosa DSM 1707

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