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Characterisation of O-Antigen Biosynthesis Genes in Vibro Anguillarum and Their Association with IS1358

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Characterisation of O-Antigen Biosynthesis Genes in Vibro Anguillarum and Their Association with IS1358 rL.rz'qÎ Characterisation of O'antigen biosynthesis genes in Vibrio anguillarum and their association with IS/358 By Kathy Eva Daniels (nee Jedani) B.Sc Hons. DiP. Ed. (Adetaide) A thesis submitted for the degree of Doctor of PhilosoPhY Department of Microbiology and Immunology UniversitY of Adelaide July, t999 ABSTRACT Víbrio anguillarunr is an important pathogen that causes the disease vibriosis in feral and cultured fish. To date 23 serotypes of. V. anguillarum have been identified. Strains from the Ol and 02 serotypes are the most frequent serot¡res isolated from disease outbreaks. The pathogenesis of V. anguillarum is not well understood nor are the virulence determinants involved in the process of infection. The best cha¡acterised virulence determinant is the iron transport system located on the large plasmid, pJMl. More recent studies have focussed on characterising other factors involved in the pathogenic process including: the haemolysin, MOMP and flagella. LPS is an important virulence factor in numerous pathogenic bacteria and has been found to contribute to the virulence of V' anguillarum. However at present the genes involved in the biosynthesis of this virulence determinant have not been isolated, it was the objective of this thesis to characterise the genes required for the synthesis of O-antigen in V. anguillarum and to gain a better understanding of the mechanisms involved in the reaffangements of O-antigen biosynthesis gene clusters inVibrio species. Two different antisera, one raised against V. anguillarum Ol and the other against the 04 serotype were generated in this study to aid in the investigation of virulence determinants. The anti V. anguillarum 04 serum reacted only to a major outer membrane protein (MOMP) of -36 kDa. MOMP varied in size between the l0 serotypes (O1-Ol0) tested. Purified MOMP from V. anguillarum Ol was N-terminal sequenced and found to be homologous to an immunogenic, highty variable protein P2, in Haemophilus influenzae and 1L sommus. The V. anguillarum Ol serum contained antibodies to both the MOMP and LPS as determined by Westem blotting. Affinity purification was used to obtain LPS specific antibodies to further investigate by immunogold electron microscopy and immunofluorescence, LPS expression in V. anguillarum Ol. IS/358, a recently characterised insertion sequence, has been shown to be linked to the O-antigen biosynthesis regions in numerous V. cholerae serotypes. IS/358 was found to be widely distributed in a number of V. anguillaru,n serotypes including Ol and 02. IS/358 was found to be present in multiple copies in serotlpes 02, O7 and 09 suggesting that this element is a site for recombination, gene duplication or that it may be capable of transposition. The IS/358 elements from the different serotypes were cloned and sequenced. Analysis of the sequence revealed either the presence of a complete ORF in serotypes containing multiple copies of the element or three small ORFs in those strains that possess only one IS1358 element. It was found by T7 promoterþolymerase expression ar¡says that serotypes 02, 07 and 09 produce a protein of 42 kDa which corresponds to the predicted molecular weight. IS1358 was found to be associated with polysaccharide biosynthesis genes in V. anguillarum Ol and 02. In V. anguillarum 02, PCR was used to amplify DNA between copies of IS/-358. These PCR products were cloned and sequenced. Analysis of the sequence indicated the presence of genes homologous to polysaccharide biosynthesis and regulation genes. A chromosomal map was generated showing the organisation of the amplified DNA with respect to the adjacent IS/358 elements. Inverse PCR and cosmid cloning were used to further map and sequence this region. ln V. anguillarum 01, cosmid cloning was used to isolate the DNA flanking the single copy of IS/358. Cosmid clones were screened with a DlGlabelled IS/-158 probe, allowing the regions adjacent to be partially cloned and sequenced. Characterisation of the O-antigen biosynthesis region in V. anguillarum Ol, designated wbh has resulted in construction of a physical chromosomal map with 24 open reading frames (ORFs) being identified. Most ORFs showed significant homology to other polysaccharide biosynthesis genes from numerous other bacteria. Genes involved in the biosynthesis of rhamnose were identified in the wbh region of. V. anguillarum 01 based on homology. h addition genes homologous to nucleotide sugar transferases and components of homopolymer O-antigen transport systems were also detected. Transposon insertions using Tn\phoA (Cm*) in the wbh rcgion were characterised and shown to result in different O-antigen phenotypes as observed by silver staining of SDS-PAGE. The differences observed in the O-antigen profile of these mutants was dependent on the location of the mutation. Analysis of a mutant with an insertion outside of the sequenced wbh region in conjunction with sequencing and subcloning of cosmids downstream of the characterised wbh region suggests that additional genes may be required for expression of LPS in V. ønguillarum Ol. However these additional genes were not directly linked to the wbh region and Southern mapping indicated that it was located within 40 kb. Sugar analysis of the LPS from the V. anguillarum Ol wildtype and transposon mutants indicates the presence of glucose, rhamnose and glucosamine in varying amounts. Genes present in the wbh region account for the rhamnose component of the LPS. kr this study thewbh region responsible for O-antigen biosynthesis was isolated and partially characterised. The operon appears to be made up of genes that were acquired from other bacteria. The presence of IS1358 indicates that it may have played a role in the acquisition or rearrangement of the polysaccharide biosynthesis genes in V. anguillarum o1. To my wonderful husband Craig, and my Mum and Dad, all of you will be forever in my heart. Acknowledgments "You never know where your going until you get there" I must start this rather difficult task by thanking Professor Paul Manning for allowing me to undertake this study in his laboratory. Next, I would like to extend a special thank you to Dr. Uwe Stroeher for his friendship, supervision and support to the end. I am eternally grateful to my "last minute" supervisor Dr. Renato Morona. I would not have finished this Ph.D without your help, knowledge and expertise- wish I could say more but you know how I feeM hope you know that I have appreciated your extreme effort! I would like to thank Dr. Debra Milton, who provided me with support from the other side of the world. I must also acknowledge her expertise in working with V. anguillarum, and thank her for the many strains, tips and pointers she has given during our acquaintance. I hope that one day we shall meet, so I can thank you in person. Many thanks! During the years of my Ph.D I met and enjoyed the company of numerous people- both students and staff. To everybody in the lab both past and present, thanks for the past five years. I would like to make special mention of Sarah, a friend and gym partner (when I wasn't l*y);Liz,Ithank you for all the small things you did that helped enormously and Luisa- thanks for your help when I really needed things to go right, especially with IFs. I must also thank Chris Cursaro for his photographic talents etc and the "computer boys" for their help at crucial times during the writing of this thesis. I would like to thank Carole Gannon for allowing me to find my true calling in life- you have provided me with an enjoyable outlet and I thank you very much, for that, and your friendship. To my dear friends on the outside, Helen and Andrea, thanks for being there when I needed to complain, cry and be disappointed. You will be forever my friends and I wish you all the luck and happiness in the world- I will miss you heaps!! To all of my family both new and old, I thank you for all understanding that we didn't see you as much as we would have liked-please know that you will never be far from our minds and are forever in our hearts. I would also like to thank you for all your support over the years (and years) of study that I have undertaken. My final acknowledgment must go to my dearest friend, my husband, Craig- I would not have finished if it weren't for you. There are not enough words in all of the dictiona¡ies to describe the love and gratitude I feel for you. I know that our life starts on a new path with the completion of our theses, and I can't wait to go down it together, and wherever it leads- starting in Canada!! I love You. Abbreviations A: adenine Ãzcoi absorbance at260nm aa: amino acid ACP: acyl carrier protein ACL: antigen carrier lipid (bactoprenol) Ap: ampicillin ATP: adenosine-5' -triphosphate bp: base pair BSA: bovine serum albumin C: cytosine CIP: calf intestinal phosphatase Cm: chloramphenicol CTP: cytosine-S' -triphosPhate C-terminal: carboxy terrminal DIG: digoxigenin DNA: deoxyribonucleic acid DNase: deoxyribonuclease dNTP: deoxyribonucleoside triphosphate ddNTP: dideoxyribonucleoside triphosphate DTT: dithiothreitol ECA: enterobacterial common antigen BDTA: ethylene-diamine-tetra-acetic acid EtBr: ethidium bromide G: guanine Gm: gentamycin GTP: guanine-5' -triphosphate HRP: horse radish peroxidase IPTG: i sopropyl- p -D-thio galactopyranoside IS: insertion sequence kb: kilobase pairs kDa: kilodalton KDO: keto-3 -deoxy-D-manno-octulosonic acid Km: kanamycin LA: luria agar LB: luria broth LPS: lipopolysaccharide LR.LPS: long range PCR mg: milligram ml: millilitre mM: millimolar
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