This dissertation, "Genetic Manipulation Systems for Laribacter Hongkongensis: a Novel Bacterium Associated With Gastroenteritis" by Suet-lai, Shirley, Ma, 馬雪麗, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled "Genetic manipulation systems for Laribacter hongkongensis, a novel bacterium associated with gastroenteritis" Submitted by Shirley Ma Suet Lai for the degree of Master of Philosophy at the University of Hong Kong in March, 2005 Laribacter hongkongensis, a novel bacterial species first isolated from the blood and empyema pus of a liver cirrhotic patient, was found to be associated with community-acquired gastroenteritis and traveller's diarrhea in a multi-centered prospective study in 2004. In this study, genetic manipulation systems were developed to facilitate further understanding of the biology and possible pathogenesis mechanisms of this bacterium. Plasmid profiles of 21 strains of human isolates of L. hongkongensis were analyzed using plasmid extraction and pulsed-field gel electrophoresis (PFGE). Four out of the 21 strains were found to harbor plasmids of size 20 kb. Two of the five plasmids of size Six different E. coli vectors and shuttle vectors were transformed into cells of L. hongkongensis using electroporation to test their functionality in the bacterium. These plasmids include two shuttle E. coli-Staphylococcus vectors, designated pALC2084 and pCL52.2 and four E. coli plasmids with either pUC or pBR322 viiiorigins of replications. Only two of the six plasmids, pBK-CMV and pCL52.2, resulted in colony formation after transformation in seven and six out of the 21 strains tested respectively. Plasmid extraction, however, did not reveal the presence of the respective transformed plasmids in those colonies, suggesting that the plasmids were integrated into the chromosome of L. hongkongensis. An EcoRI fragment of pHLHK8 that consists of the replication region was fused with pBK-CMV that harbors the kanamycin resistance gene to form pPW380. This E. coli-Laribacter shuttle vector was transformed into cells of the 21 strains of L. hongkongensis with a median transformation efficiency of 2.5510 . pPW380 was further used for the development of an inducible expression shuttle vector of L. hongkongensis. pPW380 was fused with the tetracycline inducible promoter from pALC2084 forming pPW578. Expression of green fluorescent protein (GFP) and glutathione S-transferase (GST) cloned into pPW578 after tetracycline induction were confirmed by fluorescence assay and western blotting respectively. Using pBK-CMV as a suicide vector, gene replacement of one of the flagellar rod protein genes, flgG, was achieved in L. hongkongensis. Complementation of the phenotype was attempted using the constructed pPW578. The putative flgG gene together with a 37-bp upstream sequence which consisted of the putative Shine- Dalgarno sequence was cloned into pPW578. After tetracycline induction, a swarm ring was observed for the complemented mutant but not for the mutant or the non- induced complemented mutant using semi-solid agar assay. The swarm ring was substantially smaller than that of the wild type indicating partial complementation. ixThis study has developed an E. coli-L. hongkongensis inducible expression shuttle vector, pPW578 and a gene deletion and complementation system for L. hongkongens