鮑曼不動桿菌(Acinetobacter baumannii)是一種威脅生命的醫院獲得性病菌。該細菌有很强的環境適應能力。它能夠在重症監護室被分離出來并有很高的幾率感染免疫系統受損的病人。鮑曼不動桿菌有很高的傾向獲得多重抗藥性。目前在亞洲和歐洲有多株泛抗藥性菌株被發現。一些基因組比對研究著重報告了鮑曼不動桿菌的抗藥基因片段和與抗藥性相關的基因突變。然而，抗藥基因的轉錄調控和該細菌在抗生素治療過程中引發的反應并未得到很好的研究。因此，我們運用鏈特異性轉錄組測序技術（RNA-seq）對一些抗藥菌株和非抗藥菌株在不同環境下生長的樣本進行測序，來研究該細菌，尤其是在抗生素治療中抗藥菌株的基因轉錄調控。 / 本研究運用轉錄組測序技術（RNA-seq）系統分析了十二株鮑曼不動桿菌在培養液生長狀況下的轉錄組。本次研究共收集了九株多重抗藥性菌株和三株敏感菌株，其中包括了一些快速生長的菌株和慢速生長的菌株。在快速生長的菌株中，氨基酸代謝途徑、甘油脂代謝途徑和钳铁化合物生物合成途徑被向上調控並扮演着重要角色。多重抗藥性菌株擁有更多與轉位酶（transposase）相關的抗生素抗性基因，但除此之外，在對數期的生長過程中多重抗藥性菌株與敏感菌株並未在許多其他代謝途徑中表現差異性控制。 / 三株擁有相同脉冲场凝胶电泳（PFGE）樣式但是表現出不同抗藥性的菌株分別生長於含有阿米卡星(Amikacin)、亞胺培南（Imipenem）或美羅培南（Meropenem）的培養液中，然後它們的轉錄組也被進行了研究。菌株生長在含有抗生素的培養液中時，與能量製造相關的的途徑和核醣體合成途徑被向上調控。作用機制不同的抗生素對細菌有不同的影響，阿米卡星誘發更多基因被向上調控，例如與蛋白質折疊相關的基因；碳青霉烯类抗生素誘發更多的基因被向下調控，例如甘油脂代謝途徑。然而，許多在抗生素治療過程中被緊密調控的基因功能仍舊未知。在抗生素環境生長的條件下基因調控和抗藥機制可能會更複雜。 / 最後，本研究找到一些新的與抗藥性相關的基因和单核苷酸变异（SNVs）。其中，源自於同一操縱子的大环内酯二位轉磷酸酶（macrolide2’ phosphotransferase）同源體Mph和大环内酯外排泵蛋白同源體Mel只存在並一同表達於鮑曼不動桿菌的阿米卡星抗藥株中。這兩個基因或對阿米卡星的抗藥性有一定貢獻作用。總而言之，這些成果爲將來的深度研究提供了重要依據。 / Acinetobacter baumannii is a life-threatening nosocomial pathogen, which has versatile adaptability to the environment. It can be isolated from intensive care unit (ICU) and causes high prevalence of infection among immunocompromised patients. A. baumannii has high tendency to develop multidrug resistance. Currently, pan-drug resistant strains have been reported in Asia and Europe. Several comparative genomic studies revealed the structures of drug resistant islands and antibiotic-related mutations in A. baumannii. However, the transcriptional regulation of drug resistant genes, and the multidrug resistant response of A. baumannii under the treatment of antibiotics are not well studied. By applying strand-specific RNA-sequencing on sensitive and multidrug resistant strains growing in various conditions, we aimed to study the transcriptional responses and gene regulation of A. baumannii, specifically under the antibiotic treatment. / The transcriptome of twelve A. baumannii strains, including nine multidrug resistant strains and three sensitive strains, were systematically analyzed in planktonic state by RNA-seq. Among the multidrug resistant strains there are both fast-and slow-growing strains. Amino acid metabolic pathways, glycerol lipid metabolic pathways and siderophore biosynthetic process are found to be key pathways that are up-regulated in fast-growing strains. Except that multidrug resistant strains possess more transposase-associated antibiotic resistant genes, intriguingly, only a few pathways are differentially regulated between multidrug resistant and sensitive strains during fast growth in antibiotic-free medium. / Three strains of the same PFGE pattern but with different antibiotic resistance patterns were treated by amikacin, imipenem, and meropenem, and their transcriptomes were analyzed. The energy generation-related pathways and ribosome synthesis pathway were commonly up-regulated when the strains were grown in antibiotic-treated media. Amikacin triggers more genes up-regulated, including genes responsible for protein folding, while carbapenems trigger more genes down-regulated, including glycerol lipid metabolic process, revealing the different actions of antibiotics. However, many tightly-regulated genes during antibiotic treatment were functionally unknown, suggesting that gene regulation during antibiotic response and the actual mechanisms involved could be far more complex. / Finally, this study also identified several novel genes and single nucleotide variations (SNVs) which were correlatedto antibiotic-specific resistance. A macrolide 2’ phosphotransferase homolog Mph and a macrolide efflux protein homolog Mel, which commonly exist only in A. baumannii amikacin resistant strains and are co-expressed in the same operon, may contribute to amikacin resistance. In summary, the results presented in this thesis have opened the venue for future investigations. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Qin, Hao. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 107-114). / Abstracts also in Chinese.

Pseudomonas aeruginosa is an opportunistic human pathogen that infects immunocompromised individuals such as those suffering from burns or the genetic disorder cystic fibrosis. This organism utilizes a cell-cell communication mechanism known as quorum sensing (QS) to coordinate virulence gene expression and biofilm formation. It has three interconnected QS systems, namely las, rhl and pqs. Each system is comprised of autoinducer synthesis genes, lasI, rhlI, and pqsABCDH, and the cognate regulatory genes, lasR, rhlR, and pqsR, respectively. Here, we primarily focused on understanding the regulatory mechanisms of QS, which we investigated at two levels. First, we sought to identify additional activators that regulate QS at the level of the las and rhl systems, and second, we investigated the regulation of downstream genes, particularly biofilm exopolysaccharide genes, by QS. For the first approach, we employed a mutagenesis screen to identify global QS activators. We screened a non-redundant transposon library for mutants deficient in QS-dependent phenotypes. We identified a novel regulator, GidA, a glucose-inhibited cell division protein, that selectively controls QS gene expression posttranscriptionally via RhlR-dependent and –independent pathways. For the second part, we established a regulatory link between QS and Pel exopolysaccharide. We showed that the las system represses Pel and modulates colony biofilm structure through the pqs pathway. LasR mediated colony rugosity via 4-hydroxy-2-alkylquinolines in a PqsR-independent manner, ascribing a novel function to this class of signaling molecules in P. aeruginosa. Taken together, our study highlights the complexity of QS, which involves integration of various regulatory pathways to control downstream processes in response to different environmental conditions. / Graduation date: 2012

Clostridium perfringens type A isolates producing enterotoxin (CPE) are an important cause of food poisoning and non-food-borne human gastrointestinal (GI) diseases, including antibiotic-associated diarrhea (AAD), and spontaneous diarrhea (SD). In enterotoxigenic type A isolates, the cpe gene is found on the chromosome in food poisoning isolates, but is present on a large virulence plasmid in AAD and SD type A isolates. Food poisoning cases typically exhibit shorter duration of infection and less severe GI symptoms than AAD or SD. Since previous epidemiological evidence has linked the newly discovered beta2-toxin (CPB2) to gastroenteritis in pigs, horses, and chickens, we hypothesize that the CPB2 toxin may be an accessory toxin when cpe positive type A isolates cause human AAD or SD. In the current study, the presence and expression of CPE and CPB2 were assessed in 44 C. perfringens type A human fecal isolates associated with GI diseases in the United Kingdom. Polymerase chain reaction (PCR) and restriction fragment length polymorphisim (RFLP) confirmed the presence of the cpe (32%) and cpb2 (39%) genes. Furthermore, pulsed field gel electrophoresis (PFGE) and I-CeuI RFLP PFGE Southern blot analysis was used to show the localization of the cpe and cpb2 genes, as well as to determine that there was no clonal relationship between the isolates. All surveyed cpb2-positive isolates were determined to carry their cpb2 gene on a large plasmid that was estimated to be the similar size of the cpe large plasmid. Finally, CPE and CPB2 Western blotting demonstrated that all cpe-positive isolates expressed CPE and that all cpb2-positive isolates expressed CPB2. This study identified, for the first time, the C. perfringens non-food-borne human GI disease isolates carrying both the cpe and cpb2 genes (18%), and these isolates all actively expressed both CPE and CPB2. It was also shown that, although CPE expression occurs only under sporulation conditions, CPB2 expressed both in vegetative and sporulation conditions. The CPB2 made by two of these cpe /cpb2 - positive isolates was determined to be very (-99%) similar to the deduced amino acid sequence of the biologically-active CPB2 made by the original type C isolate CWC245. Finally, the expression of CPB2 by only type A isolates carrying the cpe gene on a plasmid and not the isolates carrying a chromosomal cpe gene, could possibly explain the increased GI symptoms and disease duration associated with these non-food-borne GI diseases. Collectively, the current results support a significant association between cpb2-positive C. perfringens isolates and non-food-borne GI disease in human. / Graduation date: 2004

Most members of the Rhizobiaceae possess single copies of the poly-3-hydroxybutyrate biosynthesis genes, phbA, phbB and phbC. Analysis of the genome sequence of Bradyrhizobium japonicum reveals the presence of five homologues of the PHB synthase gene phbC as well as two homologues of the biosynthesis operon, phbAB. The presence of multiple, seemingly redundant homologues may suggest a functional importance. Each B. japonicum phbC gene was cloned and used to complement the pleiotropic phenotype of a Sinorhizobium meliloti phbC mutant; this mutant is unable to synthesize PHB, grow on certain PHB cycle intermediates and forms non-mucoid colonies on yeast mannitol medium. Two of the five putative B. japonicum phbC genes were found to complement the S. meliloti phbC mutant phenotype on D-3-hydroxybutyrate although none of them could fully complement the phenotype on acetoacetate. Both complementing genes were also able to restore PHB accumulation and formation of mucoid colonies on yeast mannitol agar to phbC mutants. In-frame deletions were constructed in three of the five phbC open reading frames in B. japonicum, as well as in both phbAB operons, by allelic replacement. One of the phbC mutants was unable to synthesize PHB under free-living conditions; one of the two phbAB operons was shown to be necessary and sufficient for PHB production under free-living conditions. These mutants also demonstrated an exopolysaccharide phenotype that was comparable to S meliloti PHB synthesis mutants. These strains were non-mucoid when grown under PHB-inducing conditions and, in contrast to wild-type B. japonicum, formed a compact pellet upon centrifugation. Interestingly, none of the mutants exhibited carbon-utilization phenotypes similar to those exhibited by S. meliloti PHB mutants. Wild-type B. japonicum accumulates PHB during symbiosis, and plants inoculated with the phbC mutants demonstrate a reproducible reduction in shoot dry mass. Analysis of bacteroid PHB accumulation in the mutant strains suggests that the phbAB operons of B. japonicum are differently regulated relative to growth under free-living conditions; mutants of the second phbAB operon demonstrated a significant reduction in PHB accumulation during symbiosis. These data suggest that the first phbAB operon is required for PHB synthesis only under free-living conditions, but is able to partially substitute for the second operon during symbiosis. Deletion of both phbAB operons completely abolished PHB synthesis in bacteroids. Analysis of the upstream regions of these genes suggest the existence of putative RpoN binding sites, perhaps indicating a potential mode of regulation and highlighting the metabolic complexity that is characteristic of the Rhizobiaceae. PHB metabolism in S. meliloti has been studied in considerable detail with two notable exceptions. No reports of the construction of either a β-ketothiolase (phbA) or a PHB depolymerase (phaZ ) mutant have ever been documented. The phaZ gene, encoding the first enzyme of the catabolic half of the PHB cycle in S. meliloti, was identified and a phaZ mutant strain was generated by insertion mutagenesis. The phaZ mutant demonstrates a Fix+ symbiotic phenotype and, unlike other PHB cycle mutants, does not demonstrate reduced rhizosphere competitiveness. Bacteroids of this strain were shown to accumulate PHB, demonstrating for the first time that S. meliloti is able to synthesize and accumulate PHB during symbiosis. Interestingly, there is no significant difference in shoot dry mass of plants inoculated with the phaZ mutant, suggesting that PHB accumulation does not occur at the expense of nitrogen fixation. The phaZ mutant strain was also used to demonstrate roles for PhaZ in the control of PHB accumulation and exopolysaccharide production. When grown on high-carbon media, this mutant demonstrates a mucoid phenotype characteristic of exopolysaccharide production. Subsequent analyses of a phoA::exoF fusion confirmed elevated transcription levels in the phaZ mutant background. In contrast, mutants of the PHB biosynthesis gene, phbC, have a characteristically dry phenotype and demonstrate reduced exoF transcriptional activity. The phaZ mutant also demonstrates a significant increase in PHB accumulation relative to the wild-type strain. Previous work on phasin mutants in S. meliloti demonstrated that they lack the ability to synthesize PHB. Transduction of the phaZ lesion into the phasin mutant background was used to construct a phaZ-phasin mutant strain. Analysis of the PHB biosynthesis capacity of this strain showed that the lack of PHB synthesis exhibited by S. meliloti phasin mutants is due to loss of PHB biosynthesis activity and not due to an inherent instability in the PHB granules themselves. A recent study suggested that some bacteria may possess an alternate pathway for acetate assimilation that would bypass the need for the glyoxylate cycle in organisms that do not possess the enzyme, isocitrate lyase. In these organisms, acetate is assimilated through the ethylmalonyl-CoA pathway, which has significant overlap with the anabolic half of the PHB cycle, including reliance on the PHB intermediate 3-hydroxybutyryl-CoA. The observation that phbB and phbC mutants of S. meliloti are unable to grow well on acetoacetate -- coupled with previously unexplained data that show a class of mutants (designated bhbA-D) are able to grow on acetate, but not on hydroxybutyrate or acetoacetate -- made it tempting to speculate that an ethylmalonyl-CoA-like pathway might be present in S. meliloti, and that this pathway might overlap with the PHB cycle at the point of 3-hydroxybutyryl-CoA. An in-frame mutation of phbA was constructed by cross-over PCR and allelic replacement. This mutant exhibited a complete abolition of growth on acetoacetate, suggesting that PhbA represents the only exit point for carbon from the PHB cycle and that an alternative ethylmalonyl-CoA-like pathway is not present in this organism. During symbiosis, rhizobial cells are dependent on the provision of carbon from the host plant in order to fuel cellular metabolism. This carbon is transported into the bacteroids via the dicarboxylate transport protein, DctA. Most rhizobia possess single copies of the transporter gene dctA and its corresponding two-component regulatory system dctBD. The completed genome sequence of B. japonicum suggests that it possesses seven copies of dctA. Complementation of Sinorhizobium meliloti dct mutants using the cosmid bank of B. japonicum USDA110 led to the identification a dctA locus and a dctBD operon. Interestingly, the B. japonicum dctABD system carried on the complementing cosmid was not able to complement the symbiotic deficiency of S. meliloti strains carrying individual mutations in either dctA, dctB, or dctD suggesting that the B. japonicum dctBD is unable to recognize either DctB/DctD or the DctB/DctD-independent regulatory elements in S. meliloti. All seven B. japonicum dctA ORFs were cloned and an analysis of their capacity to complement the free-living phenotype of a S. meliloti dctA mutant demonstrated that they all possess some capacity for dicarboxylate transport. Mutants of all seven B. japonicum dctA ORFs were constructed and an analysis of their free-living phenotypes suggested that significant functional redundancy exists in B. japonicum DctA function. Given the large number of potential dctA genes in the genome, coupled with an apparent lack of dctBD regulators, it is tempting to speculate that different DctA isoforms may be used during free-living and symbiotic growth and may be subject to different regulatory mechanisms than those of better-studied systems. A comprehensive analysis of desiccation tolerance and ion sensitivity in S. meliloti was conducted. The results of these analyses suggest that genetic elements on both pSymA and pSymB may play a significant role in enhancing cell survival under conditions of osmotic stress. The S. meliloti expR+ strains SmUW3 and SmUW6 were both shown to exhibit considerably higher desiccation tolerance than Rm1021, suggesting a role for enhanced exopolysaccharide production in facilitating survival under adverse conditions. Furthermore, scanning electron microscopy of inoculated seeds suggests that S. meliloti cells initiate biofilm formation upon application to the surface of seeds. This finding has implications for the analysis of OSS and the development of desiccation assays and may explain some of the variability that is characteristic of desiccation studies.

Most members of the Rhizobiaceae possess single copies of the poly-3-hydroxybutyrate biosynthesis genes, phbA, phbB and phbC. Analysis of the genome sequence of Bradyrhizobium japonicum reveals the presence of five homologues of the PHB synthase gene phbC as well as two homologues of the biosynthesis operon, phbAB. The presence of multiple, seemingly redundant homologues may suggest a functional importance. Each B. japonicum phbC gene was cloned and used to complement the pleiotropic phenotype of a Sinorhizobium meliloti phbC mutant; this mutant is unable to synthesize PHB, grow on certain PHB cycle intermediates and forms non-mucoid colonies on yeast mannitol medium. Two of the five putative B. japonicum phbC genes were found to complement the S. meliloti phbC mutant phenotype on D-3-hydroxybutyrate although none of them could fully complement the phenotype on acetoacetate. Both complementing genes were also able to restore PHB accumulation and formation of mucoid colonies on yeast mannitol agar to phbC mutants. In-frame deletions were constructed in three of the five phbC open reading frames in B. japonicum, as well as in both phbAB operons, by allelic replacement. One of the phbC mutants was unable to synthesize PHB under free-living conditions; one of the two phbAB operons was shown to be necessary and sufficient for PHB production under free-living conditions. These mutants also demonstrated an exopolysaccharide phenotype that was comparable to S meliloti PHB synthesis mutants. These strains were non-mucoid when grown under PHB-inducing conditions and, in contrast to wild-type B. japonicum, formed a compact pellet upon centrifugation. Interestingly, none of the mutants exhibited carbon-utilization phenotypes similar to those exhibited by S. meliloti PHB mutants. Wild-type B. japonicum accumulates PHB during symbiosis, and plants inoculated with the phbC mutants demonstrate a reproducible reduction in shoot dry mass. Analysis of bacteroid PHB accumulation in the mutant strains suggests that the phbAB operons of B. japonicum are differently regulated relative to growth under free-living conditions; mutants of the second phbAB operon demonstrated a significant reduction in PHB accumulation during symbiosis. These data suggest that the first phbAB operon is required for PHB synthesis only under free-living conditions, but is able to partially substitute for the second operon during symbiosis. Deletion of both phbAB operons completely abolished PHB synthesis in bacteroids. Analysis of the upstream regions of these genes suggest the existence of putative RpoN binding sites, perhaps indicating a potential mode of regulation and highlighting the metabolic complexity that is characteristic of the Rhizobiaceae. PHB metabolism in S. meliloti has been studied in considerable detail with two notable exceptions. No reports of the construction of either a β-ketothiolase (phbA) or a PHB depolymerase (phaZ ) mutant have ever been documented. The phaZ gene, encoding the first enzyme of the catabolic half of the PHB cycle in S. meliloti, was identified and a phaZ mutant strain was generated by insertion mutagenesis. The phaZ mutant demonstrates a Fix+ symbiotic phenotype and, unlike other PHB cycle mutants, does not demonstrate reduced rhizosphere competitiveness. Bacteroids of this strain were shown to accumulate PHB, demonstrating for the first time that S. meliloti is able to synthesize and accumulate PHB during symbiosis. Interestingly, there is no significant difference in shoot dry mass of plants inoculated with the phaZ mutant, suggesting that PHB accumulation does not occur at the expense of nitrogen fixation. The phaZ mutant strain was also used to demonstrate roles for PhaZ in the control of PHB accumulation and exopolysaccharide production. When grown on high-carbon media, this mutant demonstrates a mucoid phenotype characteristic of exopolysaccharide production. Subsequent analyses of a phoA::exoF fusion confirmed elevated transcription levels in the phaZ mutant background. In contrast, mutants of the PHB biosynthesis gene, phbC, have a characteristically dry phenotype and demonstrate reduced exoF transcriptional activity. The phaZ mutant also demonstrates a significant increase in PHB accumulation relative to the wild-type strain. Previous work on phasin mutants in S. meliloti demonstrated that they lack the ability to synthesize PHB. Transduction of the phaZ lesion into the phasin mutant background was used to construct a phaZ-phasin mutant strain. Analysis of the PHB biosynthesis capacity of this strain showed that the lack of PHB synthesis exhibited by S. meliloti phasin mutants is due to loss of PHB biosynthesis activity and not due to an inherent instability in the PHB granules themselves. A recent study suggested that some bacteria may possess an alternate pathway for acetate assimilation that would bypass the need for the glyoxylate cycle in organisms that do not possess the enzyme, isocitrate lyase. In these organisms, acetate is assimilated through the ethylmalonyl-CoA pathway, which has significant overlap with the anabolic half of the PHB cycle, including reliance on the PHB intermediate 3-hydroxybutyryl-CoA. The observation that phbB and phbC mutants of S. meliloti are unable to grow well on acetoacetate -- coupled with previously unexplained data that show a class of mutants (designated bhbA-D) are able to grow on acetate, but not on hydroxybutyrate or acetoacetate -- made it tempting to speculate that an ethylmalonyl-CoA-like pathway might be present in S. meliloti, and that this pathway might overlap with the PHB cycle at the point of 3-hydroxybutyryl-CoA. An in-frame mutation of phbA was constructed by cross-over PCR and allelic replacement. This mutant exhibited a complete abolition of growth on acetoacetate, suggesting that PhbA represents the only exit point for carbon from the PHB cycle and that an alternative ethylmalonyl-CoA-like pathway is not present in this organism. During symbiosis, rhizobial cells are dependent on the provision of carbon from the host plant in order to fuel cellular metabolism. This carbon is transported into the bacteroids via the dicarboxylate transport protein, DctA. Most rhizobia possess single copies of the transporter gene dctA and its corresponding two-component regulatory system dctBD. The completed genome sequence of B. japonicum suggests that it possesses seven copies of dctA. Complementation of Sinorhizobium meliloti dct mutants using the cosmid bank of B. japonicum USDA110 led to the identification a dctA locus and a dctBD operon. Interestingly, the B. japonicum dctABD system carried on the complementing cosmid was not able to complement the symbiotic deficiency of S. meliloti strains carrying individual mutations in either dctA, dctB, or dctD suggesting that the B. japonicum dctBD is unable to recognize either DctB/DctD or the DctB/DctD-independent regulatory elements in S. meliloti. All seven B. japonicum dctA ORFs were cloned and an analysis of their capacity to complement the free-living phenotype of a S. meliloti dctA mutant demonstrated that they all possess some capacity for dicarboxylate transport. Mutants of all seven B. japonicum dctA ORFs were constructed and an analysis of their free-living phenotypes suggested that significant functional redundancy exists in B. japonicum DctA function. Given the large number of potential dctA genes in the genome, coupled with an apparent lack of dctBD regulators, it is tempting to speculate that different DctA isoforms may be used during free-living and symbiotic growth and may be subject to different regulatory mechanisms than those of better-studied systems. A comprehensive analysis of desiccation tolerance and ion sensitivity in S. meliloti was conducted. The results of these analyses suggest that genetic elements on both pSymA and pSymB may play a significant role in enhancing cell survival under conditions of osmotic stress. The S. meliloti expR+ strains SmUW3 and SmUW6 were both shown to exhibit considerably higher desiccation tolerance than Rm1021, suggesting a role for enhanced exopolysaccharide production in facilitating survival under adverse conditions. Furthermore, scanning electron microscopy of inoculated seeds suggests that S. meliloti cells initiate biofilm formation upon application to the surface of seeds. This finding has implications for the analysis of OSS and the development of desiccation assays and may explain some of the variability that is characteristic of desiccation studies.

Bacteriophages are viruses that infect bacteria and they are the most numerous biological entities on Earth. Temperate phage can adopt two different lifestyles. In the lytic lifestyle, a phage injects its genome into the host and a controlled developmental program ensues. The phage DNA is replicated, phage genes are expressed and new viral particles are assembled. Ultimately, the host cell lyses and the phage particles are released into the environment. In the lysogenic lifestyle, a phage integrates its genome into the host chromosome, creating a prophage. The cell containing the prophage is known as a lysogen. Most prophage genes are not expressed. However, those that are encode a wide variety of functions. One function is exclusion, or the prevention of a different phage type from successfully infecting the lysogenic cell. Most exclusion systems are limited to a specific phage. Bacteriophage HK239 is unique in that it has a wide range of exclusion including Lambda, P1vir, P2, HK022, and T4rII. To learn more about HK239, the genome was sequenced and annotated. The genome is 41,538 bp in length and there are 71 open reading frames. It has a genomic organization similar to other lambda phage and is most closely related to bacteriophage HK022. No additional genes that share homology with known exclusion functions were identified through the sequence analysis of the HK239 genome. It is possible that an open reading frame for which no database matches were found may indeed encode an exclusion function.

Thesis (Ph.D.)--University of Wollongong, 2009. / Typescript. Includes bibliographical references: leaf 188-214.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

Shiga toxin-producing E. coli (STEC) including E. coli O157:H7 are potential food and water borne zoonotic bacterial pathogens capable of causing outbreaks of severe illness in humans. The virulence of E. coli O157:H7 strains may be related to the type of Stx produced and several Stx2 variants have been identified which appear to differ in their ability to cause disease. Two lineages exist within O157 strains where lineage I is associated mainly with human and bovine isolates and lineage II is associated mainly with bovine isolates. The goal of this study was to identify and characterize a lineage II EC970520 Stx2c phage and determine if variations in the phage compared to Stx2 phage found within the lineage I strain, EDL933, can result in differences in virulence observed between the lineages. This study suggests: 1) that the lineage II strain EC970520 contains a highly heterogeneous Stx2c variant phage; 2) that location of integration of the phage within the genome of a bacterium may be important for host selection; 3) that EC970520 Stx2c phage genes are lineage II specific but only a subset of EDL933 phage genes are lineage I specific; 4) that differences in the stability of phages within bacteria contribute to the evolution of new pathogens; 5) that variation in phage genes can be used to detect different strains of E. coli O157:H7 and other STEC; and 6)that the type of phage may result in phenotypic differences between lineages and occurrence of human disease. Results of this study indicate that lineage II strains may be less virulent than lineage I strains due to specific genetic differences and the ability to release phage which is important to the evolution of new pathogenic strains. / xv, 162 leaves : ill. ; 29 cm.

The 33 kilodalton (kD) manganese stabilizing protein (MSP) is intimately associated with the photolysis of water to molecular oxygen. The two main purposes of this study were: 1) to analyze previously constructed MSP mutants and 2) to subclone, express, and purify the wild-type MSP in Escherichia coli in order to investigate the relationship between structure and function of this protein.Growth rates were compared between bacterial cells harboring only the vector, the vector plus the wild-type MSP gene, and the vector plus a mutant MSP gene. No significant differences were seen. This result implies that the expression of the wild-type MSP or mutant MSP is not toxic to the cells. Plasmid DNA isolation and restriction analyses of several of the mutant clones also confirmed the presence of the correct size inserts in the vector. However, upon sequencing several mutant clones, it appeared that losses and/or rearrangements of sequences was occurring. Thus, it was concluded that MSP was not being stably maintained in E. coli.Expression of the wild-type gene was achieved in E. coli by IPTG induction of the gene in pUC120 cloned under the control of the lac promoter. The expressed protein was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS/PAGE) and confirmed by western blotting. Purification of the wild-type protein was obtained by membrane fractionation over a DEAE ion exchange column and the expression product was detected by western blotting. However, the expression product was lost in the concentration procedure and therefore is not available for reconstitution experiments.The wild-type MSP gene was also subcloned in a hybrid shuttle vector pTNTV, previously constructed in our laboratory (1). This construct was used to permit constitutive highlevel expression of the cloned gene and may prove to be an alternative vector to better express the MSP and mutant MSP in future investigations.These results demonstrate that it is possible to express the wild-type MSP gene from cyanobacteria in E. coli, but the problems of instability and recombination of the mutant genes in the vector have to be addressed before proper expression of these genes can be obtained. / Department of Biology