| Summary: | 博士 === 國立交通大學 === 生物科技系所 === 93 === The thesis covered three major approaches aimed to identify the property of bacterial virulence determinants. In the first part, we determined the entire DNA sequence of pLVPK, a 219-kb virulence plasmid harbored in Klebsiella pneumoniae. A total of 251 open reading frames were annotated. The obvious virulence-associated genes carried by the plasmid are the capsular polysaccharide synthesis regulator rmpA and its homolog rmpA2, and multiple iron-acquisition systems, including iucABCD-iutA and iroBCDN siderophore gene clusters, fepBC ABC-type transporter, and fecIRA that encodes iron uptake regulatory system. In addition, several gene clusters homologous with copper, silver, lead, and tellurite resistance genes of other bacteria were also identified. The presence of thirteen insertion sequences located mostly at the boundaries of the aforementioned gene clusters suggests that pLVPK was derived from a sequential assembly of various horizontally-acquired DNA fragments. In the second part, we analyzed the complete genome sequence of P. aeruginosa PAO1 to unravel the evolution of a group of important virulence factors, the two component systems. Gene organization and functional motif analyses of the 123 two component system (2CS) genes in Pseudomonas aeruginosa PAO1 were carried out. By comparing the phylogenetic trees built respectively for the two components, we showed that more than half of the sensor-regulator gene pairs, especially the 2CSs with OmpR-like regulators, are derivatives of a common ancestor, and have most likely co-evolved through gene pair duplication, while several of the 2CS pairs, especially those with NarL-like regulators, appeared to be relatively divergent. Correlation of the classification of sensor kinases and response regulators further provides support for these models. We have identified six congruent clades, which represent the group of the most recently duplicated 2CS gene pairs. Sequence comparison showed that certain paralogous 2CS pairs may carry a redundant function even after a gene duplication event. However, comparative analysis of the putative promoter regions of the paralogs suggested that functional redundancy could be prevented by a differential control. Finally in the third part, we analyzed 38 completed genomes using HMMER to identify the putative 2CS components and investigate the evolution of the 2CSs of OmpR-family. The distribution of OmpR-like response regulators among different genomes of different taxonomy groups also supported the hypothesis that 2CSs are originated in the last common ancestor of bacteria and subsequently passed to the other species. Mostly, the 2CS genes containing an OmpR-like regulator-encoding gene were found in the order of regulator-to-sensor (RS). The amino acid sequences around the phosphorylated histidine residue of the sensor kinases from either RS or SR (sensor-to-regulator) 2CSs were nearly identical. This suggested that the interaction of 2CS component may have constrained the sequences of the interacting domain between sensor kinase and the cognate response regulator while the ancestral components were brought together during evolution into a RS or SR gene cluster, where coordinated transcriptional control may be economically favored. The nearly invariant gene order and the conservation of catalytic domains of these 2CSs provide strong evidence for the co-evolution of 2CSs.
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