| Summary: | 碩士 === 國立交通大學 === 生物科技研究所 === 90 === A sequence exhibiting high homology with the kvgAS (Klebsiella virulence gene) which has been previously identified in the highly virulent strain K. pneumoniae CG43 were isolated by PCR-based cloning from CG43 and designated kvhAS (kvg homolog). The kvhAS was demonstrated to prevail in all the strains analyzed by dot-blotting hybridization. In addition, KvhAS share common motifs, especially the residues near the biological active sites, with the virulence-associated two-component system BvgAS and the multidrug resistance-associated two-component system EvgAS in B. pertussis and E. coli respectively.
To investigate the functional role of kvhAS in K. pneumoniae CG43, the operon structure of kvhAS was verified. In addition, the positively auto-regulatory mechanism was verified both in vitro by electrophoretic mobility shift assay and in vivo by the galU reporter system. The four kvhAS mutants, CG43-S3-kvhAS-, CG43-S3-kvhS-, CG43-S3-U9451-kvhAS-, and CG43-S3-U9451-kvhS-, were constructed by gene replacement through homologous recombination. To determine whether kvhAS is a virulence determinant in CG43 pathogenesis, a mouse peritonitis model was used to determine LD50 of the wild type CG43-S3 and the isogenic kvhAS mutant, and a slight increase of LD50 of the kvhAS mutant was observed. Moreover, heat shock (44℃) was found to activate the PkvhAS activity. While a lot of two-component systems were found to regulate the physically linked genes, the genes adjacent to the kvhAS were analyzed and annotated. The genes of dha regulon, the putative acid-resistance genes, yfdXL which show a high homology with the evgAS-regulated yfdX, and several ORFs with unknown function were identified downstream or upstream of kvhAS. The identical gene organization of kvhAS and the physically linked genes were demonstrated by analysis of the recombinant phage DNA containing kvhAS. The specific binding between the phosphorylated His6-KvhA and the adjacent promoter-containing regions were demonstrated by electrophoretic mobility shift assay. The sequences specifically bound by the phosphorylated His6-KvhA were analyzed, and the features of the 5 sequences were observed. They all contain multiple repeats of T and A, 6~8 bp of palindromes, and AT-concentrated regions. To further identify the KvhA-regulated genes, the direct-binding experiments coupled with nickel-chelating resin or agarose gel electrophoresis were carried out, but found to fail to fish out the His6-KvhA bound target promoters.
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