Studies on the interactions among genomic island modules in Klebsiella pneumoniae 1084

• Main Authors: Yi-Min Hong, 洪義閔
• Other Authors: Ying-Tsong Chen
• Format: Others
• Language: zh-TW
• Published: 2015
• Online Access: http://ndltd.ncl.edu.tw/handle/39928630655712488361

碩士 === 國立中興大學 === 基因體暨生物資訊學研究所 === 103 === Horizontal gene transfer between bacteria has been shown to be an important mechanism for exchange of genetic determinants. These confer a selective advantage to the recipient, e.g., adaption to environment and enhance pathogenicity. Bacterial genomes contain clusters of genes that are acquired by horizontal gene transfer, named genomic islands (GIs). How the acquired biosynthesis/metabolic pathways merged into the existing network of the cell remain elusive. In our previous study, we have identified a 208-kb genomic island from newly sequenced Klebsiella pneumonia 1084 genome. This 208-kb genomic island, KPHPI208, is composed of 8 genomic modules (GMs), GM1~GM8. GM1 consists of genes responsible for colibactin production. In our previous study, the colibactin-producing K. pneumonia 1084 was demonstrated to induces DNA double-strand breaks both in vitro and in vivo. In Escherichia coli, the biosynthesis of colibactin required a 4’- phosphopantetheinyl transferase (PPTase). Interestingly, PPTase also contribute to the synthesis of yersiniabactin, which is also contained as a GM in KPHPI208 (GM3). GM6 consists of genes responsible for microcin E492 (MccE492) production. MccE492 is a low molecular weight bacteriocin. It is composed of polypeptide core with C-terminal enterobactin. In this study we aimed to determine the possible regulation of gene expression among colibactin, microcin and siderophore GMs. In addition to previously constructed 1084SΔclbA, we generated mceAB knockout mutant strain ΔmceAB and double mutant strain ΔmceABΔclbA using allelic exchange. The mutant strains we also utilized by the members in our group to perform functional analysis, including antimicrobial test, siderophore secretion, and to establish the biochemical assay procedure for the toxins. We designed primers targeting the genes in the 8 GMs and other virulence genes in the chromosome. Compared to wild type strain, ΔmceAB showed decreased antimicrobial activity. While we did not see the differences of siderophore secretion between the mutant and wild type, using Real-Time PCR we were able to detect the expression of target gene in siderophore modules and other genomic modules in vivo. We also identified increased transcription of yersiniabactin GM and GM4 in cell culture infection model. Our result suggests a possible regulation among horizontally acquired genomic modules carried in the HPI.