Whole Genome Sequencing, Transcriptome Analysis of Acid Response, and Urease Gene Cluster Characterization of Klebsiella pneumoniae CG43

• Main Authors: Lin, Fang-Yu, 林芳瑜
• Other Authors: Chang, Hwan-You
• Format: Others
• Language: zh-TW
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/72275031070553698724

碩士 === 國立清華大學 === 分子醫學研究所 === 101 === Klebsiella pneumoniae is an important opportunistic pathogen that causes various human diseases such as pneumonia, urinary tract infection, meningitis, bacteremia and septicemia. In Taiwan, K. pneumoniae is the predominant pathogen responsible for pyogenic liver abscess in diabetic patients and K1 and K2 serotypes account for the majority of the isolates. K. pneumoniae CG43 was originally isolated from a patient with pyogenic liver abscess in Taiwan. It is a highly virulent K2 serotype strain. In this study, the whole genome sequence of K. pneumoniae CG43 was determined and annotated. The genome is 5,166,857 bp in length. The similarity of the CG43 genome sequence with that of K. pneumoniae NTUH-K2044 and MGH 78578 are 93 % and 94 %, respectively. K. pneumoniae CG43 has 203 open reading frames distinct from K. pneumoniae NTUH-K2044 and K. pneumoniae MGH 78578. Furthermore, because the ability of acid resistance is important for Enterobacteriaceae, we also performed transcriptome analysis of K. pneumoniae CG43 gene expression profile under acidic growth conditions. The data indicate that 4.94 % genes in CG43 genome were induced, while 18.34 % genes were repressed. Most of the up-regulated genes are associated with chaperone-related function and many of the down-regulated genes belong to maltose regulon. Besides, there are two urease gene clusters in K. pneumoniae CG43, different from most of K. pneumoniae that has only one urease gene cluster. Urease catalyzes the hydrolysis of the urea into carbon dioxide and ammonia. Besides providing nitrogen sources, the reaction can neutralize acidic environments, allowing pathogenic bacteria to survive the acidic conditions. Three types of urease mutant strains (ΔureA1, ΔureA2 and ΔureA1 ΔureA2) were constructed in CG43. Growth of ΔureA1 and ΔureA1 ΔureA2 double mutant was reduced in M9 minimal medium using urea as the sole nitrogen source. The two mutant strains lack urease activity as determined by Christensen’s Urea Agar. In addition, the growth of wild type and three urease mutant strains has no difference under acidic environment. The result suggests that urease is not a major factor contributing to acid tolerance of K. pneumoniae CG43 under normal cultural condition. To sum up, the complete CG43 genome sequence will provide critical information for future analysis of the virulence factors in the bacterium.