Roles of RcsFCDB signaling relay in Klebsiella pneumoniae CG43

• Main Authors: Peng, Kuan-Nan, 彭冠男
• Other Authors: Peng, Hwei-Ling
• Format: Others
• Language: zh-TW
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/33794545703428109095

碩士 === 國立交通大學 === 生物科技系所 === 102 === The RcsFCDB phosphorelaying system originally identified as a regulator of capsule synthesis (Rcs) is also involved in regulation of the expression of other virulence properties including flagella production, swimming motility, and O-antigen chain length control. RcsF is a periplasmic lipoprotein; RcsC and RcsD are two inner membrane proteins respectively exerting histidine kinase (Hk) and histidine phosphotransfer (Hpt) activity; RcsB is a cytoplasmic response regulator. We have previously shown that the deletion of rcsB from Klebsiella pneumoniae CG43S3 reduced the levels of not only the capsular polysaccharide (CPS) biosynthesis but also the type 3 fimbriae major pilin MrkA production and the acid stress response. Here we investigate if the regulatory activity of RcsB could be correlated to the RcsFCD phosphorelay. Firstly, we generate K. pneumoniae CG43S3ΔrcsF, CG43S3ΔrcsC, CG43S3ΔrcsD, CG43rcsD-hk, and CG43rcsD-hpt, and compare their deletion effects with those of CG43S3ΔrcsB. The results showed that CG43S3ΔrcsD exerted a similar phenotype as that of CG43S3ΔrcsB. However, the deletion of rcsF or rcsC had no apparent effect on the production of CPS or MrkA; overexpression of RcsC increased the CPS production; deletion of rcsF rendered the bacteria insensitive to polymyxin B stimulation. We have also measured the gene expression using promoter reporter assay and qRT-PCR analysis. The results revealed that PrcsB had much higher activity than the activity of PrcsB, PrcsDB, and PrcsF could be induced by weak acid while PrcsC, PrcsB, and PrcsF were polymyxin B inducible. Finally, the set up in vitro phosphorelay system revealed that the non-specific dye ProQ could be used to determine the phosphorelaying activity between the signal transferred from RcsC, RcsD to RcsB.