Construction of the clpP mutant strain of Pseudomonas aeruginosa
ZHANG Jia-qin1, RAO Hui-hua1, 2, XU Qiao-li1, 2, HUANG Chao-yang1, FANG Li-li1, MA Xiao-bo1, SONG Xiu-yu3
1.Department of Clinical Laboratory, the First Affiliated Hospital of Xiamen University, Xiamen 361003, China; 2.The First Clinical Medical College of Fujian Medical University, Fuzhou 350005, China; 3.Xiamen Central Blood Service Station, Xiamen 361003, China
Abstract:To delete clpP gene of Pseudomonas aeruginosa PAO1, the clpP gene with 3' and 5' flanking sequences was amplified by PCR using the primers clpP-F1and clpP-R1, and then the PCR product was cloned into pMD19T for construction of pCKR1. A gentamicin resistance cassette amplified from pUCGM was inserted into EcoRV sites of pCKR1 for construction of pCKR2. Then the FclpP-GM-clpPR fragment was obtained by cutting pCKR2 with EcoRⅠ/HindⅢ and gel purification, and inserted it into EcoRⅠ/HindⅢ sites of pEX18Tc to construct the homologous recombinant vector pCKR3. The P. aeruginosa clpP mutant strain was obtained by conjugations between P. aeruginosa PAO1 and E. coli SM10 transformed with pCKR3. The homologous recombinant vector pCKR3 was verified by enzymatic digestion and the final P. aeruginosa clpP mutant strain was verified by PCR analysis and DNA sequencing. The clpP gene of P. aeruginosa was successfully deleted, which laid a foundation for further study of its biological function.
[1] Qiu D, Eisinger VM, Head NE, et al. ClpXP proteases positively regulate alginate overexpression and mucoid conversion in Pseudomonas aeruginosa [J]. Microbiology, 2008,154(Pt 7): 2119-2130. [2] Dougan DA, Mogk A, Bukau B. Protein folding and degradation in bacteria: to degrade or not to degrade? That is the question[J] . Cell Mol Life Sci, 2002, 59(10): 1607-1616. [3] Gottesman S. Proteases and their targets in Escherichia coli [J] . Annu Rev Genet, 1996, 30: 465-506. [4] WANG zhen-hai,SUN ye-qing. Progress in Study on Clp Protease[J]. Pharmaceutical Biotechnology,2005,12(6):412-415. 王振海,孙野青. Clp蛋白酶研究进展[J] . 药物生物技术, 2005,12(6):412-415.英文对照 [5] Yu AY, Houry WA. ClpP: a distinctive family of cylindrical energy-dependent serine proteases[J] . FEBS Lett, 2007, 581(19): 3749-3757. [6] de Bruijn I, Raaijmakers JM. Regulation of cyclic lipopeptide biosynthesis in Pseudomonas fluorescens by the ClpP protease[J]. J Bacteriol, 2009, 191(6): 1910-1923. [7] Bjarnsholt T, Jensen PO, Fiandaca MJ, et al. Pseudomonas aeruginosa biofilms in the respiratory tract of cystic fibrosis patients[J] . Pediatr Pulmonol, 2009, 44(6): 547-558.