【作者】 孔丽萍；

【导师】 段康民；

【作者基本信息】 西北大学 ， 生物化学与分子生物学， 2008， 硕士

【摘要】 铜绿假单胞菌(绿脓杆菌,Pseudomonas aeruginosa,PA)是一种条件致病菌,其很多致病性都与毒性因子的分泌有关,其中吩嗪类复合物是很重要的毒性因子,包括:吩嗪-1-羧酸(phenazine-1-carboxylic acid,PCA)、绿脓菌素(pyocyanin)等。PCA等吩嗪类复合物在细胞内可作为电子载体,增加感染细胞内的氧化压力,使感染的目标细胞中毒死亡,绿脓菌素与囊肿性纤维化病人(CF)的肺部感染有关。PCA为绿脓菌素合成的中间体,而phzA1B1C1D1G1(phzA1)操纵子参与了PCA的合成过程。已有的研究报道表明次抑制浓度的抗生素可以作为信号分子调节致病基因的表达。本实验室前期研究表明:次抑制浓度(subinhibitory concentrations)的四环素可以调节phzA1操纵子的表达;在次抑制浓度四环素条件下,以发光杆菌的荧光素酶基因操纵子luxCDABE为报道基因,对含有phzA1操纵子报道载体的转座突变库进行了筛选。本实验对筛选出的其中三株突变体PAM0487、PAM0487R、PAM0207进行了研究。在三株突变体PAM0487、PAM0487R、PAM0207中,phzA1操纵子的表达降低。通过随机PCR、基因测序及基因比对确定了突变点在染色体上的位置。发现其中的两株突变体PAM0487、PAM0487R的突变点均位于PA0487基因启动子区内,PA0487基因为假定钼元素的转运调节基因。PAM0207位于PA0207基因启动子区内。通过测定绿脓菌素在野生型PAO1、PAM0487、PAM0487R中的生成量,发现在突变体PAM0487和PAM0487R中绿脓菌素的生成量降低,证实PA0487基因对phzA1操纵子在转录水平的正向调节作用表现在表型上。在不同浓度的四环素条件下,phzA1操纵子表达受四环素调节的现象在突变体PAM0487R中消失,说明PA0487是四环素对phzA1操纵子调节途径中的一员。由于铜绿假单胞菌中吩嗪类复合物的合成受群体感应系统(quorum-sensing,QS)的调控,本实验研究了PA0487基因与QS系统的关系,发现PA0487对群体感应系统相关基因的表达有调节作用。将Gm~r-lacZ基因插入野生型PAO1的PA0207基因中间构建了突变体PAK0207,在突变体PAK0207中phzA1操纵子的表达无明显变化,表明对phzA1具有调节作用的基因并不是PA0207。更多还原

【Abstract】 Pseudomonas aeruginosa is an opportunistic pathogen. A number of secretable virulence factors contribute to its pathogenicity, including phenazines. P. aeruginosa releases at least four phenazine compounds. They are 1-hydroxy-phenazine (1-OH-PHZ)、PCA、phenazine-1-carboxamide (PCN) and pyocyanin. Pyocyanin is an important phenazine compound, and it is suggested to be involved in lung infecion of cystic fibrosis patients. Quorum sensing network controls the production of phenazines.A novel regulator of phzA1 operon in Pseudomoas aeruginosa had been identified by screening a transponson mutagenesis library in the presence of subinhibitory concentrations of tetracycline (Tet). Three mutants that exhibited changed expression of the phzA1 operon were obtained. The transposon insertion sites were determined by arbitrary PCR and subsequent PCR product sequencing. Two sites found to be at the promoter region of gene PA0487, and one at the promoter region of gene PA0207. PA0487 was required for the expression of phzA1 operon, and in the knockout mutant some of the genes involved in quorum sensing were down-regulated. The activation of phzA1 by subinhibitory concentrations of Tet was abolished in PAM0487, probably due to that PA0487 is involved in subinhibitory antibiotic regulation pathway. In conclusion, our results indicate that PA0487 is involved in regulating the expression of phzA1, possibly through the quorum sensing system.更多还原