铜绿假单胞菌吩嗪合成新调节基因的研究

发布时间：2019-03-25 15:24

【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。
[Abstract]:Pseudomonas aeruginosa (Pseudomonas aeruginosa,PA) is a conditional pathogen. Many pathogenicity of Pseudomonas aeruginosa is related to the secretion of toxic factors, among which phenazine complexes are very important toxic factors. Phenazine-1-carboxylic acid (phenazine-1-carboxylic acid,PCA), pyocyanin (pyocyanin), etc. can be used as electronic carriers in cells to increase the oxidative pressure in infected cells and cause poisoning and death of infected target cells. Pseudomonas aeruginosa is associated with pulmonary infection in patients with cystic fibrosis (CF). PhzA1B1C1D1G1 (phzA1) operon is an intermediate in the synthesis of pyogenin, and phzA1B1C1D1G1 (phzA1) operon is involved in the synthesis of PCA. Previous studies have shown that subinhibitory concentrations of antibiotics can be used as signal molecules to regulate the expression of pathogenic genes. Previous studies in our laboratory showed that tetracycline with subinhibitory concentration of (subinhibitory concentrations) could regulate the expression of phzA1 operon. Using luciferase gene operon luxCDABE as reporter gene, the transposable library containing phzA1 operon reporting vector was screened under the condition of tetracycline subinhibitory concentration. In this study, three of the selected mutants, PAM0487,PAM0487R,PAM0207, were studied. In the three mutants PAM0487,PAM0487R,PAM0207, the expression of phzA1 operon was decreased. The position of mutation point on chromosome was determined by random PCR, gene sequencing and gene comparison. Two mutants of PAM0487,PAM0487R were found to be located in the promoter region of the PA0487 gene. The PA0487 gene was supposed to regulate the transport of molybdenum. PAM0207 was located in the promoter region of the PA0207 gene. By measuring the production of pyocyanin in wild-type PAO1,PAM0487,PAM0487R, it was found that the production of pyocyanin in mutant PAM0487 and PAM0487R decreased, which confirmed that the positive regulatory effect of PA0487 gene on phzA1 operon at the transcriptional level was shown to be phenotypic. At different concentrations of tetracycline, the tetracycline-regulated expression of phzA1 operon disappeared in mutant PAM0487R, suggesting that PA0487 is one of the regulatory pathways of tetracycline on phzA1 operon. Because the synthesis of phenazine complexes in Pseudomonas aeruginosa was regulated by quorum sensing system (quorum-sensing,QS), the relationship between PA0487 gene and QS system was studied in this study. It was found that PA0487 could regulate the expression of quorum sensing system related genes. The expression of phzA1 operon in mutant PAK0207, was not changed by inserting Gm~r-lacZ gene into the PA0207 gene of wild type PAO1, indicating that the gene that regulates phzA1 is not PA0207..
【学位授予单位】：西北大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R378

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