rsmA调控基因的筛选及其相关表型测定

Identification of Genes that Regulate rsm A and Phenotypes Affected

为了筛选对rsm A具有调控功能的相关基因及这些基因对铜绿假单胞菌致病性的影响,通过构建染色体组上整合有rsm A-lux CDABE报道子的铜绿假单胞菌菌株,并以rsm A基因表达为线索进行随机转座突变,利用发光变化筛选出对rsm A基因表达有影响的转座突变体.最终共得到约30000个克隆,经筛选得到20株发光明显变化的突变体,其中rsm A表达增强的有6个克隆,而rsm A表达减弱的有14个克隆.通过随机PCR并对产物测序及比对,最终确定7个被破坏的基因.对致病性相关表型测定,结果表明细菌运动性、绿脓菌素、鼠李糖脂和胞外多糖产量等均受到不同程度的调节.这些基因对rsm A表达有调节作用,并可能通过rsm A影响铜绿假单胞菌的多个致病因子及致病性.

RsmA is a key post-translational regulator that controls multiple virulence factors in human pathogen Pseudomonas aeruginosa. In this study, we used an rsmA-luxCDABE reporter integrated on the chromosome of P. aeruginosa to identify genes that influence the expression of rsmA. We screened a transposon mutant library of 30,000 clones for mutants that exhibited either increased or decreased expression of rsmA. Of the total of 20 confirmed mutants, 14 demonstrated a reduced expression of rsmA and 6 increased expression. Seven genes disrupted by transposon insertion were eventually identified by arbitrary PCR and sequence alignment. In agreement with the important roles of RsmA, bacterial motility, pyocyanin and extracellular polysaccharide production were affected at various degrees in these mutants. Identification of these putative regulators of rsmA provides not only new information of the complex regulatory networks that govern the pathogenicity of P. aeruginosa, but also potential new targets for controlling P. aeruginosa virulence.