Identification of novel genes associated with alginate production in Pseudomonas aeruginosa using mini-himar1 mariner transposon-mediated mutagenesis.

摘要: Pseudomonas aeruginosa is a Gram-negative, environmental bacterium with versatile metabolic capabilities. P. an opportunistic bacterial pathogen which establishes chronic pulmonary infections in patients cystic fibrosis (CF). The overproduction of capsular polysaccharide called alginate, also known as mucoidy, promotes the formation mucoid biofilms are more resistant than planktonic cells to antibiotic chemotherapy and host defenses. Additionally, conversion from nonmucoid phenotype clinical marker for onset infection CF. Alginate by endergonic process heavily taxes cellular energy. Therefore, alginate production highly regulated aeruginosa. To better understand regulation, we describe protocol using mini-himar1 transposon mutagenesis identification novel regulators prototypic strain PAO1. procedure consists two basic steps. First, transferred (pFAC) E. coli SM10/λpir into recipient PAO1 via biparental conjugation create high-density insertion mutant library, were selected on isolation agar plates supplemented gentamycin. Secondly, screened isolated colonies map site through inverse PCR DNA primers pointing outward gentamycin cassette sequencing. Using this protocol, have identified regulators, mucE (PA4033) kinB (PA5484), wild-type mucA encoding anti-sigma factor MucA master regulator AlgU (AlgT, σ22). This high-throughput can be modified other virulence-related genes causing change colony morphology.