Pseudomonas Aeruginosa Rsma Regulate Gene Screening And Regulatory Pathway Research

Pseudomonas aeruginosa is a Gram-negative bacillus that grows ubiquitously and is an important opportunistic pathogen. It is one of the three major pathogens in hospital-aquired infections. Its infection is difficult to treat because the pathogen's high resistance to various antibiotics.RsmA is an RNA binding, posttranscriptional regulatory protein. RsmA negatively controls quorum sensing by down-regulating N-acylhomoserine lactones, the quorum-sensing signal molecule. It modulates the expression of a number of virulence-related genes important for acute and chronic infections. Swarm motility and Pseudomonas quinolone signal biosynthesis has also been shown to be positively controlled by RsmA. Other phynotypes affected by RsmA include antibiotic resistance, the type three secretion system (TTSS) and extracellular enzyme production.RsmA plays an important regulatory role at the level of transcription and translation, but its own regulation is unclear. In this research, we used transposon mutagenesis to carry out a genome level screen for the genes that regulate rsmA. Several of the genes identified were invetigated further.The promoter region of the rsmA genes was fused upstream of the luminescence reporter gene cluster luxCDABE and transferred to PAO1. The strain PAO1 (CTX-rsmA) carrying the rsmA-LuxCDABE reporter on the chromosome was used for transposon mutagenesis. We investigated genes involved in the regulation of rsmA by random inserting a transposon into PAO1 (CTX-rsmA) genome and selecting mutants with a changed rsmA expression profile. Nine mutants were identified and the transposon insertion sites in these mutants were determined by semi-random PCR and subsequent DNA sequencing. The genes disrupted were determined by comparing the sequences obtained and the genome sequence using BLAST software. These strains were tested for changes in biofilm, pyocyanin, Congo-red, antibiotic resistance. One of the transposon mutants, A7, disrupted PA4732 and showed significant changes in several phenotypes. PA4732 (pgi) encodes phosphate-6-glucose isomerase (pgi), which plays a role in carbon metabolism of Pseudomonas aeruginosa. Complementation test indicates the changed phenotype could be restored to the level seen in as the wild type PAO1. A gene knockout mutant of the pgi gene was also constructed, and the results confirm that the rsmA gene is regulated by pgi. Therefore, rsmA gene seems to associate with carbon metabolism. These results provide new understanding of the regulation of virulence factors in Pseudomonas aeruginosa.