Pseudomonas Aeruginosa In The Genome Effect Phenazine Phza2 Synthetic Gene Expression Regulation Of Gene Selection And Research

Pseudomonas aeruginosa, which is a gram-negative opportunistic pathogen, remains a leading cause of infection among hospitalized patients who are burned, postoperative or immunocompromised. It was difficult to cure the infection caused by this bacterium due to its resistance to antibiotics.Phenazines are a group of small molecular compounds with redox activity produced by Pseudomonas aeruginosa. The biological functions of phenazines include being virulence factors and signal molecules which can regulate the expression of many genes. PAO1contains two homologous gene clusters, phzA1B1C1D1E1F1Gl=1(phzAl) and phzA2B2C2D2E2F2G2(phzA2), which are responsible for synthesis of phenazines Three additional genes (phzM, phzS, and phzH) encoding unique enzymes are involved in the conversion of PCA to pyocyanin,1-HP and PCN. Pyocyanin is one of the most important phenazines. Several studies demonstrated that pyocyanin contributes in variety of ways to the pathophysiological effects observed in P. aeruginosa airways infection. It is very important to study the regulation of phenazine synthesis in order to understand the mechanism of virulence.In this research, we used transposon mutagenesis to carry out a genome level screen for genes that regulate phzA2. The promoter of the phzA2gene cluster was fused upstream of the luminescence reporter gene luxCDABE and transferred to P. aeruginosa PAO1. The resulting strain CTX-phzA2-PAO1carrying the phzA2-luxCDABE reporter on its chromosome was used for transposon mutagenesis. We identified genes involved in the regulation of phzA2by random inserting a transposon into CTX-phzA2-PAO1genome and selecting mutants with a changed phzA2expression profile.14mutants were identified and the transposon insertion sites were determined by arbitrary PCR and subsequent DNA sequencing of the PCR products. These disrupted genes were determined by comparing the obtained sequences with the genome sequence of PAO1using BLAST software.Two of the14transposon mutants, P2G1and P2E4, showed significant changes in the expression of phzA2and production of pyocyanin. We constructed gene knockout mutants of PA3043and PA1387. The results showed that the expression of phzA2and production of pyocyanin did not change in PA3043knockout mutant PAO1(A3043), which suggested that PA3043was not a regulator gene of phzA2. In the other knochout mutant PAO1(△1387), both the expression of phzA2and the production of pyocyanin were reduced, in contrast to the fact that the expression of phzA2and production of pyocyanin were significantly increased in transposon mutant P2E4. To rule out polar effect on the downstream genes in the transposon mutant, we over-expressed the downstream gene PA1388in PAO1, but the expression of phzA2showed no difference compared with the wild type strain.The results indicated the regulation of phenazine biosynthesis is complex and there are still unidentified regulatory genes affecting phzA2. It is important to investigate the relationship between phzA2and these newly identified genes.