A Study On Novel Gene DsbM In Pseudomonas Aeruginosa

Pseudomonas aeruginosa is a main opportunistic human pathogen in nosocomialinfections and its intrinsical resistance to antibiotics limits the disinfection efficacy.Its pathogenesis and antibiotic resistance are due to the presence of serials virulencefactors. As antibiotics have been used extensively and constantly, P. aeruginosareflects higher multidrug resistance, and the trend is keeping ascending.In this study, a clinical isolated strain PA68was adopted to construct a mutantlibrary from which a drug resistant screen was carried out. One mutant strain withhigh streptomycin resistance was isolated and named M122. Gene cloning andsequencing indicated that the alteration of M122phenotype was due to the insertionalinactivation of the gene PA0058, which is a new gene in P. aeruginosa.In this study, the bacteriostatic experiment in vitro indicates that M122hasabnormally high aminoglycoside resistance. After transforming a plasmid containingthe PA0058into M122, the resistance phenotype restored partially. To exclude thestrain specific, PA0058gene was knocked-out in P. aeruginosia PAK. It is the firsttime that gene PA0058was identified to be involved in upward aminoglycosideresistance in P. aeruginosa.The gene PA0058encodes a234residues conserved protein of unknownfunction. We cloned and expressed this gene in Escherichia coli. The primarystructure analysis of PA0058demonstrates that it contains a CXXC motif, which isconserved in thiol:disulfide oxidoreductases. The tertiary structure prediction showsthis protein has a thioredoxin-like fold. The studies on biochemical characters ofpurified PA0058suggest that it has thiol:disulfide oxidising, reducing andisomerisation activities. The biochemical characterization suggests that PA0058is anovel disulfide oxidoreductase. Hence, the protein was designated as DsbM.DNA microarray was used to analyze the genomic expression in M122. Theresults indicated that the mutation of gene PA0058causes multiple gene expressionchange in P. aeruginosa, especially the significantly up-regulated antioxidant enzymegenes. Microarray analysis of the M122mutant showed its unusual phenotype might be related to the bacterial antioxidant defense system mediated by the oxyR regulon.The study on the functions of dsbM gene has important implications forunderstanding the mechanisms underlying aminoglycoside resistance in P. aeruginosa.It will helpful to prevent and cure the infection caused by P. aeruginosa.