Study On The Regulatory Mechanism Of DNA Topoisomerase ? On Pathogenicity Of Pseudomonas Aeruginosa PAO1

Pseudomonas aeruginosa is a gram-negative aerobic bacterium,which can cause acute and chronic infections in patients with immunodeficiency.The main reason why P.aeruginosa causes diseases is that virulence factors could cause tissue damages.These virulence factors include a phenazine compound named pyocyanin with redox activity.A transposon mutant of a high-yield pyocyanin is found in our previous researches.Based on the gene knockout method,an first recombination mutant(top A-RM)is obtained which contains incomplete top A gene,and we confirm that top A(topoisomerase ?)was required for viability of P.aeruginosa.Through indepth studies of the strain,we aim to explore the regulation effect of top A gene on production of pyocyanin.In this study,we confirmed the change of production of pyocyanin in top A-RM through transcription level,HPLC and chloroform extraction.The results showed that genes related to phenazine in top A-RM were activated and the yield of pyocyanin in top A-RM was higher than that in the wild type PAO1.In order to study the reason why the production of pyocyanin rose in top A-RM,we tested the expression of QS system which is positively correlated with phenazine synthesis.We find that QS system was not involved in the production of pyocyanin in top A-RM.In this study,by detecting the expression of genes related to DNA repair,it was found that the altered Top A in top A-RM caused DNA damage,and the bacteria needed to start the DNA repair process through the SOS reaction,namely the emergency response,to ensure the normal replication of DNA.DNA damage causes the self-cleavage of Prt R regulatory proteins,which caused de-reperession of transcriptional regulators prt N and ptr B,which were controlled by Prt R.This study found that prt N and ptr B gene expression have been up-regulated,which are respectively active the expression of genes involved in the synthesis of the bacteriocin pyocin and suppresses the ? secretion system.In addition,this study examined the expression of genes involved in reactive oxygen species(ROS)that can induce SOS reaction,and the results showed that the altered Top A did lead to the production of ROS.To explore whether the increase of pyocyanin in top A-RM is due to the alteration of Prt R.We examined the production of pyocyanin in double mutants ?prt N-top A-RM and ?ptr B-top A-RM.The studies showed that the production of pyocyanin in the former significantly decreased,while the latter did not change.The production of pyocyanin in the ?prt NC-top A-RM could restore to the level of top A-RM.The results indicated that prt N was associated with the production of pyocyanin in top A-RM.Through overexpressing prt N in the wild type PAO1 and detecting the production of pyocyanin in it,it was found that the regulation relationship existed only in the case of top A damage.In order to further study effects of Top A on the pathogenicity of P.aeruginosa,this study examined expression of genes involved in supercoil,it was found that negative supercoil increased in top A-RM.Meanwhlie,we examined its motility ability,production of extracellular polysaccharide,adhesion,production of biofilm,and antibiotic resistance in top A-RM.The increase of production of extracellular polysaccharide and adhesion ability were found.The decrease of swarming motility ability also was found.Meanwhile,the sensitivity to streptomycin,tobramycin,ciprofloxacin,and rifampicin also increased.In conclusion,Top A with pathogenicity and drug resistance of P.aeruginosa are closely associated.