Knockout Of Hfq Gene In Aeromonas Veronii And Its Effect On Persister Formation

Hfq(Host factor of the RN A bacteriophage Q?,abbreviated as Hfq)is a ubiquitous global regulator in bacteria.As an RNA chaperone,it influences the translation rate and stability of targeted transcript by mediating complementary base pairs of small RNA and messenger RNA(sRNA-mRNA).Hfq involves bacterial growth,proliferation,toxicity,and stress tolerance and other physiological and biochemical processes.Bacterial cells may escape the effects of antibiotics without undergoing genetic change,which are known as persisters.Unlike resistant bacteria,persister is a dormant phenotypic variant that weakens all life activities and is resistant to many drugs.The importance of Hfq to cell growth is presumed to be related with the formation of persister.There are many reports on Hfq,but it has not been researched in Aeromonas veronii yet.The potential function of Hfq in persistence formation was mentioned in a few studies in Escherichia coli.In this study,A.veronii C4 was selected as the material.Firstly,the hfq mutant A.veronii C4-Ahfq was constructed based on the principle of homologous recombination,and then the growth curves of C4-?hfq,as well as the minimum inhibitory concentration and minimum bactericidal concentration(MIC/MBC)for 9 antibiotic drugs were measured.The results showed that hfq knockout strain exhibited significantly delayed the growth rate and was more susceptible to antibiotics.The hfq complemented strain hfq/pBBR-hfq has the functions recovered to some extent.The four antibiotics selected in MIC/MBC assays were conducted drug susceptibility test,including chloramphenicol,tetracycline,cephalothin(CTX),and ciprofloxacin.The antibiotic.drug sensitivity curve was plotted,demonstrating that hfq knockout reduced the formation of persistence significantly compared to A.veronii wild type(WT).Finally,real-time quantitative PCR was performed in both ?hfq and wild type strains to detect the expression levels of some known persistence-related genes,including clpB,oxyR,dnaK,rpoS,oppA,oppB,phoU,smpB,relE,recA and cspD.Compared with wild type,the ?hfq strain showed significantly down-regulated expression levels of clpB,oxyR,rpoS,recA and cspD,significantly up-regulated oppB,and no significant changes in the expressions of other genes.In accordance with previous reports,it is proposed that Hfq can regulate the formation of persister through multiple cellular pathways in A.veronii,and hfq knockout results in a significant decrease in the persistence level.