Construction And Antibiotic Resistance Analysis Of Serratia Marcescens BaeSR Two-component Mutants

Serratia marcescens which can cause diseases in humans,animals and insects is an opportunistic pathogen.In clinical treatment,Serratia marcescens can cause patients to be infected with pneumonia,meningitis,septicemia,urinary tract infection and other diseases.In agricultural productions it can rot cantaloupe,zucchini,sweet pepper and other crops.Meanwhile,in economic insect breeding,it also can make silkworms die of septicemia and bees die of white-headed pupae.Therefore,the study of Serratia marcescens is great important.The infection caused by Serratia marcescens has been treated with antibiotics such as chloramphenicol and streptomycin in clinic.However,the challenge of Serratia marcescens drug resistance gets serious increasingly.It has been reported that the two-component system BaeSR can regulate the drug resistance of bacteria in many enterobacterias.So we obtained the knock-out strainΔbaeSR and the complemented strain CΔbaeSR on the basis of Serratia marcescens SCQ1 by using the one-step-cloning technology and a suicide plasmid vector system to explore the influences of two-component system BaeSR about biological functions,especially macrolides antibiotic resistance.The main findings of the experiment are as follows:1.We coloned the baeSR gene sequence from Serratia marcescens SCQ1 genome successfully and analyzed its bioinformatics.The results are as follows:the baeS gene is 1383bp totally,encoding 460 amino acids,expressing 51.7 k Da protein.The protein BaeS is histidine protein kinase,whose isoelectric point is 9.11.It is without signal peptides,no glycosylation sites and two transmembrane domains and three conserved domains,namely HAMP domain,His KA domain and HATPase domain.baeR gene base number is 717bp and encodes 238 amino acids.The BaeR protein,whose molecular weight is about 27.3k Da and its isoelectric point is 5.35.There are no signal peptides,no glycosylated sites and no transmembrane domains,but there are two conserved domains,REG domain and Trans reg-C domain.According to the sequence comparison,two compenent systems BaeSR has homology in many bacterias.2.A knock-out strainΔbaeSR and a complemented strains CΔbaeSR of Serratia marcescens were successfully constructed using a suicide plasmid vector system and a one-step cloning technique.3.Physiological and biochemical experiments were performed on the wild strain SCQ1,the knock-out strainΔbaeSR and complemented strain CΔbaeSR.The data were analysed to show that the knock-out strainΔbaeSR showed no change in growth morphology and rate compared with the wild strain.So,the two-component systemΔbaeSR was not involved in regulating the growth of Serratia marcescens SCQ1.In addition,the protease activity,lecithinase activity,haemolytic activity and motility of the knock-out strainΔbaeSR were not significantly different compared to the wild strain SCQ1.In terms of stress resistance,the knock-out strainΔbaeSR did not show similar resistance to different concentrations of Na Cl and Zn Cl₂ compared to the wild strain,but the knock-out strain showed increased sensitivity to sodium tungstate.The results of the virulence test showed that the knock-out strainΔbaeSR had a different LT₅₀ compared to the wild strain SCQ1,but there was no significant change in its LD₅₀.4.A drug sensitivity experiment was conducted to investigate whether the two-component system BaeSR of Serratia marcescens SCQ1 had any effects on resistance to aminoglycosides,rifamycins and macrolide antibiotics.The results showed that the knock-out strainΔbaeSR was 2-fold less susceptible to rifampicin,amikacin and the macrolide antibiotics azithromycin,erythromycin and clarithromycin,so it was hypothesized that the two-component system BaeSR could regulate the susceptibility of Serratia marcescens SCQ1 to macrolide antibiotics,and this was verified by quantitative polymerase chain reaction（q-PCR）.According to the experiment,the gene mac A and mac B encode specific efflux pumps of macrolide antibiotics and play an important role in macrolide antibiotic resistance;23S r RNA methyltransferase gene rlm C and rlm D could enhance rlm A methyltransferase gene which reduces the susceptibility of E.coli to macrolide antibiotics.q-PCR results showed that both genes mac A and mac B were significantly down-regulated and 23S r RNA methyltransferase genes rlm A,rlm C and rlm D were significantly up-regulated in knock-out strainΔbaeSR compared to wild strain SCQ1.So,the two-component system BaeSR can regulate the susceptibility of Serratia marcescens SCQ1 to macrolide antibiotics by regulating the genes mac A,mac B,rlm A,rlm C,and rlm D.In summary,the two-component system BaeSR of Serratia marcescens SCQ1 is not involved in regulating its own growth,biofilm formation,protease activity,lecithinase activity,haemolytic activity,motility,and resistance to Na Cl and Zn Cl₂stress;however,it is involved in regulating susceptibility to sodium tungstate,and resistance to rifampicin,amikacin and the macrolide antibiotics erythromycin,azithromycin and clarithromycin resistance.