Cloning And Functional Analysis Of Serratia Marcescens RpfF Gene

Serratia marcescens is a gram-negative bacterium widely distributed in plants,animals,and the environment,belonging to the genus Serratia.S.marcescens is an opportunistic pathogenic bacterium that infects plants,invertebrates,and vertebrates.It can infect some economic plants and important insects,such as silkworms.Moreover,S.marcescens is also one of the common pathogens of hospital infections,which can cause pneumonia,urinary tract infections,osteomyelitis,surgical wound infections,and other diseases.Meanwhile,it has a variety of antibiotic resistance,which increases the difficulty of clinical treatment.Many studies have reported that the rpfF gene exists in a variety of gram-negative bacteria.It is the key gene for the synthesis of bacterial quorum sensing signaling molecule DSF which indirectly regulates the virulence-related biological characteristics,such as extracellular protease,extracellular polysaccharide,motility,and biofilm formation.However,the rpfF gene is rarely reported in S.marcescens,and its function is still unclear.In this study,the rpfF gene was cloned using S.marcescens SCQ1 genomic DNA as a template.The rpfF gene knocked out mutant and its complemented strains were constructed by the homologous recombinant method.The relevant biological characteristics of the wild-type,knock-out,and complementation strains were detected.Combined with metabolomics analysis,the function of the rpfF gene in S.marcescens was explored.The main research methods and results are as follows:1.The complete sequences of the rpfF gene were PCR-amplified using S.marcescens SCQ1 genomic DNA as a template and sequenced.Using a variety of bioinformatics software analyses,the rpfF gene is 867 bp long,encoding 288 amino acids.The Rpf F protein has four amino acid secondary structures,includingαhelix,random coil,extension,andβturn,and the isoelectric point is 7.10.Rpf F is predicted to be a hydrophilic protein with no apparent signal peptide,no transmembrane structure,one glycosylation site,and 21 phosphorylation sites.After the prediction of conserved domains,homology,and multiple sequence alignments,we found that the Rpf F contains the enoyl-Co A hydratase domain（PRK08788）,which belongs to the crotonase-like Superfamily.These results show that the rpfF gene is highly conserved.2.The deletion mutantΔrpfF and complementation strain CΔrpfF were constructed successfully which was verified by PCR and sequencing.3.The relevant biological characteristics of the wild-type strain SCQ1,ΔrpfF,and CΔrpfF were detected.As a result,the colony morphology,protease and lecithinase hydrolysis activities,osmotic pressure,acid-base tolerance,and hemolytic activity did not change significantly when compared with the wild-type strain SCQ1.However,the logarithmic growth rate,motility,and biofilm formation ofΔrpfF were decreased and the H₂O₂ tolerance was enhanced.The antibiotic resistance test showed that theΔrpfF was more resistant to cefotaxime and ceftazidime than SCQ1,but the resistance to other antibiotics was unchanged.However,high-performance liquid chromatography analysis showed that both the wild-type strain SCQ1 and the knock-out strainΔrpfF do not produce the signal molecule BDSF,which was different from B.cepacia complex and C.turicensis.The virulence test showed that there was no significant difference in LD₅₀ ofΔrpfF compared with the wild-type strain SCQ1,but the LT₅₀ ofΔrpfF was significantly prolonged.4.Using non-targeted metabolomics to detect the primary metabolites of the mutantΔrpfF and the wild-type strain SCQ1,a total of 50 specific differential metabolites were identified,of which 26 were up-regulated and 24 were down-regulated.Through metabolite cluster analysis and metabolic pathway analysis,the 50 differential metabolites were mainly distributed in amino acid metabolism,vitamin metabolism,lipid metabolism,carbon metabolism,energy metabolism,nucleic acid metabolism,and other metabolic pathways.A total of 7 ROS-related differential metabolites were detected.Combined with the bacterial H₂O₂ resistance plate test,the enhanced antioxidant capacity ofΔrpfF may be due to the synthesis and metabolism of differential antioxidant metabolites.In conclusion,the knockout strainΔrpfF has enhanced resistance to H₂O₂ and cephalosporins.Metabolome analysis suggests that the rpfF gene may affect the antioxidant capacity and the drug resistance of S.marcescens by regulating the production and elimination of ROS.