Regulation Of ArgR And SmpB On OmpA Biogenesis In Aeromonas Veronii

Trans-translation is a ribosome rescue mechanism prevalent in bacteria,and its core molecule SmpB(Small protein B)together with tmRNA rescuing ribosomes when translation is making errors.In addition,SmpB can also regulate the two-component system,control the quality of proteins,affect the formation of persister and regulate cell cycle and so on.In this study,set as a kind of aquatic pathogen called Aeromonas veronii C4 as research target,by constructing a cDNA library of it to screen SmpB interaction protein through the method of bacterial two-hybrid assay,and set the interaction protein as the entry point to explore other signal transduction pathways that SmpB may be involved in.A cDNA library of A.veronii C4 was constructed successfully with an arginine repressor(ArgR)which interacted with SmpB was screened,and then the key sites were identified for the interaction proteins.In this study,the cDNA library was constructed with a library capacity of 1.6×106 cfu/ml.By taking SmpB as the bait protein,19 of interacted proteins were screened by bacterial two-hybrid assay,and only the ArgR had the correct reading frame after blast.The full-length protein of ArgR interacted strongly with SmpB which was verified by bacterial two-hybrid assay and GST pull-down.And then the ArgR truncation mutants ?N81 and ?C81 were constructed,by the way,the sites of Q50,S54R,D121,G134 of ArgR were mutated to A according to conserved domain alignment and conserved site analysis.The interaction domain and sites of ArgR and SmpB were also verified by bacterial two-hybrid assay.For this part,we concluded that SmpB interacts with ArgR through its C-terminal domain with the sites of G133K,D138KR;while both of the C-terminal domain or N-terminal domain of ArgR is important to interact with SmpB,and the key sites are S54R,D121,G134.Based on the results of previous studies in our laboratory,both the positively regulatory relationship and binding sites need to be identified of SmpB on ompA.OmpA is the major outer membrane protein of the gram-negative bacteria.It is a multifunctional protein that not only maintains the integrity of the cell membrane structure,but also participates in the formation of biofilms,or as a virulence factor involved in cell adhesion.The binding sites of SmpB on ompA promoter may exist in the upstream of the transcription initiation site which arrange from-125 to-106 bp,-96 to-75 bp,-59 to-49 bp,-32 to-21 bp.In this part,the ompA promoter mutant T-96GG,C-55AC,C-28HAT was fused with the fluorescent reporter gene eGFP and co-transformed with the SmpB expression vector,so the binding site of SmpB on ompA promoter could be determined by fluorescence change.In this way,we indicated that SmpB positively regulates ompA expression by binding to the C-28AT site of the ompA promoter,and that the G11S and K152 sites of SmpB are critical for this regulatory relationship.To further clarify the regulation of ArgR on ompA or the control mechanism between ArgR and SmpB on ompA.The expression vector ArgR or co-expression vector which express both SmpB and ArgR was co-transformed with the plasmid which fused the ompA promoter with eGFP,the relationship between them were identified by analyzing the change of fluorescence value.It was found that when ArgR acts alone,it downregulated the expression of ompA in the logarithmic phase but upregulated in stationary phase.In the case of the coexistence of ArgR and SmpB,the regulatory relationship changes:in the logarithmic phase,the combination of ArgR and SmpB inhibits the expression of ompA;in the stationary phase,the expression of ompA was not affected.And Real-time PCR verified that argR was highly expressed in the logarithmic and stationary phase,while smpB with low expression in the logarithmic phase but reverse in stationary phase(unpublished).It was speculated that ArgR and SmpB interacted to form fewer complexes in logarithmic phase,and finally to inhibite the expression of ompA.However,in the stationary phase,ArgR and SmpB interacted to form large complex,eliminating the regulation on ompA.The phenotypic changes caused by ompA under the control of SmpB were verified by constructing the gene knockout strain.The A.veronii C4 knockout strains AOmpA and ASmpBAOmpA were constructed based on the principle of homologous recombination,and the phenotypic changes of the knockout strains were detected.The results showed that the knockout strains AOmpA and ASmpBAOmpA grew more slowly than the wild type strain;and the ability of biofilm formation on AOmpA did not change any more,but the biofilm formation ability of ASmpBAOmpA knockout strain was significantly increased compared with wild type strain(p<0.05)but not in AOmpA;by the way,?OmpA showed more sensitive to gentamicin and more tolerant to trimethoprim,and ?SmpB is only sensitive to gentamicin,and ?SmpB?OmpA is more sensitive to gentamicin than AOmpA.In summary,we propose a novel regulatory mechanism consisting of ArgR,SmpB and OmpA,that is,ArgR and SmpB can serve as transcription factors to regulate the expression of ompA directly or synergistically,and affect a series of phenotypic changes of pathogen such as growth,biofilm formation or antibiotic resistance.The results of this study has further revealed the mechanism of which SmpB-mediated regulation on ompA in A.veronii C4,and providing a theoretical basis for further elucidation of the infectious ability and pathogenicity of A.veronii,and supporting helps for good understanding the complex transcriptional regulatory network of pathogens.