| Truperella pyogenes is an opportunistic pathogen that can infect pigs,cattle,sheep and other livestock and cause a variety of diseases and cause serious economic losses.Antibiotics are the main means of treating T.pyogenes,but recent studies have found that T.pyogenes are more and more resistant to drug resistance,and there is a trend of multidrug resistance.Pat B protein is a transmembrane protein involved in the transport of bacterial substances.Studies have shown that Pat B protein is a member of the ABC transport superfamily,which is related to bacterial multidrug resistance.To study the biological function of T.pyogenes pat B gene will help to reveal the mechanism of drug resistance and pathogenicity of T.pyogenes,and provide theoretical basis for the treatment of T.pyogenes.In this study,the clinical isolate BMH06-3 of T.pyogenes was used as the research object.Based on the whole genome sequence of BMH06-3 obtained by the previous sequencing in the laboratory,the upstream 999 bp and downstream 999 bp of the pat B gene were defined as the upper and downstream homology arms of the pat B gene,using PCR technology to amplify the upper and lower homology arms of the pat B gene with BMH06-3whole genome DNA as a template,the recombinant plasmid p BAVIK-T5-gfp△pat B was constructed with the plasmid p BAVIK-T5-gfp as the vector,and the plasmid p BAVIK-T5-gfp△pat B was transferred into BMH06-3 competent cells by electroporation,PCR technology and genome sequencing were used to verify the results.T.pyogenes BMH06-3△pat B was successfully constructed,and the changes of growth state,drug resistance phenotype,biofilm and transcription level of BMH06-3 after pat B gene deletion were analyzed.In this study,the recombinant plasmid p BAVIK-T5-gfp △ pat B was successfully constructed.The double-enzyme digestion and PCR verification of the recombinant plasmid p BAVIK-T5-gfp △ pat B indicated that the plasmid was constructed successfully.The suspected gene deletion strain was verified by PCR with the upstream primer of the upstream homology arm and the downstream primer of the downstream homology arm.The results revealed that the PCR product band decreased.The genome sequencing results showed that the pat B gene was not found,so BMH06-3△pat B was successfully constructed.The results of BMH06-3 and BMH06-3 △ pat B growth rate test revealed that the growth rate of BMH06-3 did not change significantly after pat B gene deletion.The minimum inhibitory concentrations of 15 antibiotics against BMH06-3,BMH06-3 △ pat B and T.pyogenes standard strains were determined by the micro-broth dilution method.The test results revealed that BMH06-3 was resistant to these 15 antibiotics after the deletion of the pat B gene.The resistant phenotype did not change.The biofilm forming ability of BMH06-3 and BMH06-3△pat B was determined by crystal violet staining.The test results showed that the biofilm forming ability of BMH06-3 decreased after pat B gene deletion.After the deletion of the pat B gene,the amount of bacteria in the biofilm decreased,the dead bacteria increased,and the thickness of the biofilm decreased.The results of RNAseq analysis revealed that a total of 188 genes had increased expression and 317 genes had decreased expression.Among them,the m RNA expressions of related proteins in metabolic pathways such as ABC transport superfamily,quorum sensing system and oxidative phosphorylation metabolic pathway changed.The changes of the relative m RNA expression of luxs,rbs A,rbs B,lsrk and plo may be the reason for the decrease of the biofilm formation ability of BMH06-3.In this study,pat B gene deletion strain of T.pyogenes was constructed by homologous recombination technology,and the growth rate,drug resistance phenotype,transcriptome level and biofilm formation ability of the pat B gene-deleted strain were studied,which will provided the theoretical foundation for effectively preventing and treating the infections caused by T.pyogenes. |
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