Characterization of PhoP of Mycobacterium avium and construction of its mutant harboring defective PhoP gene

Abstract:

[Objective] To understand the regulatory mechanism of PhoP of Mycobacterium avium, the function of PhoP was analyzed and a Mycobacterium avium strain with defective PhoP gene was constructed. [Methods] DNA fragment of PhoP DNA-binding domain (PhoPC) amplified by PCR was cloned into the pGEX-4T-3 vector, and then the recombinant plasmid was transformed into Escherichia coli BL21(DE3) for expression of GST-PhoPC protein. PhoPC protein was prepared through removal of GST-tag from GST-PhoPC protein by thrombin cleaving. The promotor regions of PhoP, MAV0127, PhoU and Amt were amplified by PCR, respectively. Electrophoretic mobility shift assay (EMSA) was carried out to analyze the binding activity of PhoPC to these promotor fragments. For construction of a Mycobacterium avium strain with defective PhoP gene, PhoP homologous recombinant DNA fragment with defective mutation was obtained by ligasing the upper-stream and down-stream regions of PhoP gene, which were amplified by PCR. The PhoP recombinant DNA fragment was cloned into the suicide plasmid pGMB151. The recombinant plasmid was then transferred into cells of Mycobacterium avium by using electroporation for homologous recombination. The strains harboring defective PhoP gene were selected and identified by PCR. [Results] EMSA results show that PhoPC protein could bind to the promotors of PhoP, MAV0127 and Amt genes, but not bind to the promotor of PhoU. A deletion of 309 bp of the defective PhoP gene was confirmed by PCR and DNA sequencing. [Conclusion] PhoP can regulate the transcription of its downstream genes MAV0127 and Amt, and also regulate the transcription of PhoP gene itself. However, PhoP does not participate in regulating PhoU two-component system. A Mycobacterium avium strain with defective PhoP gene was successfully constructed, which contributes to further study on the role of PhoP in transcription regulation in Mycobacterium avium.