Transmission of Yesinia pestis relies primarily on the bite of flea vectors,which is closely related to the biofilm formation of this pathogen[1].Y.pestis synthesizes heavily the attached biofilms,a population of bacterial colonies embedded in self-produced exopolysaccharide matrix,in the flea proventriculus[1].The biofilm formation leads to the blockage of fleas,making the fleas feel hungry and repeatedly attempt to feed,
Flea-borne transmission is a recent evolutionary adaptation that distinguishes the deadly Yersinia pestis from its progenitor Y.pseudotuberculosis,a mild pathogen transmitted via the food-borne route.Y.pestis synthesizes biofilms in the flea gut,which is important for fleaborne transmission.Yersinia biofilms are bacterial colonies surrounded by extracellular matrix primarily containing a homopolymer of N-acetyl-D-glucosamine that are synthesized by a set of specific enzymes.Yersinia biofilm production is tightly regulated at both transcriptional and post-transcriptional levels.All the known structural genes responsible for biofilm production are harbored in both Y.pseudotuberculosis and Y.pestis,but Y.pestis has evolved changes in the regulation of biofilm development,thereby acquiring efficient arthropod-borne transmission.
Objective This study is to verify the use of rich BHI medium to substitute synthetic media for gene regulation studies in Yersinia pestis. Methods The transcriptional regulation of rovA by PhoP or via temperature upshift, and that of pla by CRP were investigated when Y. pestis was cultured in BHI. After cultivation under 26 °C, and with temperature shifting from 26 to 37 °C, the wild-type (WT) strain or its phoP or crp null mutant (ΔphoP or Δcrp, respectively) was subject to RNA isolation, and then the promoter activity of rovA or pla in the above strains was detected by the primer extension assay. The rovA promoter-proximal region was cloned into the pRW50 containing a promoterless lacZ gene. The recombinant LacZ reporter plasmid was transformed into WT and ΔphoP to measure the promoter activity of rovA in these two strains with the β-Galactosidase enzyme assay system. Results When Y. pestis was cultured in BHI, the transcription of rovA was inhibited by PhoP and upon temperature upshift while that of pla was stimulated by CRP. Conclusion The rich BHI medium without the need for modification to be introduced into the relevant stimulating conditions (which are essential to triggering relevant gene regulatory cascades), can be used in lieu of synthetic TMH media to cultivate Y. pestis for gene regulation studies.
ZHANG Yi QuanMA Li ZhiWANG LiGAO HeTAN Ya FangGUO Zhao BiaoQIU Jing FuYANG Rui FuZHOU Dong Sheng
