Characterization Of The Phosphate-specific Transport System In Cronobacter Sakazakii BAA-894

The phosphorus is the essential elements for cell growth,thus its absorption and utilization play an important role for the growth of bacteria.In Escherichia coli,there existed phosphate transport system(Pst)with high Km value and Pit system with low Km values,to absorb and transfer inorganic phosphorus(Pi),respectively.While for Cronobacter sakazakii,the important food-borne pathogen,the role of Pst system had not been reported.The topic was studied in the current study.Firstly,the pst mutant strain was constructed by homologous recombination,and then the influence of the pstSCAB operon on the membrane structures and pathogenicity of C.sakazakii under different phosphate concentration conditions were investigated.The main conclusions are as follows:I.By the protein blast about the genes relativing to phosphate transport between C.sakazakii BAA-894 and E.coli MG1655,there were only 28 genes in BAA-894 have homologous genes among 50 genes in MG1655.All four genes homologous proteins of pstSCAB operon were founded in BAA-894,and all homologys are above 90%.These data showed that it is different in phosphate transport between C.sakazakii and E.coli and PstSCAB operon in C.sakazakii may play a very important role.II.The pst mutant strain was constructed using sequence alignment in C.sakazakii BAA-894 and E.coli W3110.The pstSCAB operon has no obvious influences on the growth between wildtype and mutant no matter in C.sakazakii BAA-894 or E.coli W3110 in LB media.In LP media,the pstSCAB operon will restrain the growth in W3110 not in BAA-894.There were significant difference in a two-component regulatory system between C.sakazakii BAA-894 and E.coli W3110 which maybe relate to the mutant of rpoS in C.sakazakii BAA-894.III.We analysed the extracellular polysaccharide,the structure of the flagellum using optical microscope and electron microscope.Under LP condition,the bacteria secreted more extracellular polysaccharide(EPS),synthesized more flagella to adapt to environmental changes.When pstSCAB operon was deleted,the mutant secreted less EPS and biosynthesized less flagella than wildtype in C.sakazakii BAA-894.These showed that pstSCAB operon effected the secretion of EPS and flagella synthesis.IV.Under LP condition,the lost of the pstSCAB operon increased the membrane permeability and stimulated the aggregation of cells,but reduced the biofilm formation and the adhesion in C.sakazakii BAA-894.This suggested the pstSCAB operon influenced bacteria biofilm-forming ability and adhesion ability.V.Through analysis of the transcriptome,it was confirmed that the absence of pstSCAB operon and phosphate down-regulated the transcription of genes synthesizing bacterial fimbriae,flagella and EPS.By analyzing the differentially regulated genes in mutants,we ensured the differences in phosphate regulation between C.sakazakii and E.coli,and the global influence of the pstSCAB operon in C.sakazakii.