| Cronobacter species are opportunistic pathogen and can cause necrotizing enterocolitis and meningitis in neonates.In this study,C.sakazaki BAA-894 was used as a background strain.By observing its phenotype and extracellular secretions,it was found that there is a big difference between Cronobacter sakazakii and E.coli.Qualitation and quantitation of EPS were conducted to explore the effects of internal regulation and changes in lipopolysaccharide structure on the production of EPS by C.sakazaki BAA-894.The transcriptome data were used to analyze phenotypic experimental phenomena and changes of expression of other genes.Different growth environments and the resistance analysis of Cronobacter sakazakii under the structure of lipopolysaccharide were carried out.The results are listed below:(1)M9 is the basic medium,while LB is rich in a variety of amino acids.When C.sakazakii grow on M9 medium,it will secretes highly transparent and sticky substances that around the colonies.Through tannin mordant staining and high performance liquid chromatography,these substances were proved to be the colanic acid which is a kind of exopolysaccharide of C.sakazakii.But when growing on M9 medium,C.sakazakii will not secretes colanic acid.Following the construction of wcaDE deletion mutant of colanic acid synthase in C.sakazakii BAA-894,the secretion of colalate disappeared.Five mutants of rcsA,rcsB,rcsC,rcsD and rcsF of RCS phosphate transfer system were constructed.It was found that the synthesis of colanic acid was regulated by this system.(2)Useing LB medium as control,when growing in M9 medium,the transcriptional level of the gene clusters of colanic acid biosynthesis are significantly up-regulated corresponding to the colanic acid quantitation and staining;genes related to flagellar formation and chemotaxis are all down-regulated;genes of amino acid biosynthesis are more widely regulated.C.Sakazakii BAA-894 will reduce the synthesis of accessory structures such as flagella and cell motility to save energy for growth in nutrient-deficient environment,and selectively produces amino acids to meet the nitrogen source required for its own growth.(3)By adding a mixed amino acid to the M9 medium and quantifying the colanic acid with tannin mordant,it was found that the yield of the colanic acid was decreased.So the amino acid deficiency causes C.sakazakii to secrete a large amount of colanic acid.The drug resistance of C.sakazakii BAA-894 on different media was tested by antibiotic disk diffusion assay.It was found that C.sakazakii BAA-894 was more sensitive to antibiotics on M9 medium,whereas the content of amino acids has little effect on the resistance of bacteria.(4)ESA-RS04103 is the gene encoding O-antigen ligase in C.sakazakii BAA-894.When ESA-RS04103 was deleted from the C.sakazaki BAA-894 chromosome,the secretion of colanic acid decreased;the self-aggregation,hydrophobicity,outer membrane permeability and membrane formation of the bacteria were significantly changed.The drug resistance of ?ESA-RS04103 on different media was tested by antibiotic disk diffusion assay.The results showes that ?ESA-RS04103 was less sensitive to chloramphenicol,neomycin and novobiocin than C.sakazakii BAA-894.In addition,mutant has lower resistance to erythromycin and clarithromycin,the resistance to other antibiotics are greater than on LB medium.In M9 medium,the deficiency of ESA_RS18930 in C.sakazakii leads to changes in transcriptional level comparaing with wild type.Gene clusters of colanic acid biosynthesis are down-regulated,which was consistent with the quantitation and staining results;genes of flagella formation and chemotaxis are up-regulated;genes involved in antibiotic resistence are also differentially expressed.As a result,O-antigen deficiency will reduces colanic acid secretion,increases bacterial flagella formation and motility and leads to change in drug resistance. |
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