Study On Osmoregulated Periplasmic Glucans Gene Of Shiagella Flexneri And Salmonella

The gene mdoB, mdoC, mdoD, mdoG and mdoH play an important role in synthesis of osmoregulated periplamic glucans(OPGs). The six OPG defective mutants of Shigella flexneri 2457T were constructed for studying the functions of mdo genes in OPG synthesis byλ-Red recombination system. The effect of osmolarity on synthesis of OPG was also detected. In this study, the OPGs were constructed and purified, their backbone constitutes and substitutes were conferred. At the same time, the growth and biofilm formation in media and wash waters were described for both Shigella flexneri 2457T(S 2457T) and its defective mutants, and its Salmonella SL1344 defective opgGH mutant. In addition, the virulence was checked by Caco-2 cell model, as well as the antibiotics sensitivity, heat shock and pH tolerance. The main results are as follows:(1) The gene mdoB, mdoC, mdoD, mdoG, mdoH and mdoGH of S 2457T were in vivo replaced with the help ofλ-Red recombinant system; mdo gene deletion mutants were gained to study the functions of those genes during OPG synthesis.(2) The OPGs of Shigella, Salmonella and E. coli were isolated and purified to study the characteristics of OPGs. The succinic acid and phosphate substitutes in opgB, opgC and opgD mutants were quantified. The charged and neutral OPGs were separated by DEAE cellulose anion exchange chromatography.(3) The functions of gene mdoB, mdoC, mdoD, mdoG, mdoH and mdoGH have been determined relating to the synthesis of OPGs. The mutants, opgG, opgH and opgGH of Shigella were proven defectiveness in synthesis of OPGs.(4) The growth of S 2457T and its defective mutants, SL1344 and its defective opgGH mutant in media and low nutrition low osmolarity conditions were determined. The opgGH mutants grew at a reduced growth rate under low osmolarity. When the osmolarity was increased to the normal level (0.15mol/L NaCl), the growth phenotype of mutants was resumed to the level of wild type. In wash waters, the growth of Salmonella wild type showed more stable than the Shigella wild type.(5) The biofilm formations of Shigella, Salmonella and their mutants have been quantified. Salmonella wild type can form little biofilm in wash waters except spinach wash water and green celery wash water, the growth of Salmonella was good in all wash waters at the same time. Salmonella opgGH mutant formed biofilm only in media. The Shigella wild type and all mutants formed biofilm in cabbage wash water and in 1/8TSB medium. There was no significant effect of increasing osmolarity on the biofilm formation.(6) The virulence of S 2457T and mutants was detected by Caco-2 cell model. The OPG defective mutants did not reduce in cell infection compared to the wild type.(7) The antibiotic ability, heat tolerane, the pH and 0.5%SDS effect on growth of S 2457T and mutants have been studied; the mutant opgD of Shigella is the most resistant to heat shock in the stress tolerance experiments. All the bacteria were inhibited by 0.5% SDS, while the mutant opgGH of Shigella could survive in a wider range of pH than other mutants and wild type.