【作者】 刘柳；

【导师】 樊明涛； Arvind Bhagwat； 孟江洪；

【作者基本信息】 西北农林科技大学 ， 食品科学， 2009， 博士

【摘要】 基因mdoB、mdoC、mdoD、mdoG和mdoH是与渗透调节周质葡聚糖的合成密切相关的几个重要基因。本研究以福氏志贺菌2457T野生型为出发菌株,利用λ-Red系统重组技术,构建了福氏志贺菌2457T的mdoB、mdoC、mdoD、mdoG、mdoH以及mdoGH的缺陷型突变体,研究了这些基因在渗透调节周质葡聚糖合成中的功能以及培养基中渗透压对渗透调节周质葡聚糖合成的影响;分离、提取并纯化了渗透调节周质葡聚糖,测定了葡聚糖的组成和取代基,分析了葡聚糖内各单糖的连接方式。此外,还研究了福氏志贺菌2457T野生型及渗透调节周质葡聚糖合成缺陷型opgB、opgC、opgD、opgG、opgH和opgGH突变体、沙门氏菌SL1344及其渗透调节周质葡聚糖合成缺陷型opgGH突变体在标准培养基和洗菜水中的生长及生物膜的产生能力;测定了福氏志贺菌2457T及其突变体对人类结肠癌细胞(Caco-2细胞)的毒力,检测了福氏志贺菌2457T及其突变体的耐药性、抗热击能力、抗酸碱度能力等。本研究主要获得以下结果:(1)本试验使用λ-Red系统,分别成功地敲除了福氏志贺菌2457T染色体上的mdoB、mdoC、mdoD、mdoG、mdoH和mdoGH基因,获得了6株缺陷型突变体,为研究它们在福氏志贺菌2457T渗透调节周质葡聚糖合成中的功能奠定了基础;(2)本试验成功提取并纯化了福氏志贺菌、沙门氏菌和大肠杆菌的渗透调节周质葡聚糖,研究了渗透调节周质葡聚糖的性质,定量测定了福氏志贺菌opgB、opgC和opgD突变体及野生型中渗透调节周质葡聚糖的磷酸取代基和琥珀酸取代基,并对带有不同电荷的渗透调节周质葡聚糖进行了鉴别;(3)确定了基因mdoB、mdoC、mdoD、mdoG和mdoH在合成渗透调节周质葡聚糖中的功能,明确了渗透压对渗透调节周质葡聚糖产量的影响,证明了基因mdoG、mdoH和mdoGH的缺失导致福氏志贺菌突变体不能合成渗透调节周质葡聚糖;(4)研究了渗透压对福氏志贺菌2457T及其6株渗透调节周质葡聚糖合成缺陷型突变体、沙门氏菌SL1344及其渗透调节周质葡聚糖合成缺陷型opgGH突变体生长的影响,结果显示将低营养培养基的渗透压提高到正常水平时,福氏志贺菌2457T和沙门氏菌SL1344及其突变体对数生长期提前;沙门氏菌野生型在洗菜水中得到的生长曲线比福氏志贺菌野生型生长曲线稳定;(5)测定了福氏志贺菌2457T和沙门氏菌SL1344及其突变体在不同的洗菜水中生物膜的产量,研究了生物膜产生的规律,沙门氏菌SL1344尽管在洗菜水中生长良好,但只在菠菜水和芹菜水中产生生物膜;沙门氏菌突变体opgGH在所有洗菜水中都不产生生物膜;福氏志贺菌2457T野生型和突变体仅在白菜水中产生了大量的生物膜。提高渗透压后,对各种菌株在不同洗菜水中产生生物膜的影响并不明显;(6)检测了福氏志贺菌2457T及其渗透调节周质葡聚糖合成缺陷突变体对Caco-2细胞的毒力,证明野生型和突变体感染Caco-2细胞的能力没有明显的差异;(7)研究了福氏志贺菌2457T及其渗透调节周质葡聚糖合成缺陷型突变体的抗热击性和抗药性,以及在含0.5% SDS的培养基和在不同pH值条件下的生长状况,其中基因mdoD缺陷型抗热击能力最明显;0.5% SDS可抑制所有菌株在1/8LBNS培养基中的生长;突变体opgGH得以生长的pH值范围明显大于其它突变体及野生型的pH值范围。更多还原

【Abstract】 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.更多还原