EFFECTS OF GROWTH TEMPERATURE, 47-MEGADALTON PLASMID, AND CALCIUM DEFICIENCY ON THE OUTER-MEMBRANE PROTEIN PORIN AND LIPOPOLYSACCHARIDE COMPOSITION OF YERSINIA-PESTIS EV76
EFFECTS OF GROWTH TEMPERATURE, 47-MEGADALTON PLASMID, AND CALCIUM DEFICIENCY ON THE OUTER-MEMBRANE PROTEIN PORIN AND LIPOPOLYSACCHARIDE COMPOSITION OF YERSINIA-PESTIS EV76
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DOI：
10.1128/iai.42.3.1092-1101.1983
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发表时间：
1983-01-01
影响因子：
3.1
通讯作者：
HANCOCK, REW
中科院分区：
文献类型：
--
作者：
DARVEAU, RP;CHARNETZKY, WT;HANCOCK, REW
几种致病性决定因子的表达。已知鼠疫菌依赖于体外生长温度。其中之一，钙依赖性与47兆道尔顿质粒的存在有关。研究了培养温度、培养基中Ca和47兆道尔顿质粒对酵母菌外膜蛋白和脂多糖组成的影响。鼠疫菌EV 76。当细胞在37 ℃下生长时，与26度相反。C，观察到LPS组成的变化和外膜蛋白（蛋白E）的量的减少。从37 ℃孵育的细胞获得的LPS。与从26 ℃生长的细胞获得的LPS相比，C具有较低的2-酮基-3-脱氧辛酸酯比例、较低的PO 43-：2-酮基-3-脱氧辛酸酯比例和在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳[SDS-PAGE]上增加的凝胶迁移率。C.由于其生长温度相关的丰度，蛋白E的性质进行了研究。该蛋白具有与其他肠杆菌孔蛋白相似的物理性质，包括在低温下溶解时在SDS-PAGE中明显形成寡聚体，酸性等电点和与肽聚糖的强非共价缔合。蛋白E被纯化，并显示在平面脂质膜中形成水通道，在1 M KCl中电导为1.1 nS。除了生长温度相关的改变外膜的LPS和孔蛋白组分，在2维PAGE中的3个斑点的量被证明是相关的温度或在生长过程中的Ca的存在。这些斑点之一含有2种主要的可热修饰的蛋白质的残余未修饰部分，尽管在100 ℃下溶解，但这些蛋白质不能移动到它们在凝胶上的热修饰位置。电泳前在室温下孵育10 min。另外2个点是另一种外膜蛋白（J）的热修饰和未修饰形式，其未出现在37 ℃生长的细胞的等电聚焦凝胶中。C.显然，这些斑点在二维分析中的出现与外膜所来源的细胞的LPS组成有关，并反映了LPS-蛋白质相互作用或Ca-蛋白质相互作用。
The expression of several virulence determinants of Y. pestis is known to be dependent on the in vitro growth temperature. One of these, Ca dependence is associated with the presence of a 47 megadalton plasmid. The effects of incubation temperature, Ca in the growth medium and the presence of the 47 megadalton plasmid on the outer membrane protein and the lipopolysaccharide [LPS] composition of Y. pestis EV76 were examined. When cells were grown at 37.degree. C as opposed to 26.degree. C, a change in LPS composition and a decrease in the amount of an outer membrane protein (protein E) were observed. The LPS obtained from cells incubated at 37.degree. C had a lower proportion of 2-keto-3-deoxyoctanate, a lower PO43-:2-keto-3-deoxyoctanate ratio and an increased gel mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE] when compared with LPS obtained from cells grown at 26.degree. C. Because of its growth temperature-related abundance, the nature of protein E was investigated. This protein had physical properties similar to those of other enterobacterial porins, including apparent formation of an oligomer in SDS-PAGE when solubilized at low temperature, acidic isoelectric point and strong noncovalent association with the peptidoglycan. Protein E was purified and shown to form an aqueous channel in planar lipid membranes with a conductance of 1.1 nS in 1 M KCl. In addition to growth temperature-related alterations in the LPS and porin components of the outer membrane, the amount of 3 spots in 2-dimensional PAGE was shown to be related to the temperature or the presence of Ca during growth. One of these spots contained residual unmodified portions of 2 major heat-modifiable proteins which failed to shift to their heat-modified positions on gels, despite solubilization at 100.degree. C for 10 min before electrophoresis. The other 2 spots were the heat-modified and unmodified forms of another outer membrane protein (J) which did not appear in the isoelectric focusing gel of cells grown at 37.degree. C. Evidently, the appearance of these spots in 2-dimensional analyses is related to LPS composition of the cells from which the outer membrane is derived and reflects LPS-protein interactions or Ca-protein interactions.