Altered localization of HrpZ in Pseudomonas syringae pv syringae hrp mutants suggests that different components of the type III secretion pathway control protein translocation across the inner and outer membranes of gram-negative bacteria
Altered localization of HrpZ in Pseudomonas syringae pv syringae hrp mutants suggests that different components of the type III secretion pathway control protein translocation across the inner and outer membranes of gram-negative bacteria
复制标题
DOI：
10.1128/jb.179.12.3866-3874.1997
复制
发表时间：
1997-06-01
影响因子：
3.2
通讯作者：
Collmer, A
中科院分区：
文献类型：
--
作者：
Charkowski, AO;Huang, HC;Collmer, A
丁香假单胞菌丁香61（Pss 61）通过hrp簇编码的III型蛋白分泌途径分泌HrpZ harpin 9个hrc基因代表hrp基因的子集，其也在动物病原性耶尔森氏菌、志贺氏菌和沙门氏菌属的III型毒力蛋白分泌系统中保守，hrpJ和hrpU操纵子含有7个hrc基因（将hrcQ（A）和hrcQ（B）计为一个基因），所有这些都具有参与鞭毛生物发生和分泌的额外同源物，并且其中五个编码预测的内膜蛋白。hrpZ和hrpZ操纵子分别编码HrcC和HrcJ，其与外膜相关，通过免疫印迹十二烷基硫酸钠-聚丙烯酰胺凝胶分析影响hrpJ和hrpU操纵子中所有hrc基因的内插子突变体以及TnphoA诱导的hrcC和hrcJ突变体，以确定对数生长中期培养物中HrpZ的改变的定位，所述凝胶用各种细胞级分进行电泳。hrpJ和hrpU操纵子突变体揭示了一种新的表型，即在总培养物中HrpZ的积累部分减少hrcC和hrcJ突变体培养物积累了与野生型相同总量的HrpZ，但HrpZ未结合，在所有测试的菌株中，只有hrcC突变体在周质中积累了大量的HrpZ，如通过原生质球选择性释放所示。hrpU和hrpC操纵子中的非极性突变的分析支持极性突变获得的结果。这些观察结果表明，尽管存在分泌缺陷，但在Pss 61的细胞质中仍维持恒定的HrpZ库，hrpJ和hrpU操纵子编码Sec的替代物。（一般蛋白输出）途径对于跨内膜的易位，hrpC操纵子中的基因对于跨外膜的易位是必需的，并且Pss 61 Hrp系统允许研究III型蛋白分泌中的两个遗传上可区分的阶段。
Pseudomonas syringae pv. syringae 61 (Pss61) secretes the HrpZ harpin by a type III protein secretion pathway encoded br a cluster of hrp (hypersensitive response and pathogenicity) and hrc genes, The nine hrc genes represent a subset of hrp genes that are also conserved in the type III virulence protein secretion systems of animal pathogenic Yersinia, Shigella, and Salmonella spp, The hrpJ and hrpU operons contain seven hrc genes (counting hrcQ(A) and hrcQ(B) as one gene), all with additional homologs involved in flagellar biogenesis and secretion, and five of which encode predicted inner membrane proteins, The hrpZ and hrpZ operons encode HrcC and HrcJ, respectively, which are associated with the outer membrane, Interposon mutants affected in all of the hrc genes in the hrpJ and hrpU operons and TnphoA-induced hrcC and hrcJ mutants were assayed for altered localization of HrpZ in mid-log-phase cultures by immunoblotting sodium dodecyl sulfate-polyacrylamide gels that were run with various cell fractions. The hrpJ and hrpU operon mutants revealed a novel phenotype of partially reduced accumulation of HrpZ in the total culture (despite wild-type levels of hrpZ operon transcription), all of which was cell bound and equivalent in level to that of cell-bound HrpZ in the wild type, The hrcC and hrcJ mutant cultures accumulated the same total amount of HrpZ as the wild type, but the HrpZ was fell bound, Among all the strains tested, only the hrcC mutant accumulated significant amounts of HrpZ in the periplasm, as indicated by selective release through spheroplasting. Analysis of nonpolar mutations in the hrpU and hrpC operons support the results obtained with polar mutations. These observations indicate that a constant pool of HrpZ is maintained in the cytoplasm of Pss61 despite secretion deficiencies, that the hrpJ and hrpU operons encode an alternative to the Sec (general protein export) pathway For translocation across the inner membrane, that genes in the hrpC operon are necessary for translocation across the outer membrane, and that the Pss61 Hrp system permits study of two genetically distinguishable stages in type III protein secretion.