Hutcheson S W, Bretz J, Sussan T, Jin S, Pak K
Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland 20742, USA.
J Bacteriol. 2001 Oct;183(19):5589-98. doi: 10.1128/JB.183.19.5589-5598.2001.
In Pseudomonas syringae strains, the hrp-hrc pathogenicity island consists of an HrpL-dependent regulon that encodes a type III protein translocation complex and translocated effector proteins required for pathogenesis. HrpR and HrpS function as positive regulatory factors for the hrpL promoter, but their mechanism of action has not been established. Both HrpR and HrpS are structurally related to enhancer-binding proteins, but they lack receiver domains and do not appear to require a cognate protein kinase for activity. hrpR and hrpS were shown to be expressed as an operon: a promoter was identified 5' to hrpR, and reverse transcriptase PCR detected the presence of an hrpRS transcript. The hrpR promoter and coding sequence were conserved among P. syringae strains. The coding sequences for hrpR and hrpS were cloned into compatible expression vectors, and their activities were monitored in Escherichia coli transformants carrying an hrpL'-lacZ fusion. HrpS could function as a weak activator of the hrpL promoter, but the activity was only 2.5% of the activity detected when both HrpR and HrpS were expressed in the reporter strain. This finding is consistent with a requirement for both HrpR and HrpS in the activation of the hrpL promoter. By using a yeast two-hybrid assay, an interaction between HrpR and HrpS was detected, suggestive of the formation of a heteromeric complex. Physical interaction of HrpR and HrpS was confirmed by column-binding experiments. The results show that HrpR and HrpS physically interact to regulate the sigma(54)-dependent hrpL promoter in P. syringae strains.
在丁香假单胞菌菌株中，hrp-hrc致病岛由一个依赖HrpL的调节子组成，该调节子编码III型蛋白转运复合体和致病所需的转运效应蛋白。HrpR和HrpS作为hrpL启动子的正调控因子，但其作用机制尚未明确。HrpR和HrpS在结构上与增强子结合蛋白相关，但它们缺乏接收结构域，且似乎不需要同源蛋白激酶来发挥活性。研究表明hrpR和hrpS以操纵子形式表达：在hrpR的5'端鉴定出一个启动子，逆转录酶PCR检测到hrpRS转录本的存在。hrpR启动子和编码序列在丁香假单胞菌菌株中保守。将hrpR和hrpS的编码序列克隆到兼容的表达载体中，并在携带hrpL'-lacZ融合的大肠杆菌转化体中监测它们的活性。HrpS可作为hrpL启动子的弱激活剂，但其活性仅为在报告菌株中同时表达HrpR和HrpS时检测到的活性的2.5%。这一发现与hrpL启动子激活需要HrpR和HrpS两者一致。通过酵母双杂交试验，检测到HrpR和HrpS之间的相互作用，提示形成了异源复合体。通过柱结合实验证实了HrpR和HrpS的物理相互作用。结果表明，HrpR和HrpS通过物理相互作用来调节丁香假单胞菌菌株中依赖σ54的hrpL启动子。
