Nissan Gal, Manulis Shulamit, Weinthal Dan M, Sessa Guido, Barash Isaac
Department of Plant Sciences, Faculty of Life Sciences, Tel-Aviv University, Tel-Aviv 69978, Israel.
Mol Plant Microbe Interact. 2005 Jul;18(7):634-43. doi: 10.1094/MPMI-18-0634.
HrpL, an alternative sigma factor, activates the transcription of the Hrp regulon by its binding to a common "hrp box" promoter. Based on computational techniques, the hrp box previously was defined as a consensus bipartite cis element, 5'-GGAACC-N(15-16)-CCACNNA-3'. The present report combines a quantitative in vivo assay for measuring Hrp promoter activity with site-specific mutagenesis to analyze the effect of consensus and nonconsensus nucleotides on promoter activity. The analysis was carried out with Hop effectors of the tumorigenic bacterium Pantoea agglomerans pv. gypsophilae, in which HrpL is indispensable for gall formation. Mutational analysis indicates that the hrp box consensus can be divided into crucial and noncrucial nucleotides. The first 5 nucleotides (nt) of the--35 consensus motif (GGAAC) and the 3 nt of the--10 motif (ACNNA) are crucial, whereas other consensus and adjacent nonconsensus nucleotides exert a significant effect on the promoter's strength. With spacing of 13 or 17 nt between the two motifs, significant activity was still retained. Gel shift assays indicated that deletion of GG from the--35 consensus motif eliminated HrpL binding, whereas mutations in the--10 consensus motif or modification of the spacing, which eliminates promoter activity, did not elicit any effect. The degeneracy in Hrp promoters of four hrp and type III effector genes of P agglomerans pv. gypsophilae indicated significant differences in promoter activity, whereas increasing the promoter strength of the Hop effector, HsvG, resulted in overexpression of gall formation.
HrpL是一种替代性的σ因子,它通过与一个共同的“hrp框”启动子结合来激活Hrp调控子的转录。基于计算技术,hrp框先前被定义为一个共有二分顺式元件,即5'-GGAACC-N(15-16)-CCACNNA-3'。本报告将用于测量Hrp启动子活性的定量体内分析与位点特异性诱变相结合,以分析共有和非共有核苷酸对启动子活性的影响。该分析是在致瘤细菌成团泛菌嗜石膏亚种的Hop效应子上进行的,其中HrpL对于形成瘿瘤是不可或缺的。突变分析表明,hrp框共有序列可分为关键和非关键核苷酸。-35共有基序的前5个核苷酸(nt)(GGAAC)和-10基序的3个nt(ACNNA)是关键的,而其他共有和相邻的非共有核苷酸对启动子强度有显著影响。两个基序之间间隔13或17 nt时,仍保留显著活性。凝胶迁移分析表明,从-35共有基序中缺失GG消除了HrpL结合,而-10共有基序中的突变或间隔的改变(这消除了启动子活性)并未产生任何影响。成团泛菌嗜石膏亚种的四个hrp和III型效应子基因的Hrp启动子中的简并性表明启动子活性存在显著差异,而增加Hop效应子HsvG的启动子强度会导致瘿瘤形成的过表达。