Ravagnani A, Jennert K C, Steiner E, Grünberg R, Jefferies J R, Wilkinson S R, Young D I, Tidswell E C, Brown D P, Youngman P, Morris J G, Young M
Institute of Biological Sciences, University of Wales, Aberystwyth, Ceredigion SY23 3DD, UK.
Mol Microbiol. 2000 Sep;37(5):1172-85. doi: 10.1046/j.1365-2958.2000.02071.x.
The spo0A genes of Clostridium beijerinckii NCIMB 8052 and Clostridium cellulolyticum ATCC 35319 were isolated and characterized. The C-terminal DNA-binding domains of the predicted products of spo0A from these two organisms, as well as 16 other taxonomically diverse species of Bacillus and Clostridium, show extensive amino acid sequence conservation (56% identity, 65% similarity over 104 residues). A 12-amino-acid motif (SRVERAIRHAIE) that forms the putative DNA recognition helix is particularly highly conserved, suggesting a common DNA target. Insertional inactivation of spo0A in C. beijerinckii blocked the formation of solvents (as well as spores and granulose). Sequences resembling Spo0A-binding motifs (TGNCGAA) are found in the promoter regions of several of the genes whose expression is modulated at the onset of solventogenesis in Clostridium acetobutylicum and C. beijerinckii. These include the upregulated adc gene, encoding acetoacetate decarboxylase (EC 4.1.1. 4), and the downregulated ptb gene, encoding phosphotransbutyrylase (EC 2.3.1.c). In vitro gel retardation experiments using C. acetobutylicum adc and C. beijerinckii ptb promoter fragments and recombinant Bacillus subtilis and C. beijerinckii Spo0A suggested that adc and ptb are directly controlled by Spo0A. The binding affinity was reduced when the 0A boxes were destroyed, and enhanced when they were modified to conform precisely to the consensus sequence. In vivo analysis of wild-type and mutagenized promoters transcriptionally fused to the gusA reporter gene in C. beijerinckii validated this hypothesis. Post-exponential phase expression from the mutagenized adc promoter was substantially reduced, whereas expression from the mutagenized ptb promoter was not shut down at the end of exponential growth.
分离并鉴定了拜氏梭菌NCIMB 8052和溶纤梭菌ATCC 35319的spo0A基因。这两种微生物以及其他16种分类学上不同的芽孢杆菌属和梭菌属物种的spo0A预测产物的C端DNA结合结构域显示出广泛的氨基酸序列保守性（在104个残基上有56%的同一性，65%的相似性）。形成推定DNA识别螺旋的一个12个氨基酸的基序（SRVERAIRHAIE）特别高度保守，表明存在共同的DNA靶标。拜氏梭菌中spo0A的插入失活阻断了溶剂（以及孢子和聚-β-羟基丁酸）的形成。在丙酮丁醇梭菌和拜氏梭菌中，在溶剂生成开始时表达受到调节的几个基因的启动子区域中发现了类似Spo0A结合基序（TGNCGAA）的序列。这些基因包括上调的编码乙酰乙酸脱羧酶（EC 4.1.1.4）的adc基因和下调的编码磷酸转丁酰基酶（EC 2.3.1.c）的ptb基因。使用丙酮丁醇梭菌adc和拜氏梭菌ptb启动子片段以及重组枯草芽孢杆菌和拜氏梭菌Spo0A进行的体外凝胶阻滞实验表明，adc和ptb直接受Spo0A控制。当0A框被破坏时，结合亲和力降低，而当它们被修饰以精确符合共有序列时，结合亲和力增强。在拜氏梭菌中对与gusA报告基因转录融合的野生型和诱变启动子进行的体内分析验证了这一假设。诱变的adc启动子在指数后期的表达大幅降低，而诱变的ptb启动子在指数生长结束时的表达并未关闭。
