Malakooti J, Wang S P, Ely B
Department of Biological Sciences, University of South Carolina, Columbia 29208, USA.
J Bacteriol. 1995 Aug;177(15):4372-6. doi: 10.1128/jb.177.15.4372-4376.1995.
Caulobacter crescentus differentiates prior to each cell division to form two different daughter cells: a monoflagellated swarmer cell and a nonmotile stalked cell. Thus, one might expect that developmentally expressed genes would be regulated by mechanisms different from those used to regulate the expression of the biosynthetic genes. To determine a consensus promoter sequence for genes involved in biosynthetic or housekeeping functions, DNA fragments containing the regulatory regions of the ilvD, ilvR, cysC, pleC, and fdxA genes were cloned. S1 nuclease protection mapping and primer extension techniques were used to identify the transcription initiation sites. Comparison of the regulatory regions of these genes with those of the published sequences of the ilvBN, rrnA, trpFBA, dnaA, dnaK, hemE, and rsaA genes has resulted in the identification of a putative promoter consensus sequence. The -35 region contains the sequence TTGACGS, which is similar to the Escherichia coli -35 region, while the -10 region, GCTANAWC, has a more balanced GC content than the corresponding region in E. coli. Oligonucleotide-directed site-specific mutagenesis of both the ilvBN and pleC promoters indicates that mutations that make a promoter more like the consensus result in increased promoter activity, while mutations decreasing similarity to the consensus result in decreased promoter activity.
新月柄杆菌在每次细胞分裂前进行分化,形成两种不同的子细胞:单鞭毛游动细胞和无运动能力的柄细胞。因此,人们可能会预期,发育表达基因的调控机制与用于调控生物合成基因表达的机制不同。为了确定参与生物合成或管家功能的基因的共有启动子序列,克隆了包含ilvD、ilvR、cysC、pleC和fdxA基因调控区的DNA片段。使用S1核酸酶保护图谱和引物延伸技术来鉴定转录起始位点。将这些基因的调控区与已发表的ilvBN、rrnA、trpFBA、dnaA、dnaK、hemE和rsaA基因序列的调控区进行比较,从而确定了一个推定的启动子共有序列。-35区包含序列TTGACGS,与大肠杆菌的-35区相似,而-10区GCTANAWC的GC含量比大肠杆菌的相应区域更为均衡。对ilvBN和pleC启动子进行寡核苷酸定向位点特异性诱变表明,使启动子更接近共有序列的突变会导致启动子活性增加,而降低与共有序列相似性的突变则会导致启动子活性降低。