Mohan S, Dubnau D
Department of Microbiology, Public Health Research Institute, New York, NY 10016.
J Bacteriol. 1990 Jul;172(7):4064-71. doi: 10.1128/jb.172.7.4064-4071.1990.
comC specifies a protein product that is required for genetic competence in Bacillus subtilis. The probable transcriptional start site of comC has been localized by high-resolution primer extension analysis and shown to be preceded by an appropriately positioned sequence that resembles the consensus promoter for the sigma A form of RNA polymerase. Low-resolution S1 nuclease transcription mapping was used to identify the comC terminator, which is located near a palindromic element recognizable in the DNA sequence. Deletion analysis of the sequence upstream from the likely promoter identified a region required in cis for the expression of comC. An overlapping, and possibly identical, sequence was shown to inhibit the expression of competence and of several late competence genes, when present in multiple copies. This was interpreted as due to the titration of a positively acting competence transcription factor (CTF) by multiple copies of the promoter-bearing fragment. In crude lysates of B. subtilis grown to competence, a DNA-binding activity that appeared to be specific for the comC promoter fragment was detected by gel retardation assays. This activity, postulated to be due to CTF, was detected only following growth in competence medium, only in the stationary phase of growth, and was dependent on the expression of ComA, a known competence-regulatory factor. In the presence of the mecA42 mutation, the ComA requirement for CTF activity was bypassed, and CTF activity could be detected in lysates prepared from a strain grown in complex medium. This behavior suggested that either the expression or the activation of CTF was regulated in a competence-specific manner. Comparison of the putative CTF-binding site defined by deletion analysis with a similarly positioned sequence upstream from the start site of the late competence gene comG revealed that both sequences contained palindromes, with 5 of 6 identical base pairs in each arm. It is suggested that these palindromic sequences comprise recognition elements for CTF binding and that CTF binding must occur for the appropriate expression of late competence genes.
comC 基因编码一种蛋白质产物，该产物是枯草芽孢杆菌遗传感受态所必需的。通过高分辨率引物延伸分析确定了 comC 基因可能的转录起始位点，并表明其前面有一个位置合适的序列，该序列类似于 RNA 聚合酶 σA 形式的共有启动子。利用低分辨率 S1 核酸酶转录图谱鉴定了 comC 基因的终止子，其位于 DNA 序列中可识别的回文元件附近。对可能启动子上游序列的缺失分析确定了一个顺式作用的区域，该区域是 comC 基因表达所必需的。当以多拷贝存在时，一个重叠且可能相同的序列被证明会抑制感受态和几个晚期感受态基因的表达。这被解释为是由于携带启动子的片段多拷贝对正向作用的感受态转录因子（CTF）的滴定作用。在生长至感受态的枯草芽孢杆菌粗裂解物中，通过凝胶阻滞试验检测到一种似乎对 comC 启动子片段具有特异性的 DNA 结合活性。推测这种活性是由 CTF 引起的，仅在感受态培养基中生长后、仅在生长的稳定期才能检测到，并且依赖于已知的感受态调节因子 ComA 的表达。在 mecA42 突变存在的情况下，对 CTF 活性的 ComA 需求被绕过，并且在从复杂培养基中生长的菌株制备的裂解物中可以检测到 CTF 活性。这种行为表明 CTF 的表达或激活是以感受态特异性方式调节的。通过缺失分析确定的假定 CTF 结合位点与晚期感受态基因 comG 起始位点上游类似位置的序列进行比较，发现这两个序列都包含回文结构，每条臂中有 6 个碱基对中的 5 个相同。有人提出这些回文序列包含 CTF 结合的识别元件，并且 CTF 结合对于晚期感受态基因的适当表达是必需的。
