Mutational analysis of DegU regulating genetic competence and exo enzyme production in B. subtilis

对推定的德谷HTH区进行了丙氨酸扫描诱变。结果，我们获得了5个突变体N183A、I192A、T196A、H200A和I205A，它们的表达水平与ComK靶点之一comG的degud -deficient突变体相似。Western分析显示，突变体DegU蛋白的稳定性与野生型DegU相似。用凝胶阻滞法检测突变体DegU蛋白的comk启动子结合，结果表明5个突变体DegU蛋白都失去了dna结合活性。接下来，我们测试了这些突变DegU蛋白对pre - lacz表达的影响。我们观察到突变的DegU蛋白比comG-lacZ表达更倾向于导致pre - lacz表达的严重降低。因此，我们在这些菌株中引入了多拷贝degR，这增强了磷酸化DegU的稳定性，导致pre - lacz的表达增加。在此背景下，仍有10株菌株表现出极低的pre - lacz表达水平。Western分析表明，除I204A外，这些突变体DegU蛋白在孢子培养基中生长的细胞中是稳定的。在凝胶阻滞实验中，我们发现在aprE启动子的作用下，德谷蛋白形成阶梯状和多段复合物。与野生型DegU相比，缺乏与comK启动子结合能力的5个突变体DegU蛋白对aprE启动子的结合亲和力也降低。此外，4个突变体DegU蛋白K195A、N199A、V201A和S202A对aprE启动子的结合亲和力降低。我们通过携带不同核苷酸变化的comK- lacz融合确定了DegU在comK启动子上识别的序列。结果确定degu识别序列为5‘-GNNATTTA-N8-TAAATNNC-3’。通过我们对DegU的丙氨酸扫描分析，对dna结合重要的氨基酸在磷酸化过程中没有发生很大的变化，因此，DegU识别的顺式序列在磷酸化过程中不会发生变化。在aprE启动子中发现了几个degu结合序列的候选序列，这些序列的改变降低了aprE- lacz融合对degu的依赖性。少

Alanine-scanning mutagenesis of the putative HTH region of DegU was carried out. As a result, we obtained five mutants, N183A, I192A, T196A, H200A and I205A, showing expression levels similar to that in a degU-deficient mutant with respect to comG, one of the targets of ComK. Western analysis revealed the stability of the mutant DegU proteins similar to that of wild-type DegU. Examination of comK-promoter-binding of the mutant DegU proteins with gel retardation assay demonstrated that all the five mutant DegU proteins lost a DNA-binding activity. Next, we tested the effects of these mutant DegU proteins on aprE-lacZ expression. It was observed that the mutant DegU proteins tended to cause more severe reduction of aprE-lacZ expression than comG-lacZ expression. Thus, into these strains we introduced multicopy degR, which enhances stability of phosphorylated DegU, leading to an increase in aprE-lacZ expression. In this background still ten strains showed very low levels of aprE-lacZ expr … More ession. Western analysis showed that these mutant DegU proteins were stable in cells growing in sporulation medium with the exception of I204A. We found that DegU formed ladder-like and multimer complexes in gel retardation assay using the aprE promoter. The five mutant DegU proteins lacking the binding ability to the comK promoter also showed a reduced binding-affinity to the aprE promoter compared to wild-type DegU. In addition, four mutant DegU proteins, K195A, N199A, V201A and S202A showed reduced binding-affinity to the aprE promoter. We determined sequences recognized by DegU on the comK promoter by comK-lacZ fusions carrying various nucleotide changes. As a result, DegU-recognized sequence was determined to be 5'-GNNATTTA-N8-TAAATNNC-3'. The amino acids important for DNA-binding did not largely change upon phosphorylation by our alanine-scanning analysis of DegU, therefore, the DegU-recognized cis-sequences would not be changed upon phosphorylation. In the aprE promoter several candidates for the DegU-binding sequence were found and alteration of those sequences reduced DegU-dependency of the aprE-lacZ fusions. Less