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

丙氨酸扫描突变可能是DECU的hTH区。结果，我们获得了5个突变体，N183A、I192A、T196A、H200A和I205A，其表达水平与脱谷氨酸缺失突变体中Comg的表达水平相似，Comg是ComK的靶标之一。Western分析表明，突变的Degu蛋白与野生型Degu蛋白的稳定性相似。用凝胶滞留实验检测突变的DECU蛋白与comK启动子的结合，结果表明5个突变的DECU蛋白都失去了DNA结合活性。接下来，我们测试了这些突变的DECU蛋白对APRE-LacZ表达的影响。研究发现，突变的DECU蛋白导致的APRE-LacZ表达的降低比Comg-LacZ的表达更严重。因此，我们在这些菌株中引入了多拷贝DECR，它增强了磷酸化DECU的稳定性，导致APRE-LacZ表达增加。在此背景下，仍有10株菌株表现出非常低的apre-lacZ Expr…水平更多的圣餐。Western分析表明，除I204A外，这些突变的DECU蛋白在产孢液中生长的细胞中都是稳定的。在APRE启动子的凝胶滞留实验中，我们发现DEGU形成了梯状和多聚体复合体。缺乏与comK启动子结合能力的5个突变的DECU蛋白与APRE启动子的结合亲和力也低于野生型DECU。此外，4个突变的DECU蛋白K195A、N199A、V201A和S202A与apre启动子的结合亲和力降低。我们通过携带各种核苷酸变化的comK-LacZ融合，确定了comK启动子上DECU识别的序列。结果确定脱氧核糖核酸识别序列为5‘-GNNatTTA-N8-TAAATNNC-3’。通过我们对DECU的丙氨酸扫描分析，对DNA结合至关重要的氨基酸在磷酸化后没有很大变化，因此，DECU识别的顺式序列在磷酸化后不会改变。在APRE启动子中发现了几个去谷氨酸结合序列的候选者，这些序列的改变降低了APRE-LacZ融合的去依赖性。较少

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