Nakano M M, Zhu Y, Lacelle M, Zhang X, Hulett F M
Department of Biochemistry and Molecular Biology, Oregon Graduate Institute of Science and Technology, Beaverton, OR 97006, USA.
Mol Microbiol. 2000 Sep;37(5):1198-207. doi: 10.1046/j.1365-2958.2000.02075.x.
The two-component regulatory proteins ResD and ResE are required for anaerobic nitrate respiration in Bacillus subtilis. ResD, when it undergoes ResE-dependent phosphorylation, is thought to activate transcriptionally anaerobically induced genes such as fnr, hmp and nasD. In this report, deletion analysis of the fnr, hmp and nasD promoter regions was carried out to identify cis-acting sequences required for ResDE-dependent transcription. The results suggest that the hmp and nasD promoters have multiple target sequences for ResDE-dependent regulation and that fnr has a single target site. Gel mobility shift assays and DNase I footprinting analyses were performed to determine whether ResD interacts directly with the regulatory regions of the three genes. Our results indicate that ResD specifically binds to sequences residing upstream of the hmp and nasD promoters and that phosphorylation of ResD significantly stimulates this binding. In contrast, a higher concentration of ResD is required for binding to the fnr promoter region and no stimulation of the binding by ResD phosphorylation was observed. Taken together, these results suggest that ResD activates transcription of fnr, hmp and nasD by interacting with DNA upstream of these promoters. Our results suggest that phosphorylation of ResD stimulates binding to multiple ResD binding sites, but is much less stimulatory if only a single binding site exists.
双组分调节蛋白ResD和ResE是枯草芽孢杆菌厌氧硝酸盐呼吸所必需的。ResD在经历依赖于ResE的磷酸化时，被认为可转录激活厌氧诱导基因，如fnr、hmp和nasD。在本报告中，对fnr、hmp和nasD启动子区域进行了缺失分析，以鉴定ResDE依赖性转录所需的顺式作用序列。结果表明，hmp和nasD启动子具有多个ResDE依赖性调控的靶序列，而fnr有一个单一的靶位点。进行了凝胶迁移率变动分析和DNase I足迹分析，以确定ResD是否直接与这三个基因的调控区域相互作用。我们的结果表明，ResD特异性结合于hmp和nasD启动子上游的序列，并且ResD的磷酸化显著刺激这种结合。相比之下，结合到fnr启动子区域需要更高浓度的ResD，并且未观察到ResD磷酸化对结合的刺激作用。综上所述，这些结果表明ResD通过与这些启动子上游的DNA相互作用来激活fnr、hmp和nasD的转录。我们的结果表明，ResD的磷酸化刺激与多个ResD结合位点的结合，但如果仅存在一个结合位点，则刺激作用要小得多。
