Selective Gene Regulation by Manganese and Iron in Bacillus subtilis

9317425赫尔曼这个项目试图确定两种对环境重要的元素铁和锰在遗传特征良好的土壤微生物枯草芽孢杆菌中的基因调控中的作用。在初步研究中，确定了铁或锰或两者都能抑制的基因融合。其中三个金属调控基因被克隆并利用分子遗传学进行了分析。在枯草杆菌中，铁的调节似乎涉及一种与肠道毛皮蛋白在DNA结合特异性上相似的调节蛋白。到目前为止，还没有任何革兰氏阳性生物的毛皮同源物被描述出来。与肠道毛皮蛋白不同，枯草杆菌铁调节蛋白似乎歧视锰。在肠道生物中，锰可以模拟铁，抑制铁载体的生物合成和其他铁调节功能。相比之下，枯草杆菌铁载体的产生对锰的抑制不敏感。相反，我们检测到的高度敏感的锰特异转录调控提示了一种新型金属调控系统的存在。这一系统可能部分解释了锰离子对芽孢杆菌和其他革兰氏阳性生物次生代谢物产生的许多显著影响。在芽孢杆菌中，铁和锰似乎控制着不同但重叠的调节基因。例如，我们已经确定了几组启动子融合，它们对铁而不是锰或对锰而不是铁有选择性的反应。在一个案例中，我们从基因上分离了铁和锰的反应。这些结果表明，这些元件对转录具有特异性和独立性的调控作用。然而，允许细胞选择性地检测这些元件并介导适当的转录反应的机制在很大程度上是未知的。我们将定义铁和锰对基因调控的全球影响，并确定选择性基因调控的分子基础。使用蛋白质凝胶电泳仪，我们将识别金属调控蛋白质组，并确定不同金属离子调节子之间的串扰程度。利用转座子Tn917-LacZ分离金属调控基因，并确定金属调控的DNA序列决定因素。这将为相关金属调节蛋白的遗传和生化鉴定提供必要的工具。对这些调控蛋白的表征有望阐明蛋白质如何区分化学上相似的金属离子，以实现选择性转录控制。这笔赠款提供有关细菌如何对其环境做出反应的信息，特别是关于金属存在的信息。***

9317425 Helmann This project seeks to define the role of two environmentally important elements, iron and manganese, in gene regulation in the genetically well-characterized soil microbe Bacillus subtilis. In preliminary studies, gene fusions repressible by iron or manganese or both were identified. Three of these metal-regulated genes were cloned and analyzed using molecular genetics. In B. subtilis, iron regulation appears to involve a regulatory protein similar in DNA binding specificity to the enteric Fur protein. There have been no Fur homologs described to date from any gram positive organisms. Unlike the enteric Fur protein, the B. subtilis iron-regulatory protein appears to discriminate against manganese. In the enteric organisms manganese can mimic iron and repress siderophore biosynthesis and other iron-regulated functions. In contrast, production of sideropheres in B. subtilis is insensitive to manganese repression. Conversely, our detection oh highly sensitive manganese-specific transcriptional control suggests the presence of a novel type of metalloregulatory system. This system may account, in part, for the many striking effects of manganous ion on production of secondary metabolities in Bacilli and other gram positive organisms. In Bacilli, iron and manganese appear to control distinct, but overlapping regulons. For example, we have identified sets of promoter fusions that respond selectively to iron but not manganese or to manganese but not iron. In one case, we have genetically separated the iron and manganese responses. These results suggest that these elements exert specific and independent transcriptional control. However, the mechanisms allowing cells to selectively detect these elements and mediated appropriated transcriptional responses are largely unknown. We will define the global effects of iron and manganese on gene regulation and determine the molecular basis of selective gene regulation. Using protein gel electrophores is, we will identify sets of metalloregulated proteins and determine the extent of cross-talk among the different metal ion regulons. Metalloregulated genes will be isolated using transposon Tn917-lacZ and the DNA sequence determinants of metalloregulation defined. This will provide the tools necessary for the genetic and biochemical identification of the relevant metalloregulatory proteins. Characterization of these regulatory proteins promises to shed light on how proteins can discriminate chemically similar metal ions to effect selective transcriptional control. %%% This grant provides information on how bacteria respond to their environment, especially as regards the presence of metals. ***