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. ***
