| Research Abstract |
The Escherichia coil H-NS protein is one of the major constituents of the nucleoid structure. This protein has been implicated not only in the compact organization of the nucleoid structure, but also in the global regulation of gene expression. In this study, on systematic mutational analysis of hns, three distinct functional domains were found in H-NS, which appear to be responsible for DNA-binding, transcriptional repression and dimerization, respectively. Mutations in the C-terminal domain resulted in a loss of its DNA-binding ability, suggesting that this domain is directly involved in its binding to DNA.The N-terminal domain was suggested to be involved in the ability to repress transcription. The relatively central portion of H-NS was found to be involved in protein-protein interaction, especially dimerization. The functional significance for each domain was also clarified. In addition, expression of the bgl operon was found to be affected by only a subset of hns mutations in a highly allele-specific manner. This finding is also addressed with regard to a unique regulatory mechanism (i.e. silencing) for the bgl operon, which is partly mediated by H-NS.
The Escherichia coli bgl operon is of interest since its expression is silent (phenotypically Bgl^-), at least under standard laboratory conditions. To identify a trans-acting factor(s) that is presumably relevant to the regulation of bgl, I screened mutants exhibiting Bgl+ phenotype. By a random insertion mutagenesis with mini-Tn10, a novel type of mutation was obtained. In this case, the insertion mutation was found to be just in front of the leuO gene encoding a putative LysR-like DNA-binding protein. Genetic analyses revealed that an overproduction of LeuO in the wild-type cells causes the phenotype of Bgl^+.
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