The induction of gene transcription in response to environmental signals is a central but still poorly understand aspect of genetic regulation. The spore-forming bacterium Bacillus subtilis will activate a diverse collection of sporulation-associated genes when nutritionally deprived and provides an excellent system with which to study transcriptional regulatory mechanisms which are responsive to the nutritional environment. I am currently studying the transcription of three sporulation-associated genes; the early-induced sporulation genes. spoOH and spoVG and a gene involved in the synthesis of a peptide antibiotic, tycA. All appear to be under a common form of negative control that is dependent on a genetic locus known as abrB. Intracellular levels of GTP also appear to play a role in the transcriptional regulation of sporulation genes. The main objectives outlined in this proposal are to: 1) define cis-acting transcriptional regulatory sites through mutational analysis of the spoVG and spoOH gene promoters. We will also examine the expression of other genes which we would expect to be influenced by abrB (i.e., the gene encoding the neutral protease npr). 2) identify regulators that are associated with GTP- and abrB- dependent negative control with the ultimate goal of establishing a pathway linking the metabolic events which promote sporulation with the activation of gene transcription. We will begin by isolating mutations which cause the expression of spoVG, tycA, and spoOH to be constitutive and/or independent of negative control. 3) isolate the abrB gene(s) by taking advantage of a transposon Tn 917 insertion we have discovered in the abrB locus. Cloned DNA flanking the transposon will be used to perform diploid analysis in order to determine if the abrB locus is composed of more than one gene. We will also use this DNA to obtain the intact abrB gene(s) which will then be sequenced in order to determine the primary structure of its product(s). The cloned DNA will be used to create a abrB deletion mutant. We will also use the abrB DNA to monitor the expression of abrB and to determine the genetic requirements for its expression. The abrB product(s) will be overproduced in B subtilis cells to examine the effect of high AbrB levels on the transcription of spoVG spoOH and tycA as well as the effect on sporulation. The abrB DNA will be mutagenized and reintroduced into B. subtilis in hopes of obtaining and characterizing abrB mutations which interfere with sporulation initiation.
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