REGULATION OF SPORULATION-ASSOCIATED GENE TRANSCRIPTION

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.