Nakano 9722885 The Gram-positive soil bacterium Bacillus subtilis activates diverse processes for its survival upon exposure to various kinds of stress. Recent findings that B. subtilis grows anaerobically support the use of B. subtilis as a system to study gene regulation in response to oxygen limitation. Since B. subtilis is often used as a host in the fermentation industry, the study of anaerobiosis in B. subtilis may provide an opportunity to improve upon its use in fermentation as well as providing insights into regulation of gene expression in response to a changing environment. B. subtilis grows anaerobically by two different pathways, nitrate respiration using nitrate as an electron acceptor and fermentation in the absence of alternative electron acceptors. This research focuses on regulation of gene expression in cells undergoing nitrate respiration. Fnr, a member of catabolite activator protein (CAP) family, is known as a global anaerobic regulator in nitrate respiration. Fnr is required for the transcription of genes whose products function in nitrate respiration, such as narGHJl, encoding respiratory nitrate reductase, and narK, which is involved in nitrite extrusion. The accumulated studies of Escherichia coli demonstrate that the activity of Fnr as a transcriptional regulator is modulated by anaerobiosis through a mechanism mediated by iron bound to cysteine residues. B. subtilis Fnr may also be activated by this mechanism, but transcription of the fnr gene is highly induced by oxygen limitation unlike the case in E. coli. The anaerobic induction of fnr transcription requires the ResD-ResE two-component signal transduction system that also regulates aerobic respiration. Thus mutations in resD or resE impair both aerobic and anaerobic respiration. The major goal of this research is to determine how the ResDE system is activated and how fnr transcription is induced by the ResDE-dependent mechanism. In order to understand the activation of the ResDE pathway, a suppresser muta nt that bypasses the requirement of the putative histidine kinase, ResE, in nitrate respiration will be characterized by sequencing the wild-type and the mutant allele of the gene. The part of the fnr promoter region that is required for anaerobic regulation will be defined by deletion or mutational analysis using PCR. The wild-type and mutant fnr promoters will be used to examine if ResD binds to the fnr promoter by gel retardation assay and DNA footprinting. Searches for other possible regulators of fnr transcription will be carried out by using transposon and plasmid integration mutagenesis as well as by looking for ResDE-controlled promoter activities in an SP( phage-borne library of B. subtilis promoter-lacZ fusions.
