Structure and expression of the cytochrome aa3 regulatory gene ctaA of Bacillus subtilis

Abstract

Mutations that define the ctaA gene of Bacillus subtilis block cytochrome aa3 formation and sporulation. We have recently described the isolation and initial characterization of the ctaA locus. Analysis of in vivo mRNA transcripts by RNase protection experiments located the 5' and 3' termini of the ctaA transcript, confirming a monocistronic structure. By using a nuclease protection assay, an increase in the abundance of steady-state ctaA mRNA was observed during the initiation of sporulation, followed by a decrease during subsequent stages. Transcripts originating from the ctaA gene were most abundant 2.0 h after the end of exponential growth. This pattern of ctaA mRNA accumulation was confirmed by coupling the transcription of the ctaA gene to lacZ in an integrative plasmid vector. Expression of ctaA was not repressed by glucose and was independent of the spoOA and spoOH (sigH) gene products. Postexponential expression was found to be dependent on the product of the strC gene. The expression of ctaA appears to be regulated in a growth stage-specific manner. The transcriptional start site, identified by high-resolution S1 nuclease protection experiments, was preceded by a single uA-dependent promoter sequence.Bacillus subtilis responds to nutrient deprivation by undergoing a series of metabolic and morphological changes that culminate in formation of a dormant endospore (13, 14), a process that requires a normally regulated and functional system of energy metabolism (24a). Sufficient perturbation of any of these metabolic functions abolishes sporulation proficiency (24a). It is of interest to determine if changes in energy-generating processes are under the control of regulators that mediate the initiation of sporulation.Genes for biosynthesis of the electron transfer component menaquinone (menCD [20]) and for several tricarboxylic acid cycle enzymes (citB [6], citG [101, and sdh [17]) are controlled at the level of transcription. Expression of these genes increases during the transition stage from exponentialto stationary-phase growth; however, each is influenced by somewhat different metabolic conditions. Genes encoding electron transport components, tricarboxylic acid cycle enzymes, and sporulation functions may share common regulatory features, and the metabolic context in which sporulation is initiated is in part defined by components of the respiratory chain. Significant variations in the composition and organization of the B. subtilis electron transport chain are observed in response to environmental conditions (5, 9,

Transcriptional regulation of the Bacillus subtilis menp1 promoter

“…Direct measurements of intracellular NADH would test this hypothesis. These responses of menp1 are similar to those observed for transcription of ctaA (22) and for accumulation of caa 3 oxidase (16). The poor match to consensus (two of six) in the Ϫ35 region of menp1, together with the perfect match (six of six) in the Ϫ10 region, suggests that menp1 is a good candidate to be a highly regulated promoter.…”

Two ResD-Controlled Promoters Regulate ctaA Expression in Bacillus subtilis

“…The two transcriptional start sites observed here and whose products are shown in Fig. 4A (lanes 2 to 7) correspond to the start sites previously proposed (15) in a study that mapped the downstream promoter start site by high-resolution S1 nuclease mapping using RNA from cells at stage T 2 .…”

“…The ResDE system is required for transcription of the following genes and operons involved in aerobic respiration; the resABCDE operon (23), encoding proteins similar to those involved in cytochrome c biogenesis (resABC) (6) and ResD-ResE (resDE) (23); the petCBD operon, encoding subunits of the cytochrome bf complex; the ctaBCDEF operon (12), encoding CtaB, which is required for the synthesis of heme O from heme B (ctaB) (25) and structural genes for cytochrome caa 3 (ctaCDEF) (22) and ctaA (23); and a gene required for heme A biogenesis (24,25) and hence for the synthesis of the heme A-containing terminal cytochrome oxidases aa 3 and caa 3 . Recognition of phenotypic traits shared by resD and ctaA mutants (15) led to a study that revealed that ResD has an essential role in the activation of in vivo expression of the ctaA promoter (23). Phenotypic similarities shared by resD and ctaA mutants, among others, included a sporulation defect and the absence of the heme A-containing terminal oxidases aa 3 and caa 3 .…”

“…A1 and A2 are situated upstream of the Ϫ35 promoter region, and A3 is downstream of the Ϫ10 region of the ctaA promoter previously identified (15). Deletion experiments revealed that binding site A1 did not influence the in vivo expression of the ctaA gene (29), suggesting that site A1 may be involved in the regulation of the divergent ctaB promoter, which also requires ResD for expression (12).…”

Catabolite Regulation of the Bacillus subtilis ctaBCDEF Gene Cluster

“…At the cta locus, there are six genes, ctaABCDEF. ctaA and ctaB encode two enzymes required for the biosynthesis of heme A, the prosthetic group of both aa 3 and caa 3 (26,27,42).…”