The level of fumarase activity in Bacillus subtilis depends on the nutritional environment; in rich medium low vegetative levels increase towards the end of the exponential phase, whereas in minimal glucose medium levels are relatively high throughout growth. Analysis of the enzyme levels in spoO mutants has revealed that a functional spoOH gene is required for the efficient expression of fumarase in both media. This highlights a regulatory role for the spoOH gene product not only in control of postexponentially expressed genes, but also during vegetative growth in defined medium. S1 transcript mapping reveals three transcriptional startpoints for the fumarase structural gene (citG) in B. subtilis. The upstream promoter region P1, which appears to contain two transcriptional startpoints, is functional in both Escherichia coli and B. subtilis. Promoter P2, which is located closer to the structural gene, is only functional in B. subtilis. Transcription from this promoter is strictly dependent on a functional spoOH gene; this gene has recently been shown to encode a minor sigma factor.

The fumarase gene (citG) of Bacillus subtilis is transcribed from two promoter regions, cilGpl and citGp2 (P1 and P2); the P2 promoter is used by the EUH form of RNA polymerase. In order to study the role of P1 and P2 in citG expression, the promoter region and various deletion derivatives that effectively separate P1 and P2 were fused to the Escherichia coli ,-galactosidase gene (lacZ) and introduced into the chromosome in single copy at the amyE locus. P1 functioned to provide a relatively low and stable basal level of fumarase activity throughout growth. In contrast, P2 activity was found to vary over at least a 50-fold range and was responsible for regulating fumarase activity during growth and sporulation in a rich medium and in response to changes in carbon source. To further investigate the role of sigma H in fumarase regulation, citGp2-lacZ fusions were introduced into a strain in which the expression of the chromosomal spoOH gene was under the control of the isopropylthiogalactopyranoside-inducible spac promoter. Induction of p,P did not lead to P2 induction, suggesting that cilG expression is not regulated at the level of spoOH transcription.The tricarboxylic acid (TCA) cycle in Bacillus subtilis plays a dual role in metabolism, providing both energy (when glycolysis is unable to fulfill the cell's energy requirements) and biosynthetic intermediates. The cycle can be regarded as two pathways: the tricarboxylic pathway (citrate to 2-ketoglutarate) and the dicarboxylic pathway (succinyl coenzyme A to oxaloacetate). These pathways are subject to independent but overlapping regulation (15,27). Levels of fumarase, a component of the dicarboxylic acid pathway, increase during vegetative growth in a rich medium to peak between 1 and 2 h after the end of exponential growth (9). Levels are highest in a lactate minimal medium (when both anabolic and energy-generating functions of the cycle are of prime importance) and are depressed by rapidly metabolizable carbon sources such as glucose and casamino acids (15).The fumarase gene (citG) has been cloned and sequenced (22,24,25), and it has been shown to be transcribed from two promoter regions, citGpl (P1) and citGp2 (P2); citGpl appears to be a complex of two overlapping promoters, Pla and Plb (9). citGpl and citGp2 are separated by a short open reading frame (orfA), which has the potential to encode a small (61-residue) polypeptide. Transcription from citGp2 is dependent on a functional spoOH gene, with the consequence that spoOH is required for efficient fumarase expression (9). spoOH encodes an alternative sigma factor (sigma H), which together with the RNA polymerase core enzyme directs transcription from citGp2 (32). Thus, the analysis of the transcriptional control of fumarase in B. subtilis is complicated by the presence of multiple promoters and the involvement of differentially regulated multiple holoenzyme forms of RNA polymerase.To tackle this problem, fusions of separated citGpl and citGp2 promoters to the Escherichia coli lacZ gene were constructed, s...

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