The tricarboxylic acid cycle occurs within the mitochondria of the yeast Saccharomyces cerevisiae. A nuclear gene encoding the tricarboxylic acid cycle enzyme citrate synthase has previously been isolated (M. Suissa, K. Suda, and G. Schatz, EMBO J. 3:1773-1781, 1984) and is referred to here as CIT1. We report here the isolation, by an immunological method, of a second nuclear gene encoding citrate synthase (CIT2). Disruption of both genes in the yeast genome was necessary to produce classical citrate synthase-deficient phenotypes: glutamate auxotrophy and poor growth on rich medium containing lactate, a nonfermentable carbon source. Therefore, the citrate synthase produced from either gene was sufficient for these metabolic roles. Transcription of both genes was maximally repressed in medium containing both glucose and glutamate. However, transcription of CIT1 but not of CIT2 was derepressed in medium containing a nonfermentable carbon source. The significance of the presence of two genes encoding citrate synthase in S. cerevisiae is discussed.

We cloned the wild-type allele of the spolID locus of Bacillus subtilis. This DNA region was shown to be transcribed beginning within an hour after the onset of sporulation. The amount of spoIID mRNA present in ceUs at 1 h after the end of growth was more than 50-fold greater than it was in growing cells; the pool of this mRNA decreased steadily after 1.5 h after the end of growth. spoIlD mRNA was present in stationary-phase cells of sporulation mutants with lesions in the spoOJ and spolIB genes but was absent in cells carrying spoOB, spoOH, spoIA, spollE, spoliG, or spollIA mutations. In vitro runoff transcription with the Ef55, Ec37, Ea32, and Eu29 forms of RNA polymerase indicated that only the Ea29 form was able to transcribe the spoliD gene.This result is consistent with results of studies with the Spo-mutants, because only mutants that produced Ea29 were able to produce spollD mRNA in vivo. In the course of this work, two additional transcription units were discovered in the DNA region neighboring the spolID gene. One of these was expressed during vegetative growth; the other was expressed early during sporulation and corresponded to an in vitro transcript produced by the Eu29 form of RNA polymerase.Temporal control of gene expression during sporulation of Bacillus subtilis is thought to occur by sequential replacement of the sigma factor component of RNA polymerase (17,18). This mechanism can account for simultaneous activation and silencing of large groups of genes, even if they are scattered around the chromosome. In the last several years, this hypothesis has received strong experimental support. At least five forms of RNA polymerase (Ea55, Ea7, EJ32, Er29, Eu28) that differ only in their sigma factors are present in vegetative or sporulating cells or both. Each enzyme has specific promoter recognition sequences which permit it to transcribe only a certain group of genes (15). These genes are transcribed in vivo at characteristic times during growth or during sporulation or both.In vegetative cells, most of the RNA polymerase holoenzyme contains a sigma subunit designated {J55 (apparent molecular weight, 55,000). In an in vitro system, the purified form of this enzyme transcribes genes expressed in vivo during growth, such as the tms and veg genes (16, 20). Ear37 and E&2 are minor forms of RNA polymerase in vegetative cells. In vitro, these forms of RNA polymerase transcribe genes that are expressed at the end of the logarithmic growth phase or very early during sporulation (e.g., spoVG, ctc, and the subtilisin E gene) (15,27,31). Another minor form of vegetative cell RNA polymerase, Ea28, transcribes a small number of growth genes (13). The Ea29 form of RNA polymerase is found only in sporulating cells, and its appearance is developmentally regulated (30). It is most abundant about 3 h after sporulation begins; when it appears it seems to replace all known vegetative cell sigma factors (14, 30). Ea29 directs transcription of the L gene (14), as well as ctc (27) and, very weakly, the veg gene (14), ...

Saccharomyces cerevisiae contains two genes, CITI and CIT2, encoding functional citrate synthase (K.-S. Kim, M. S. Rosenkrantz, and L. Guarente, Mol. Cell. Biol. 6:1936-1942). We show here that CIT2 encodes a nonmitochondrial form of citrate synthase. The DNA sequence of CIT2 presented provides a possible explanation for why the CIT2 product, unlike the CITI product, fails to be imported into mitochondria. While the products of these two genes are highly homologous, they diverge strikingly at their amino termini. The amino terminus of the CITI primary translation product extends 39 residues beyond the amino termini of Escherichia coli and porcine citrate synthases. This extension consists of a typical mitochondrial targeting motif. The amino terminus of the CIT2 primary translation product extends 20 residues beyond the amino termini of the E. coli and porcine enzymes. The CIT2-encoded extension is not homologous to that of CITI, resulting in a nonmitochondrial localization of the product. The CIT2-encoded extension, however, does bear certain similarities to mitochondrial targeting sequences. The possible role of this sequence in targeting this CIT2 product to a nonmitochondrial organelle is discussed.

The citB gene of Bacillus subtilis codes for aconitase (D. W. Dingman and A. L. Sonenshein,. By direct measurements of citB mRNA levels and by measurements of P-galatdase activity in a strain carrying a citE-lacZ fusion, we have examined the expression of citB during growth and sporulation. When cells were grown in nutrient broth sporulation medium, citB mRNA appeared in mid-to-lateexponential phase and disappeared by the second hour of sporulation. This timing corresponded closely to the kinetics of appearance of aconitase enzyme activity. Decoyinine, a compound that induces sporulation in a defined medium, caused a rapid simultaneous increase in aconitase activity and citB transcription. After decoyinine addition, the rate of increase in aconitase activity in a 2-ketoglutarate dehydrogenase (citk) mutant and in a citrate synthase (cit4) mutant was significantly less than in an isogenic wild-type strain. This is apparently due to a failure to deplete 2-ketoglutarate and accumulate citrate. These metabolites mit act as negative and positive effectors of citB expression, respectively. Mutations known to block sporulation at an early stage (spoOH and spoOB) had no appreciable effect on citB expression or aconitase activity. These results suggest that appearance of aconitase is stimulated by conditions that induce sporulation but is independent of certain gene products thought to act at an early stage of sporulation.Aconitase [EC 4.2.1.3; citrate (isocitrate) hydrolase], a tricarboxylic acid (TCA) cycle enzyme, appears to be subject to at least two forms of regulation in Bacillus subtilis. Catabolite repression of aconitase activity occurs whenever a rapidly metabolizable carbon source (e.g., glucose or mannose) is present in the medium (6). Catabolite repression is known to lower overall TCA cycle activity (6) and is thought to be a major regulating force for sporulation (20,33). For catabolite repression of aconitase to be complete, however, the medium must also contain a good source of 2-ketoglutarate (2-KG) (20,30). In the absence of a good carbon source, a high intracellular concentration of 2-KG has no detectable effect on aconitase activity (19 When cells of B. subtilis exhaust a complex medium and begin to sporulate, aconitase activity (which is at a very low level during growth) increases substantially 1 to 3 h after the onset of sporulation (14,16,36). This behavior might be taken to mean that aconitase is subject to sporulation control or that the signal that initiates sporulation also activates aconitase expression. Since aconitase activity in this case appears after the end of growth, it is possible that its regulation during sporulation is distinct from its regulation during growth in a defined medium. * Corresponding author. Mutants of B. subtilis lacking aconitase activity require glutamate (or glutamine) for growth and, after exhaustion of a complex medium, become blocked at stage 0 or I of sporulation (15,39). This again suggests a linkage between sporulation and aconitase expression. Three closel...

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The yeast nuclear gene CIT1 encodes mitochondrial citrate synthase, which catalyses the first and rate-limiting step of the tricarboxylic acid (TCA) cycle. Transcription of CIT1 is subject to glucose repression. Mutations in HAP2, HAP3 or HAP4 block derepression of a CIT1-lacZ gene fusion. The HAP2,3,4 transcriptional activator also activates nuclear genes encoding components of the mitochondrial electron transport chain, and thus it co-ordinates derepression of two major mitochondrial functions. Two DNA sequences resembling the consensus HAP2,3,4-binding site (ACCAATNA) are located at approximately -310 and -290, upstream of the CIT1 coding sequence. Deletion and mutation analysis indicates that the -290 element is critical for activation by HAP2,3,4. Glucose-repressed expression of CIT1 is largely independent of HAP2,3,4, is repressed by glutamate, and requires a DNA sequence between -367 and -348. Evidence is presented for a second HAP2,3,4-independent activation element located just upstream and overlapping the -290 HAP2,3,4 element.

The activity of aconitase in Bacillus subtilis is greatly reduced in cells cultured in media containing rapidly metabolized carbon sources (e.g., glucose). Thus, expression of this enzyme appears to be subject to a form of catabolite repression. Since the product of the citB gene of B. subtilis is required for aconitase activity, we cloned the wild-type allele of this gene and used this DNA as a probe for transcription of citB in cells grown in various media. The steady-state level of RNA that hybridized to this probe was about 10-fold higher in B. subtilis cells grown in citrate-glutamine medium than in cells grown in glucose-glutamine medium. This result correlates well with the steady-state levels of aconitase activity. Two transcripts were shown to initiate within the cloned DNA; the steady-state level of one of these transcripts varied in the same way as did aconitase activity when cells were grown in media containing different carbon sources. This is the first demonstration of regulation by the carbon source of the level of a vegetative-cell transcript in B. subtilis.In Bacillus subtilis, the first three enzymes of the citric acid cycle, i.e., citrate synthase, aconitase, and isocitrate dehydrogenase, are required both for biosynthesis of glutamate (through 2-ketoglutarate) and for utilization of nonfermentable energy sources (e.g., citrate, lactate, and various amino acids). The levels of these citric acid cycle enzymes in B. subtilis are reduced about twofold in the presence of a rapidly metabolized carbon source such as glucose (27,28,43). The residual level of activity apparently suffices for biosynthesis of amino acids and other compounds. The levels of citrate synthase (18, 28, 29), aconitase (27,28,51), and, in some strains, isocitrate dehydrogenase (19,29,43) are further reduced when a rapidly metabolized carbon source and a source of glutamate are both supplied. Glutamate alone or in combination with a poor carbon source (e.g., citrate) does not lead to reduction in the levels of these enzymes (28).Unlike the situation with Escherichia coli, regulation of carbon source utilization in Bacillus spp. does not appear to involve cyclic AMP (2,6,30,49) or the phosphoenolpyruvate phosphotransferase sugar transport system (33). Attempts to isolate pleiotropic mutants that would have phenotypes equivalent to those of mutants of catabolite gene activator protein in E. coli (9) have not yet been successful (7,(13)(14)(15). Moreover, regulation by the carbon source of the levels of various vegetative enzymes in B. subtilis may not be governed by a single mechanism. For example, accumulation of only the initial phosphorylated intermediates of various readily utilized sugars is sufficient for reduction of acetoin dehydrogenase levels (33) but not for regulation of the levels of inositol dehydrogenase (22) or D-gluconate transport enzymes (10).To explore the mechanism of synergistic regulation of aconitase levels by glucose and glutamate, we have isolated a fragment of B. subtilis DNA which transforms an aconitas...

The yeast CIT1 (mitochondrial citrate synthase) gene is subject to glucose repression and is further repressed by glucose plus glutamate. Based on deletion analysis of a CIT1-lacZ gene fusion, DNA sequences between -548 and -273 are required for full expression of CIT1. The region of transcription initiation and the putative TATA element are located at -150 to -100 and -195 respectively. A restriction fragment containing DNA sequences between -457 and -211 conferred activation and glucose-glutamate regulation when placed in either orientation upstream of a UAS-less heterologous yeast gene. Deletion of DNA sequences between -291 and -273 specifically eliminated derepression of CIT1, and destroyed one of two closely-spaced, potential binding sites for the HAP2,3,4 transcriptional activator protein. Ten-base-pair block substitutions in the region -367 to -348 reduced glucose-repressed expression. Thus, it appears that distinct DNA sequences upstream of CIT1 activate expression in glucose-repressed and derepressed cells. Possible mechanisms of regulation by glutamate plus glucose, are discussed.