Bacillus subtilis citB gene is regulated synergistically by glucose and glutamine

Abstract

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...

Role of CcpA in Regulation of the Central Pathways of Carbon Catabolism in Bacillus subtilis

“…In contrast to glycolytic enzymes, TCA cycle enzymes are repressed at a high glucose concentration in the presence of glutamate. The proteomic approach provided an overall view on the synthesis of almost all TCA cycle enzymes and was in good agreement with previous reports on enzyme activities and transcription of TCA cycle genes (15,23,34). Differential repression efficiency, suggesting a fine-tuning of expression, was described for citZ and citB, which are more strongly repressed than citC (23) (Fig.…”

Transcriptional regulation of Bacillus subtilis citrate synthase genes

“…Transcriptional regulation of citA and citZ in response to different carbon sources. The enzyme activities of aconitase and citrate synthase and the transcription of the aconitase gene (citB) are synergistically repressed by glucose and glutamate (10,26). As shown in Table 2, we found that expression of citA-lacZ was more than 6-fold lower when glucose was the sole carbon source than when succinate was the sole carbon source.…”

“…Regulation of the citB gene has been studied in detail and found to involve several mechanisms of transcriptional control (5, 6). In defined medium, citB transcription is synergistically repressed by glucose and glutamate, but it is highly derepressed in a medium with citrate as the sole carbon source (26). This repression has been associated with a region of dyad symmetry upstream of the promoter region (5,6).…”

Nitrogen and Oxygen Regulation of Bacillus subtilis nasDEF Encoding NADH-Dependent Nitrite Reductase by TnrA and ResDE

“…B. subtilis cells grown overnight on DS (Difco sporulation) agar medium were used to inoculate 2ϫ YT (yeast extract-tryptone) liquid medium (12) supplemented with 1% glucose and 20 mM phosphate buffer (pH 7.0), which contained either 0.2% KNO 3 or 10 mM KNO 2 , to an optical density at 600 nm of 0.02. Alternatively, cells grown aerobically overnight in TSS liquid medium (18) with 0.2% NH 4 Cl were used to inoculate TSS medium containing 0.2% KNO 3 with or without 0.2% NH 4 Cl. Anaerobic growth of cultures was carried out as described previously (16).…”