Characterization of the major citrate synthase of Bacillus subtilis

Abstract

The major citrate synthase of Bacillus subtilis (CS-II) was purified to near homogeneity and shown to correspond to the product of the citZ gene. Accumulation of CS-II during exponential growth and stationary phases paralleled expression of the citZ gene. The physical and kinetic properties of CS-II were similar to those of citrate synthase enzymes from Bacillus megaterium and from eukaryotic cells but differed from those of citrate synthases from many gram-negative bacteria.Citrate synthase (CS), the first enzyme of the Krebs cycle, is required for synthesis of glutamate from glycolytic intermediates and is, therefore, a critical enzyme in cellular biosynthesis (23). Moreover, the role of the Krebs cycle in production of ATP and reducing power makes this enzyme a major contributor to cellular energetics. In Bacillus subtilis, CS is needed for both growth (in the absence of a source of glutamate) and spore formation (5-7).B. subtilis has two distinct, homologous CS genes, citA and citZ, expressed during growth and early stationary phase (7,8). A null mutation in citA has little effect on growth, CS enzyme activity, or sporulation, but an in-frame deletion mutation in citZ causes partial glutamate auxotrophy, a Ͼ90% loss of CS enzyme activity, and a significant defect in sporulation. A citA citZ double mutant is an absolute glutamate auxotroph, has undetectable CS activity, and sporulates more poorly than does either a citA or citZ single mutant. These results, along with comparative measurements of citA and citZ mRNA levels, have indicated that citZ encodes the major CS of B. subtilis (8).Here we report the biochemical and kinetic properties of purified CS-II, the citZ gene product. Previous studies of B. subtilis CS activity were based on crude extracts (5, 9), now known to contain a mixture of CS-I, the product of citA, and CS-II. Several CSs from gram-negative bacteria have been purified and their kinetic and biochemical properties have been studied in detail (14,16,17,21,22), but only one CS enzyme from a gram-positive bacterium (Bacillus megaterium) has been studied previously in its homogeneously purified form (18). Bacillus sp. strain C4 CS has been studied after partial purification (19).To purify B. subtilis CS-II without contamination by CS-I, we used a citA null mutant strain, SJB9, a derivative of JH642 (7). Purification followed the method of Robinson et al. (18) with minor modifications (see the legend to Fig. 1 and Table 1). B. subtilis CS activity in crude extracts and in pure preparations was found to be rapidly lost at 4ЊC unless protected by 20% glycerol. In the presence of 20% glycerol and 100 mM KCl, the purified B. subtilis enzyme retained its activity for more than 6 months at 4ЊC and for about 2 years at Ϫ20ЊC.The N-terminal amino acid sequence (MTATRGLEGV VATTSSVSSII) and the sequence of an internal tryptic peptide (MLTEIGEVEN) were identical to those predicted from the DNA sequence of the citZ gene (7). These results very strongly suggest that citZ encodes CS-II. Molecular mass and olig...

Flux through Citrate Synthase Limits the Growth of EthanologenicEscherichia coliKO11 during Xylose Fermentation

“…Addition of acetaldehyde decreased the NADH/NAD ϩ ratio by increasing the pool of electron acceptors, which potentially increased the function of the native citrate synthase in vivo. This hypothesis was confirmed, in part, by using the B. subtilis citZ gene encoding an NADHinsensitive citrate synthase (24,25). Expression of citZ in KO11 stimulated growth and ethanol production almost twofold and substantially reduced the need to supply high levels of complex nutrients.…”

“…The primary citrate synthase in gram-positive bacteria is allosterically regulated by ATP and is relatively insensitive to NADH (25). Since an overabundance of ATP is not anticipated during xylose fermentation (42), expression of B. subtilis citZ in KO11 would be expected to increase carbon flow into Primers were used to clone the citZ gene (including a ribosomal binding site) into pCR2.1-TOPO to produce pLOI2514.…”

Lack of Protective Osmolytes Limits Final Cell Density and Volumetric Productivity of Ethanologenic Escherichia coli KO11 during Xylose Fermentation

“…The primary citrate synthase in B. subtilis (CitZ) is insensitive to NADH and is allosterically regulated by ATP (24). Expression of B. subtilis citZ also doubled cell mass, increased volumetric productivity (Fig.…”

Bacillus subtilis Metabolism and Energetics in Carbon-Limited and Excess-Carbon Chemostat Culture

“…In addition to transcriptional regulation, several TCA cycle enzymes are responsive to the energetic state of the cell (33), most prominently the major isoform of citrate synthase, which is a key enzyme of the cycle and is competitively inhibited by ATP (20). If ATP-dependent activation or reduced inhibition of enzymes contributes significantly to the control of TCA cycle activity, one would expect high TCA cycle fluxes in Plimited culture with twofold-lower intracellular ATP concentrations than those in C-or N-limited cultures.…”