CcpA-Dependent Regulation of Bacillus subtilis Glutamate Dehydrogenase Gene Expression

Abstract

The Bacillus subtilis rocG gene, encoding catabolic glutamate dehydrogenase, was found to be subject to direct CcpA-dependent glucose repression. The effect of CcpA required the presence of both the HPr and Crh proteins. The primary CcpA binding site was identified by mutational analysis and DNase I footprinting. In the absence of inducers of the Roc pathway, rocG was still expressed at a low level due to readthrough transcription. CcpA-dependent repression of rocG readthrough transcription proved to contribute to the slow growth rate of B. subtilis cells in glucose-glutamate medium. Increased readthrough expression of rocG was shown to be partially responsible for the growth defect of ccpA strains in glucose-ammonium medium.Interconversions of ␣-ketoglutarate and glutamate are major links between carbon and nitrogen metabolism. These reactions are catalyzed by several enzymes: glutamate synthase [␣-ketoglutarate ϩ glutamine ϩ NAD(P)H 3 2ϫ glutamate ϩ NAD ϩ (P)], glutamate dehydrogenase (GlutDH) [␣-ketoglutarate ϩ NH 3 ϩ NAD(P)H^glutamate ϩ NAD ϩ (P)], and glutamate-dependent aminotransferases [glutamate ϩ keto acid (or aldehyde)^␣-ketoglutarate ϩ amino acid].In Bacillus subtilis, two genes code for GlutDH proteins. The rocG gene product is the major catabolic GlutDH, while gudB encodes an intrinsically inactive GlutDH (7). Spontaneous gain-of-function mutations in gudB generate a second catabolic GlutDH, GudB1 (7). No anabolic glutamate dehydrogenase is present in B. subtilis, and all de novo synthesis of glutamate is catalyzed by glutamate synthase (3).The B. subtilis rocG gene is a member of the RocR regulon (5). RocR also controls the rocABC and rocDEF operons, whose products catalyze degradation of arginine to glutamate (9,16,17). All three roc transcription units have SigL-dependent promoters, require RocR and AhrC proteins for expression, and are induced by arginine, ornithine, or proline. The RocG-catalyzed reaction (glutamate ϩ NAD ϩ 3 ␣-ketoglutarate ϩ NH 3 ϩ NADH) can be viewed as the final step in the utilization of arginine, ornithine, and proline, providing rapidly metabolizable carbon-or nitrogen-containing compounds for biosynthesis.Many B. subtilis genes involved in utilization of alternative carbon sources are subject to carbon catabolite repression mediated by the CcpA protein (8,11,25,34). We show here that rocG expression is also subject to CcpA-dependent carbon catabolite repression. In the course of this work, we discovered that rocG is transcribed not only from its own SigL-dependent promoter but also by readthrough from an upstream gene. In fact, the known growth defect of ccpA mutants in glucoseammonium medium (13,30,33) can be partially relieved by blocking readthrough transcription of rocG. MATERIALS AND METHODSBacterial strains and culture media. The B. subtilis strains used in this study are listed in Table 1 or described in the table footnotes. The media and growth conditions for B. subtilis and Escherichia coli strains were described previously (7). TSS minimal medium was suppleme...

Hierarchical expression of genes controlled by theBacillus subtilisglobal regulatory protein CodY

“…Oligonucleotides were obtained from Integrated DNA Technologies (Table S2). Construction of strains was performed as previously described (17,60) or as described in SI Materials and Methods.…”

Carbon Catabolite Control of the Metabolic Network inBacillus subtilis

“…Inactivation of the crh alone does not affect CCR or CCA, but the residual CCR observed in pstH1 mutants disappears when the crh is disrupted or a crh1 mutation (replacement of Ser-46 with Ala) is introduced. 20,23,32,33,41,45,55,58,61,63,65,82,83) The question of how P-Ser-HPr and P-Ser-Crh contribute to CCR and CCA with different efficiencies has not been clearly answered yet, although a Crh-specific function in the regulation of expression during growth on substrates other than carbohydrates was recently revealed, 84) probably because of the drastically higher amount of HPr than of Crh during growth on carbohydrates. 85) Also, P-Ser-Crh displays altered binding to CcpA to effect catabolite control.…”

“…On the other hand, the rocG gene, encoding catabolic glutamate dehydrogenase, has been found to be subject to direct CcpA-dependent glucose repression. 63) The glutamate pool is low in ccpA mutants due to a loss of CCR of rocG, which contributes to a slow growth rate in glucose/glutamate medium.…”

“…Of these, acoABCL, levDEFG-sacC, and rocG have been experimentally proven to carry the respective cre sequences. 25,58,59,63) (ii) Enhancement of pyruvate assimilation and shutdown of the TCA cycle and respiration…”

Glutamate Dehydrogenases: Enzymology, Physiological Role and Biotechnological Relevance

“…The actual physiological reaction of each GDH depends on several factors, as the kinetic constants of the enzyme for its different substrates or the environment where the cell is developed may widely vary. In general, NADP + -GDHs usually operate in the biosynthetic direction, that is, synthesizing glutamate by the assimilation of ammonia into 2-OG [6,31,39,40,45,67,68], whereas NAD + -GDHs have primarily a catabolic function, yielding ammonia and 2-oxoglutarate from the oxidative catabolism of glutamate [23,24,39,68] (Table 1). Sometimes, both enzymes are present in the same organism, and play a different physiological role due to their different kinetic properties or their different time or place of expression [27,51,69,70].…”