Catabolite Repression of the Citrate Fermentation Genes in Klebsiella pneumoniae : Evidence for Involvement of the Cyclic AMP Receptor Protein

Abstract

Klebsiella pneumoniae is able to grow anaerobically with citrate as a sole carbon and energy source by a fermentative pathway involving the Na ؉ -dependent citrate carrier CitS, citrate lyase, and oxaloacetate decarboxylase. The corresponding genes are organized in the divergent citC and citS operons, whose expression is strictly dependent on the citrate-sensing CitA-CitB two-component system. Evidence is provided here that the citrate fermentation genes are subject to catabolite repression, since anaerobic cultivation with a mixture of citrate and glucose or citrate and gluconate resulted in diauxic growth. Glucose, gluconate, and also glycerol decreased the expression of a chromosomal citS-lacZ fusion by 60 to 75%, whereas a direct inhibition of the citrate fermentation enzymes was not observed. The purified cyclic AMP (cAMP) receptor protein (CRP) of K. pneumoniae bound to two sites in the citC-citS intergenic region, which were centered at position ؊41.5 upstream of the citC and citS transcriptional start sites. Binding was apparently stimulated by the response regulator CitB. These data indicate that catabolite repression of the citrate fermentation genes is exerted by CRP and that in the absence of repressing carbon sources the cAMP-CRP complex serves to enhance the basal, CitB-dependent transcription level.Klebsiella pneumoniae, a member of the Enterobacteriaceae, is able to grow with citrate as a sole carbon and energy source under anoxic conditions (for a review, see reference 4). Citrate fermentation involves uptake by a Na ϩ -dependent citrate carrier (32, 44), cleavage into acetate and oxaloacetate by citrate lyase, and decarboxylation of oxaloacetate to pyruvate by the Na ϩ ion pump oxaloacetate decarboxylase (12, 13). The conversion of pyruvate to acetate, formate, CO 2 , and H 2 (7, 38) is catalyzed by enzymes generally involved in anaerobic pyruvate catabolism. The citrate-specific fermentation genes form a cluster of two divergent operons (5, 6). The citCDEFG operon encodes citrate lyase ligase (CitC), the citrate lyase subunits (CitD, CitE, and CitF), and triphosphoribosyl-dephospho-coenzyme A synthase (CitG), which catalyzes the formation of a precursor of the citrate lyase prosthetic group (36, 37). The citS-oadGAB-citAB operon encodes the citrate carrier CitS, the oxaloacetate decarboxylase subunits (OadG, OadA, and OadB), and a two-component system composed of the sensor kinase CitA and the response regulator CitB (Fig. 1A). The CitA-CitB system was shown to be essential for the expression of both operons (6). CitA presumably functions as a sensor of extracellular citrate, since the periplasmic domain of this membrane-bound protein binds citrate with high affinity (K d Ϸ 6 M) and specificity (23). CitB acts as a transcriptional activator of the citS and the citC operons and binds to two sites in the 193-bp citC-citS intergenic region (Fig. 1) in a phosphorylation-dependent fashion (24).Maximal expression of a chromosomally integrated translational citSЈ-ЈlacZ fusion requires citrate, anoxic...

Glycerol-3-Phosphate-Induced Catabolite Repression inEscherichia coli

“…We postulate that G3P inhibits EIIA Glc -P-mediated stimulation of adenylate cyclase, thus lowering the cAMP concentration. A recent publication described the catabolite repression of citrate fermentation genes in Klebsiella pneumoniae by glycerol and gluconate, among other compounds (25). The authors of that report also noted the discrepancy between the state of dephosphorylation of EIIA Glc and the repressing effect of non-PTS sugars while maintaining the importance of the cAMP-CAP system in this type of catabolite repression.…”

The Pseudomonas putida Crc Global Regulator Controls the Expression of Genes from Several Chromosomal Catabolic Pathways for Aromatic Compounds

“…This enzyme has been studied mainly in Klebsiella pneumoniae, where it participates in citrate fermentation under anaerobic conditions. Expression of the oxaloacetate decarboxylase in K. pneumoniae is subject to catabolite repression by the CRP protein (22). A protein showing high similarity to E. coli and K. pneumoniae CRP is present in P. aeruginosa and in P. putida and has been called Vfr (1).…”

CitI, a Transcription Factor Involved in Regulation of Citrate Metabolism in Lactic Acid Bacteria

“…Studies of this adaptive response in K. pneumoniae (4)(5)(6)16), B. subtilis (22,23), and W. paramesenteroides (14 and this work) have shown that the expression of the proteins required for the transport and degradation of citrate is regulated at the transcriptional level. To date, K. pneumoniae CitA, B. subtilis CitT, and W. paramesenteroides CitI have been identified as transcription factors specifically involved in citrate metabolism.…”

“…This system regulates the expression of two operons, citCDEFG and CitS-oadGAB-citAB, which are involved in the transport and metabolism of citrate. Expression of these operons is induced under anoxic conditions in the presence of citrate and is subject to catabolic repression (6,16). In Bacillus subtilis, the CitS/T two-component system (a member of the CitA/B family) is involved in the regulation of the expression of genes encoding citrate transporters required for the utilization of citrate in aerobic conditions (22,23).…”

“…In these bacteria, two-component systems regulate the expression of citrate utilization genes. In particular, the CitA/B two-component system of K. pneumoniae has been extensively studied (4)(5)(6)16). This system regulates the expression of two operons, citCDEFG and CitS-oadGAB-citAB, which are involved in the transport and metabolism of citrate.…”