Three enzymes are specifically required for uptake and catabolism of citrate by Klebsiella pneumoniae under anaerobic conditions: a Na+ -dependent citrate carrier (CitS), citrate lyase (CitDEF), and the Na+ pump oxaloacetate decarboxylase (OadGAB). The corresponding genes are clustered on the chromosome, with the citCDEFG genes located upstream and divergent to the citS-oadGAB genes. We found that expression of citS from its native promoter in Escherichia coli requires the DNA region downstream of oadB. Nucleotide sequence analysis of this region revealed the presence of two adjacent genes, citA and citB. By sequence similarity, the predicted CitA and CitB proteins were identified as members of the two-component regulatory systems. The sensor kinase CitA contained, in the N-terminal half, two putative transmembrane helices which enclosed a presumably periplasmic domain of about 130 amino acids. The C-terminal half of the response regulator CitB harboured a helix-turn-helix motif typical of DNA-binding proteins. K. pneumoniae citB null mutants were unable to grow anaerobically with citrate as the sole carbon and energy source (Cit- phenotype). When cultivated anaerobically with citrate plus glycerol, all of the citrate-specific fermentation enzymes were synthesized in the wild type, but not in the citB mutants. This showed that citS, oadGAB and citDEF required the CitB protein for expression and therefore are part of a regulon. In the wild type, synthesis of CitS, oxaloacetate decarboxylase and citrate lyase was dependent on the presence of citrate, sodium ions and a low oxygen tension. In a citA null mutant which expressed citB constitutively at high levels, none of these signals was required for the formation of the citrate fermentation enzymes. This result suggested that citrate, Na+, and oxygen exerted their regulatory effects via the CitA/CitB system. In the presence of these signals, the citAB gene products induced their own synthesis. The positive autoregulation occurred via co-transcription of citAB with citS and oadGAB.

SummaryThe two-component regulatory system CitA /CitB is essential for induction of the citrate fermentation genes in Klebsiella pneumoniae. CitA represents a membrane-bound sensor kinase consisting of a periplasmic domain¯anked by two transmembrane helices, a linker domain and the conserved kinase or transmitter domain. A fusion protein (MalE±CitAC) composed of the maltose-binding protein and the CitA kinase domain (amino acids 327±547) showed constitutive autokinase activity and transferred the g-phosphate group of ATP to its cognate response regulator CitB. The autokinase activity of CitA was abolished by an H350L exchange, and phosphorylation of CitB was inhibited by a D56N exchange, indicating that H-350 and D-56 represent the phosphorylation sites of CitA and CitB respectively. In the presence of ATP, CitB±D56N formed a stable complex with MalE±CitAC.

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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...