The Na+-dependent citrate carrier of Klebsiella pneumoniae (CitS) is a member of the 2-hydroxycarboxylate transporter family. Within the highly conserved helix Vb region, Asn-185 of CitS was mutated to Val and Glu-194 was mutated to Gln. The wild-type and mutant proteins were synthesised in Escherichia coli DH5alpha or C43(DE3) as biotinylated or His-tagged CitS-fusions, respectively. The synthesis and purification procedure yielded 6.5 mg pure CitS per litre culture. The fusion proteins were characterised with E. coli cell suspensions or after reconstitution of the purified enzymes into proteoliposomes. The E194Q mutation had almost no effect on the kinetics of Na+ or citrate transport. In contrast, aberrant citrate transport kinetics were found for the N185V mutant. The apparent K(m) value for the citrate species H-citrate(2-) was increased about nine-fold, whereas the apparent Vmax value and the effect of Na+ on the transport kinetics were comparable to the wild-type. Asn-185 of CitS appears therefore to participate in the binding of H-citrate(2-).

A prominent region of the Na(+)-dependent citrate carrier (CitS) from Klebsiella pneumoniae is the highly conserved loop X-XI, which contains a putative citrate binding site. To monitor potential conformational changes within this region by single-molecule fluorescence spectroscopy, the target cysteines C398 and C414 of the single-Cys mutants (CitS-sC398, CitS-sC414) were selectively labeled with the thiol-reactive fluorophores AlexaFluor 546/568 C(5) maleimide (AF(546), AF(568)). While both single-cysteine mutants were catalytically active citrate carriers, labeling with the fluorophore was only tolerated at C398. Upon citrate addition to the functional protein fluorophore conjugate CitS-sC398-AF(546), complete fluorescence quenching of the majority of molecules was observed, indicating a citrate-induced conformational change of the fluorophore-containing domain of CitS. This quenching was specific for the physiological substrate citrate and therefore most likely reflecting a conformational change in the citrate transport mechanism. Single-molecule studies with dual-labeled CitS-sC398-AF(546/568) and dual-color detection provided strong evidence for a homodimeric association of CitS.

The biosynthesis of the 2-(5؆-phosphoribosyl)-3-dephospho-coenzyme A (CoA) prosthetic group of citrate lyase (EC 4.1.3.6), a key enzyme of citrate fermentation, proceeds via the initial formation of the precursor 2-(5؆-triphosphoribosyl)-3-dephospho-CoA and subsequent transfer to apo-citrate lyase with removal of pyrophosphate. In Escherichia coli, the two steps are catalyzed by CitG and CitX, respectively, and the corresponding genes are part of the citrate lyase gene cluster, citCDEFXG. In the homologous citCDEFG operon of Klebsiella pneumoniae, citX is missing. A search for K. pneumoniae citX led to the identification of a second genome region involved in citrate fermentation which comprised the citWX genes and the divergent citYZ genes. The citX gene was confirmed to encode holo-citrate lyase synthase, whereas citW was shown to encode a citrate carrier, the third one identified in this species. The citYZ genes were found to encode a two-component system consisting of the sensor kinase CitY and the response regulator CitZ. Remarkably, both proteins showed >40% sequence identity to the citrate-sensing CitA-CitB two-component system, which is essential for the induction of the citrate fermentation genes in K. pneumoniae. A citZ insertion mutant was able to grow anaerobically with citrate, indicating that CitZ is not essential for expression of citrate fermentation genes. CitX synthesis was induced to a basal level under anaerobic conditions, independent of citrate, CitB, and CitZ, and to maximal levels during anaerobic growth with citrate as the sole carbon source. Similar to the other citrate fermentation enzymes, CitX synthesis was apparently subject to catabolite repression.Many species of enterobacteria, such as Klebsiella pneumoniae and Escherichia coli, are able to utilize citrate under anoxic, fermentative conditions. Whereas K. pneumoniae can grow with citrate as the sole carbon and energy source (for a review, see reference 6), E. coli is dependent on the presence of an oxidizable cosubstrate (18), due to the lack of oxaloacetate decarboxylase. The initial step in all known citrate fermentation pathways is the Mg 2ϩ -dependent cleavage of citrate to acetate and oxaloacetate, a reaction catalyzed by citrate lyase (2, 10, 33).In K. pneumoniae, the structural genes for citrate lyase are part of the citCDEFG operon, which is located divergent to citS (Fig. 1). The proteins deduced from citC, citD, citE, and citF are citrate lyase ligase and the ␥, ␤, and ␣ subunits of citrate lyase, respectively (7). The citC operon is induced under anoxic conditions in the presence of citrate and Na ϩ ions. Its expression is strictly dependent on the citrate-sensing CitACitB two-component regulatory system (8,16,20) and is subject to catabolite repression, presumably by the cyclic AMP receptor protein (19).In E. coli, a citCDEFXG gene cluster between 13.9 and 14.2 min (Fig. 1) that exhibited high similarity to the K. pneumoniae citrate lyase cluster (5) but differed by the presence of an additional gene, designated citX (...

The genome of Klebsiella pneumoniae contains at least three different genes encoding citrate transporters. Recently, a third and hitherto unknown gene encoding a citrate transport system ( citW) was identified. Escherichia coli transformed with a plasmid expressing citW was able to grow on citrate as sole carbon and energy source, identifying CitW as a citrate carrier. In this report, we provide evidence that further specifies CitW as a Na(+)-independent citrate/citrate and citrate/acetate exchanger. Kinetic analysis of citrate uptake at different pH values identified Hcitrate(2-) as the transported citrate species, with a K(m) of 25 microM. Since citW is expressed under anoxic conditions and acetate is the main end-product of citrate fermentation in K. pneumoniae, citrate/acetate exchange might be its in vivo function. Sequence similarity searches identified CitW (454 amino acids, 48.15 kDa) as a member of the 2-hydroxycarboxylate transporter family (TC 2.A.24). The substrate specificity seems to partially contradict this phylogenetic classification, but appears logical with respect to the putative functional role of CitW in the citrate fermentation pathway of K. pneumoniae.