Identification of a Gene Cluster in Klebsiella pneumoniae Which Includes citX , a Gene Required for Biosynthesis of the Citrate Lyase Prosthetic Group

Abstract

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 (...

Transcriptional Regulation of the Citrate Gene Cluster of Enterococcus faecalis Involves the GntR Family Transcriptional Activator CitO

“…In a central position, we found the citD, citE, and citF genes, which encode the three citrate lyase subunits, ␥ (acyl carrier protein [ACP]), ␤ (citryl-ACP oxaloacetate lyase), and ␣ (acetyl-ACP: citrate ACP-transferase), respectively. In addition, in this region we identified the citC, citX, and citG accessories genes, which encode the acetate:SH-citrate lyase ligase, apo-citrate lyase phosphoribosyl-dephospho-coenzyme A transferase, and triphosphoribosyl-dephospho-coenzyme A synthase, respectively (7,8,(48)(49)(50).…”

The 2-Hydroxycarboxylate Transporter Family: Physiology, Structure, and Mechanism

“…In the opposite direction and upstream of the citS gene are the genes for the biosynthesis of the citrate lyase complex in the same order as observed in the genomes of the lactic acid bacteria discussed above (CDEFXG). In the K. pneumoniae cluster, the citX gene is missing, but it is located distantly in the genome next to the gene coding for the citrate/acetate exchanger CitW (125). The location of citX correlates with the presence of citW in the genome.…”

The Tricarballylate Utilization ( tcuRABC ) Genes of Salmonella enterica Serovar Typhimurium LT2

“…As noted above, the tcuB gene was formerly known as citB and had an undetermined role in citrate catabolism, and the tcuC gene was previously identified as citA (a citrate transporter) (35,36). On the basis of the work reported here, the names of these genes have been changed to tcuB and tcuC, respectively, to reflect their true role in S. enterica physiology and to distinguish them from other cit genes in the literature (32). The inability of E. coli to use tricarballylate was used to identify the full complement of S. enterica genes required for the catabolism of this tricarboxylic acid.…”