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

Abstract

The genes of Salmonella enterica serovar Typhimurium LT2 encoding functions needed for the utilization of tricarballylate as a carbon and energy source were identified and their locations in the chromosome were established. Three of the tricarballylate utilization (tcu) genes, tcuABC, are organized as an operon; a fourth gene, tcuR, is located immediately 5 to the tcuABC operon. The tcuABC operon and tcuR gene share the same direction of transcription but are independently transcribed. The tcuRABC genes are missing in the Escherichia coli K-12 chromosome. The tcuR gene is proposed to encode a regulatory protein needed for the expression of tcuABC. The tcuC gene is proposed to encode an integral membrane protein whose role is to transport tricarballylate across the cell membrane. tcuC function was sufficient to allow E. coli K-12 to grow on citrate (a tricarballylate analog) but not to allow growth of this bacterium on tricarballylate. E. coli K-12 carrying a plasmid with wild-type alleles of tcuABC grew on tricarballylate, suggesting that the functions of the TcuABC proteins were the only ones unique to S. enterica needed to catabolize tricarballylate. Analyses of the predicted amino acid sequences of the TcuAB proteins suggest that TcuA is a flavoprotein, and TcuB is likely anchored to the cell membrane and probably contains one or more Fe-S centers. The TcuB protein is proposed to work in concert with TcuA to oxidize tricarballylate to cis-aconitate, which is further catabolized via the Krebs cycle. The glyoxylate shunt is not required for growth of S. enterica on tricarballylate. A model for tricarballylate catabolism in S. enterica is proposed.Tricarballylate (1,2,3-propanetricarboxylate) is structurally related to citric acid; hence, its catabolism is thought to proceed via the tricarboxylic acid (Krebs) cycle (Fig. 1). Tricarballylate is the causative agent of grass tetany, a disease in ruminants characterized by a pronounced magnesium ion deficiency. Reports in the literature have established a strong correlation between high levels of trans-aconitate (trans-1-propene-1,2,3-tricaboxylate) in the rumen with the incidence of the disease (6). In the rumen, trans-aconitate is rapidly converted to tricarballylate in a single reductive step (Fig. 2) (25). It is not clear how tricarballylate causes grass tetany; however, it is known that tricarballylate is a good chelator of magnesium ions and that it enhances excretion of this important divalent metal ion (33). In addition, tricarballylate is known to be a potent inhibitor of aconitase, a key enzyme of the Krebs cycle (26,29,33). This effect on aconitase makes tricarballylate particularly toxic to organisms that rely on the Krebs cycle for energy generation and are unable to catabolize tricarballylate into nontoxic metabolites.In the rumen, Selenomonas ruminantium is the primary producer of tricarballylate (25). Not surprisingly, this bacterium has been the target of studies aimed at blocking the accumulation of this compound. The bacterium Acidaminococcus ferm...

Bacterial ApbC Protein Has Two Biochemical Activities That Are Required for in Vivo Function

“…Stains lacking apbC and yggX (or purF) are thiamine auxotrophs likely because of poor cluster occupancy of ThiH/ThiC (47,48,80). Complementation of a third growth defect required function of the high flux tricarballylate catabolic metabolic pathway (35,37,49,50). Strains lacking yggX and apbC are unable to use tricarballylate as a carbon and energy source, and presumably, this is from poor [Fe-S] cluster occupancy of TcuB (35).…”

Archaeal ApbC/Nbp35 Homologs Function as Iron-Sulfur Cluster Carrier Proteins

“…An abpC mutant cannot grow with tricarballylate as a carbon and energy source, which may be due to a defect in assembling or repairing [4Fe-4S] clusters in the membrane-bound TcuB protein (21,22). ApbC is a 40-kDa cytoplasmic protein with Walker A and B nucleotide-binding domains and two conserved carboxy-terminal cysteine residues separated by two amino acids (Cys-X-X-Cys).…”

Decreased Transport Restores Growth of a Salmonella enterica apbC Mutant on Tricarballylate

“…Heterologous expression of tcuC in MG1655 results in sufficient transport of citrate to allow growth (33,42). Thus, the growth of E. coli strain MG1655 on citrate provided a means of measuring the transport activities of TcuC variants.…”

“…Genetic analysis determined that a Tn10d(cat) insertion in metT was ϳ40% cotransducible by phage P22 with the remaining four suppressor mutations, placing them near the tcu operon. Sequence analysis determined that each of the four mutations was a single base change in the tcuC gene, encoding the tricarballylate transporter (33). The mutations resulted in variant TcuC proteins with G137S (tcuC51), C198S (isolated twice) (tcuC54), and W319R (tcuC52).…”

“…Importantly, the tricarballylate utilization genes tcuABC are carried in an operon, and a model for tricarballylate metabolism has been described (31,33). In this model (Fig.…”