The Escherichia coli citrate carrier CitT: a member of a novel eubacterial transporter family related to the 2-oxoglutarate/malate translocator from spinach chloroplasts

Abstract

Under anoxic conditions in the presence of an oxidizable cosubstrate such as glucose or glycerol, Escherichia coli converts citrate to acetate and succinate. Two enzymes are specifically required for the fermentation of the tricarboxylic acid, i.e., a citrate uptake system and citrate lyase. Here we report that the open reading frame (designated citT) located at 13.90 min on the E. coli chromosome between rna and the citrate lyase genes encodes a citrate carrier. E. coli transformed with a plasmid expressing citT was capable of aerobic growth on citrate, which provides convincing evidence for a function of CitT as a citrate carrier. Transport studies with cell suspensions of the transformed strain indicated that CitT catalyzes a homologous exchange of citrate or a heterologous exchange against succinate, fumarate, or tartrate. Since succinate is the end product of citrate fermentation in E. coli, it is likely that CitT functions in vivo as a citrate/succinate antiporter. Analysis of the primary sequence showed that CitT (487 amino acids, 53.1 kDa) is a highly hydrophobic protein with 12 putative transmembrane helices. Sequence comparisons revealed that CitT is related to the 2-oxoglutarate/malate translocator (SODiT1 gene product) from spinach chloroplasts and five bacterial gene products, none of which has yet been functionally characterized. It is suggested that the E. coli CitT protein is a member of a novel family of eubacterial transporters involved in the transport of di- and tricarboxylic acids.

FIG. 1

Cosubstrate-dependent citrate fermentation by E. coli. The enzymes involved in the conversion of citrate to succinate are (1) citrate lyase, (2) malate dehydrogenase, (3) fumarase, and (4) fumarate reductase.

FIG. 2

E. coli genes required for citrate fermentation and comparison with the corresponding K. pneumoniae genes. Genes shaded in dark gray represent those K. pneumoniae genes involved in citrate fermentation which are also present in E. coli; genes shaded in light gray are those present only in the E. coli or only in the K. pneumoniae cluster. All other indicated genes are presumably not directly involved in citrate fermentation.

FIG. 3

Amino acid sequence alignment of the E. coli (Ec) CitT protein with the 2-oxoglutarate/malate translocator from spinach (Spinacia oleraceae [So]) chloroplasts and five eubacterial gene products. Identical residues present in at least four of the sequences are framed in black; conservative exchanges are framed in gray. Asterisks indicate residues conserved in all sequences. Putative transmembrane helices within the CitT sequence are overscored. Details on the aligned sequences can be found in the text and in Table 1. Hi, Haemophilus influenzae; Bs, Bacillus subtilis; Hp, Helicobacter pylori.

FIG. 4

Uptake and efflux of [1,5-¹⁴C]citrate by cell suspensions of E. coli BL21(DE3) containing either pET24b or pET24-citT. The transport experiments were performed as described in Materials and Methods.

FIG. 5

Uptake and efflux of [1,5-¹⁴C]citrate by cell suspensions of E. coli BL21(DE3) containing pCitS_His-3. The transport experiments were performed as described in Materials and Methods.

FIG. 6

Uptake and efflux of dl-[1,4-¹⁴C]tartrate by cell suspensions of E. coli BL21(DE3) containing either pET24b or pET24-citT. The transport experiments were performed as described in Materials and Methods.