Mutational Analysis of Ligand Recognition by Tcp, the Citrate Chemoreceptor of Salmonella enterica Serovar Typhimurium

Abstract

The chemoreceptor Tcp mediates taxis to citrate. To identify citrate-binding residues, we substituted cysteine for seven basic or polar residues that are chosen based on the comparison of Tcp with the well-characterized chemoreceptors. The results suggest that Arg-63, Arg-68, Arg-72, Lys-75, and Tyr-150 (and probably other unidentified residues) are involved in the recognition of citrate.The closely related enteric bacteria Escherichia coli and Salmonella enterica serovar Typhimurium have multiple transmembrane receptors that mediate chemotactic responses to amino acids, non-PTS sugars, and other attractants and repellents (2,11,25,26,34,35). Some receptors (Tar for aspartate, Tsr for serine, and Trg for ribose and galactose) are found in both species. Others are species specific. Belonging to the latter class is the Salmonella serovar Typhimurium-specific chemoreceptor Tcp, which mediates taxis to citrate and a divalent cation-citrate complex and away from phenol (39). Salmonella serovar Typhimurium, but not E. coli, can utilize citrate as a sole carbon source.Ligand recognition by Tar and Tsr has been studied extensively using mutagenesis (13, 21, 22, 30, 38), chemical modification (14, 15, 17), X-ray crystallography (28, 40), and computer simulation (19). Mutations at the aspartate-binding residues of Tar cause defects in the aspartate-sensing ability without affecting the repellent-sensing ability or other receptor functions (13,21,30,38), although in some cases the maltosesensing ability is also affected due to a partial overlap of the binding sites for aspartate and the complex of maltose and maltose-binding protein (13). Tcp is homologous to Tar and Tsr, but it recognizes the non-amino acid ligand citrate. Identification of the citrate-binding residues of Tcp should further our understanding of the molecular logic underlying ligand recognition in this family of receptors.The residues involved in ligand binding in Tar and Tsr ( Fig. 1A and B) are located in the two helices (␣1 and ␣4) that extend through the cytoplasmic membrane as the first and the second transmembrane regions (TM1 and TM2), respectively (28, 40). In Tar, three Arg residues (residues 64, 69, and 73) within the helix ␣1 interact with the ␣-or ␤-carboxyl groups of aspartate (40) (Fig. 1A and B). The corresponding Arg residues of Tsr are predicted to interact with the ␣-carboxyl or the hydroxyl group of serine (19,22) (Fig. 1A and B). This triplet is perfectly conserved in Tcp as residues 63, 68, and 72 (Fig. 1B), which are expected to interact with the carboxyl groups or the hydroxyl group of citrate. Tyr-149 of Tar, which is located near the top of the helix ␣4 and interacts with the carboxyl groups of the ligand via water molecules (40), is also conserved in Tcp as Tyr-150. Because citrate has no amino group (Fig. 1C), it is reasonable that the Thr residue (154 in Tar and 156 in Tsr), which interacts with the amino group of aspartate or serine, is not conserved in Tcp (39). Since citrate has a polar hydroxyl group and three carboxyl gr...

Differential Recognition of Citrate and a Metal-Citrate Complex by the Bacterial Chemoreceptor Tcp

“…Strain HCB436 (⌬tsr-7021 ⌬(tar-cheB)2234 ⌬trg-100 zbd::Tn5 thr leu his met rpsL136) (33), which lacks CheB and A, alignment of the amino acid sequences around the ligand-binding site of the bacterial chemoreceptors of Salmonella (indicated with subscript S) and E. coli (indicated with subscript E ). Bold letters indicate the residues involved in recognition of citrate that have been studied in the previous work (14). Underlined letters of Tcp indicate residues involved in the ligand recognition, which was demonstrated by random mutagenesis in this study.…”

“…Plasmid pSU18 (34), which is derived from pACYC184, was used as a vector carrying any mutant tcp gene throughout this work. The plasmids pSNC11, pSNC12, pSNC13, pSNC14, pSNC15, pSNC16, pSNC17, and pSNC18, all of which have been constructed in the previous work (14), carry the tcp, tcp-R63C, tcp-R68C, tcp-R72C, tcp-K75C, tcp-R78C, tcp-Y150C, and tcp-K157C genes, respectively, under the control of the lac promoter.…”

“…27 and 28). In the previous study, we substituted Cys for the four conserved basic or polar residues, as well as three other basic ones, in the Tcp periplasmic domain, which is otherwise devoid of Cys (14). The results demonstrated that the five residues (Arg 63 , Arg 68 , Arg 72 , Lys 75 , and Tyr 150 ) are involved in the recognition of citrate.…”

“…The difference in chemotactic ability of the two enterobacterial species corresponds to the presence and absence of the chemoreceptor Tcp, which mediates chemotaxis toward citrate and away from phenol (13). Although wild-type E. coli has no citrate transport system and cannot respond to citrate, E. coli cells expressing Tcp as a sole chemoreceptor respond to citrate (13,14). This suggests that the response to citrate does not require citrate transport or a periplasmic binding protein, unlike E. coli taxis to maltose, which requires maltose-binding protein (MBP) 2 in addition to the chemoreceptor Tar (15)(16)(17), although there is no direct evidence that Tcp directly binds citrate.…”

Salmonella enterica Serovar Typhimurium Colonizing the Lumen of the Chicken Intestine Grows Slowly and Upregulates a Unique Set of Virulence and Metabolism Genes

“…This suggests that no flagellin was produced in the chick lumen and, with the lack of expression of chemotaxis genes (cheAWZ, tcp, tsr), suggests that there is no major chemoattractant in the lumen which Salmonella bacteria would move toward. The downregulation in expression of tcp and tsr (41) suggests that neither citrate (tcp) nor serine (tsr) is present in the lumen. Stecher et al (74) have shown that motility increases closer to the mucosa in the inflamed mouse intestine, although flagellation was less important in the noninflamed gut.…”