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

Abstract

The chemoreceptor Tcp of Salmonella enterica serovar Typhimurium can sense citrate and a metal-citrate complex as distinct attractants. In this study, we tried to investigate the molecular mechanism of this discrimination. That citrate binds directly to Tcp was verified by the site-specific thiol modification assays using membrane fractions prepared from Escherichia coli cells expressing the mutant Tcp receptors in which single Cys residues were introduced at positions in the putative ligand-binding pocket. To determine the region responsible for the ligand discrimination, we screened for mutations defective in taxis to magnesium in the presence of citrate. All of the isolated mutants from random mutagenesis with hydroxylamine were defective in both citrate and metalcitrate sensing, and the mutated residues are located in or near the ␣1-␣2 and ␣3-␣4 loops within the periplasmic domain. Further analyses with site-directed replacements around these regions demonstrated that the residue Asn 67 , which is presumed to lie at the subunit interface of the Tcp homodimer, plays a critical role in the recognition of the metal-citrate complex but not that of citrate. Various amino acids at this position differentially affect the citrate and metal-citrate sensing abilities. Thus, for the first time, the abilities to sense the two attractants were genetically dissected. Based on the results obtained in this study, we propose models in which the discrimination of the metal-citrate complex from citrate involves cooperative interaction at Asn 67 and allosteric switching.Citrate is a constituent of the tricarboxylic acid cycle operating under aerobic conditions, which provides many of the precursors required for biosynthesis and energy metabolism (1). Therefore, this compound is of physiological significance for many organisms including bacteria. Salmonella enterica serovar Typhimurium (hereafter referred to as Salmonella) has citrate transport systems responsible for utilization of citrate as a sole source of carbon and energy, but such a system is absent in the closely related species Escherichia coli (1). Consistent with the citrate utilizing ability, Salmonella shows positive chemotaxis toward citrate, whereas E. coli is not attracted by citrate (2, 3). In general, chemotaxis of E. coli and Salmonella involves an elaborate signal transduction system, in which the transmembrane chemoreceptors, in response to various chemoeffectors, regulate the activity of the histidine kinase CheA and hence that of the response regulator CheY that controls the rotational sense of the flagellar motor (4 -9). 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 citr...

McpQ is a specific citrate chemoreceptor that responds preferentially to citrate/metal ion complexes

“…McpQ is not the first specific citrate receptor reported. In contrast to E. coli , Salmonella typhimurium is able to grow on citrate (Iwama et al ., ). This may be linked to the fact S. typhimurium shows chemoattraction towards citrate whereas E. coli is not attracted (Ingolia and Koshland, ).…”

Stimulus Perception in Bacterial Signal-Transducing Histidine Kinases

“…This can be exemplified by the sensors for C 4 -dicarboxylates: the C 4 -dicarboxylate is perceived by a PAS-like domain in DcuS from E. coli, a four-helix bundle domain in Tar (aspartate) MCP of E. coli, and the CACHE domain in the succinate sensing DctB of Rhizobium leguminosarum (6,114,218,219). Osmosensing is mediated by an even larger diversity of sensor proteins that even belong to completely different families: E. coli EnvZ is a prototypical periplasmic-sensing HK, while its functional homolog from Xenorhabdus nematophilus is a small intramembrane-sensing HK (see below).…”

Physiologically relevant divalent cations modulate citrate recognition by the McpS chemoreceptor

“…This receptor employs a TarH type of domain for citrate sensing (Interpro signature IPR003122) which forms a 4-helix bundle structure (Yeh et al, 1996). Iwama et al (2006) showed that this receptor binds citrate both in its free form and in complex with magnesium ions. The authors demonstrate that free and Mg 2þ bound citrate bind to the same site at Tcp but in two distinct manners involving different sets of amino acids.…”

“…CitA was found to bind free citrate and with some reduced affinity also the citrate-Mg 2þ complex. The third class of extracytoplasmic citrate binding proteins are chemoreceptors of which Tcp is the most studied example (Yamamoto and Imae, 1993;Iwama et al, 2000;Iwama et al, 2006). This receptor employs a TarH type of domain for citrate sensing (Interpro signature IPR003122) which forms a 4-helix bundle structure (Yeh et al, 1996).…”

“…Binding at Tcp occurred at the same site but in two distinct manners involving different sets of amino acids (Iwama et al, 2006). Root exudates contain a significant amount of bivalent cations, such as Ca 2þ and Mg 2þ (Lipton et al, 1987;Qin et al, 2007), which form a complex with citrate.…”