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

Tomonori Iwama,Yasuaki Ito,Hisaaki Aoki,Hiroshi Sakamoto,Shuzo Yamagata,Keiichi Kawai,Ikuro Kawagishi

doi:10.1074/jbc.m601038200

Tomonori Iwama, Yasuaki Ito + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m601038200

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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 metal-citrate sensing, and the mutated residues are located in or near the alpha1-alpha2 and alpha3-alpha4 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.

Highlights

The rotational sense of the flagellar motor (4 –9)
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], there is no direct evidence that Tcp directly binds citrate
Citrate Binds Directly to Tcp—It has not been unambiguously determined whether citrate binds directly to the Tcp protein like aspartate to Tar or through a certain binding protein like maltose to Tar through MBP

Summary

RESULTS

Each mutant Tcp protein was expressed in the receptorless E. coli strain HCB339 (⌬MCP), and the resulting cells were examined for the abilities of responding to citrate alone and magnesium in the presence of citrate by the capillary accumulation assay (Fig. 3). In the absence of citrate, cells with any Tcp receptor showed a small diffused round-shaped colony (data not shown). It is considered that the difference in colony shape between cells expressing the R68C receptor and those expressing wild-type Tcp may be related to the ability of sensing magnesium or magnesium-citrate. The candidates were further subjected to swarming assays with tryptone semisolid agar in the absence of citrate to examine whether the mutants retained general receptor functions such as repellent sensing, transmembrane signaling, and adaptation.

Sensing abilities of the mutated Tcp receptors

Tryptone swarming ability

DISCUSSION