Identification of a Vibrio cholerae chemoreceptor that senses taurine and amino acids as attractants

Vibrio cholerae, the etiological agent of cholera, was found to be attracted by taurine (2-aminoethanesulfonic acid), a major constituent of human bile. Mlp37, the closest homolog of the previously identified amino acid chemoreceptor Mlp24, was found to mediate taxis to taurine as well as L-serine, L-alanine, L-arginine, and other amino acids. Methylation of Mlp37 was enhanced upon the addition of taurine and amino acids. Isothermal titration calorimetry demonstrated that a purified periplasmic fragment of Mlp37 binds directly to taurine, L-serine, L-alanine and L-arginine. Crystal structures of the periplamic domain of Mlp37 revealed that L-serine and taurine bind to the membrane-distal PAS domain in essentially in the same way. The structural information was supported by characterising the in vivo properties of alanine-substituted mutant forms of Mlp37. The fact that the ligand-binding domain of the L-serine complex had a small opening, which would accommodate a larger R group, accounts for the broad ligand specificity of Mlp37 and allowed us to visualise ligand binding to Mlp37 with fluorescently labelled L-serine. Taken together, we conclude that Mlp37 serves as the major chemoreceptor for taurine and various amino acids.

classical biotype and 45 in El Tor. In a previous study, we named the mlp genes (from mlp1 to mlp45) according to their locations in the chromosomes of the V. cholerae El Tor biotype 21 . Such complex chemotaxis systems have been found in various bacteria other than E. coli 22 . We have shown that Mlp24 (VC2161 in the El Tor biotype and VC0395_A1741 in the classical biotype), formerly called McpX 14 , is a chemoreceptor for various amino acid attractants such as l-arginine, l-proline and l-serine 21 . Deletion of the mlp24 gene, however, does not eliminate taxis toward amino acids, suggesting that at least one additional amino acid chemoreceptor exists.
In this study, we examined bile taxis of a classical biotype strain of V. cholerae and found that taurine (2-aminoethylsulfonate; NH 2 CH 2 CH 2 SO 3 H) serves as a strong attractant, primarily at elevated temperature. Considering its chemical similarity to amino acids, we suspected that taurine might be sensed by some of the amino acid chemoreceptors. Indeed, we found that Mlp37, the closest homolog of Mlp24, serves as a chemoreceptor for taurine as well as serine, arginine and other amino acids. We determined the crystal structures of the ligand-binding domain of Mlp37 in complex with l-serine and taurine to reveal the molecular mechanism enabling Mlp37 to accommodate these structurally distinct ligands, l-serine and taurine. Taking advantage of the structural information, we succeeded, for the first time, in labelling a bacterial chemoreceptor, in this case Mlp37, with a fluorescent serine ligand in vivo.

V. cholerae is attracted by taurine, a major constituent of bile. Consistent with a previous report
showing that an El Tor biotype strain exhibits chemotaxis to bile 15 , we found that the classical biotype strain O395N1 in the O1 serogroup, showed an attractant response to bile (Fig. 1a). In contrast, the E. coli strain RP437 and the Vibrio parahaemolyticus strain LM4070 did not show any significant response to bile ( Supplementary  Fig. S1a,b). We examined which ingredient(s) of bile act as attractants, testing cholic acid, deoxycholic acid, taurine, taurocholic acid and taurodeoxycholic acid. Glycine, another component of bile, was excluded as it was already known to attract V. cholerae 21 and E. coli 23 . Taurine elicited a strong attractant response from V. cholerae (Fig. 1b), but it did not attract E. coli, V. parahaemolyticus, or Vibrio alginolyticus ( Supplementary Fig. S1g-i). It should be noted that the latter three species showed robust attractant responses to l-serine ( Supplementary  Fig. S1d-f). Taurocholic acid and taurodeoxycholic acid also served as weak attractants, whereas cholic acid and deoxycholic acid did not ( Fig. 1b and Supplementary Fig. S1c). These results suggest that taurine is a key constituent of bile that attracts V. cholerae.
Identification of a chemoreceptor involved in taurine taxis of V. cholerae. The chemical nature of taurine (2-aminoethanesulfonate) and the broad ligand specificity of the amino acid chemoreceptor Mlp24 21 prompted us to examine whether that receptor can also sense taurine. We first examined methylation of Mlp24 (67.7 kDa) upon addition of taurine. Methylation of an MCP increases its mobility upon SDS-PAGE, which can be detected by immunoblotting 24 . Unlike l-serine, a high concentration (10 mM) of taurine enhanced methylation only slightly ( Supplementary Fig. S2d) 21 . Furthermore, all the other functional and biochemical assays we carried out showed that the contribution of Mlp24 to taurine taxis, if any, is limited ( Supplementary Fig. S2a-c). We therefore turned our attention to Mlp37, the closest homolog of Mlp24. The mlp37 gene (VCA0923 in El Tor/ VC0935_0316 in classical) encodes a polypeptide with a calculated molecular mass of 70.3 kDa. Mlp37 has 49% identity and 63% similarity to Mlp24 in amino acid sequence and like Mlp24, contains a CACHE domain 25 and a pre-CACHE motif 21 . We found that the N-terminal extension of 28 amino acid residues is missing in Mlp37 orthologs in other Vibrio species (Supplementary Fig. S3). We therefore deemed this extension dispensable for function and cloned the 'truncated' coding region of Mlp37 (residues 29 to 652; 67.1 kDa) into the vector pAH901 21  sequence encoding the FLAG tag was fused to the sequence encoding the C-terminus of Mlp37 to yield plasmid pMlp37. The V. cholerae strain O395N1 was transformed with pMlp37. Resulting transformants were tested for methylation of the Mlp37-FLAG protein. Mlp37 became methylated in the presence of taurine, l-serine ( Fig. 2a), glycine, l-alanine, l-cysteine or l-arginine, whereas l-glutamate, one of the weakest attractants, had no effect ( Supplementary Fig. S4a,b). These results suggest that Mlp37 mediates attractant responses to taurine, l-serine and a number of other amino acids. We therefore hypothesised that Mlp37 plays a major role in taurine taxis. mlp37 is required for taxis to taurine and amino acids. To confirm the role of Mlp37 in chemotaxis, we constructed the mlp37-deleted strain Vmlp37 (O395N1 ∆mlp37). In the capillary assay for chemotaxis, the mutant strain showed greatly reduced responses toward taurine (Fig. 2b). Its tactic responses to each of the twenty proteinogenic amino acids are shown in Table S1. Chemotactic responses of the ∆mlp37 mutant to l-serine, l-alanine, l-cysteine, l-arginine, l-asparagine, l-threonine, l-lysine and l-valine and glycine were markedly decreased. Most of these defects were complemented by expressing Mlp37 in trans from plasmid pMlp37 ( Fig. 2c and Supplementary Table S1). These results, together with the methylation results ( Fig. 2a), strongly argue for the involvement of Mlp37 in attractant responses to taurine and various amino acids.
Binding of taurine and amino acids to the periplasmic fragment of Mlp37. To examine whether attractants bind directly to the periplasmic domain of Mlp37, we employed isothermal titration calorimetry (ITC) with a periplasmic fragment of Mlp37 (residues 58-303; named Mlp37p). The titration curves of purified Mlp37p (10 μ M) with taurine ( Fig. 3a) show direct binding with a dissociation constant (K d ) of 3.2 μ M, calculated by assuming that an Mlp37p monomer binds one taurine molecule. ITC analyses also showed that l-serine, l-alanine and l-arginine bind directly to Mlp37p with K d values of 3.6 μ M, 2.7 μ M and 5.6 μ M, respectively (Fig. 3b, Supplementary Fig. S4c,d). In contrast, l-glutamate, which was not sensed by Mlp37 in the capillary assay, did not show appreciable binding to Mlp37p ( Supplementary Fig. S4e). The affinity of Mlp37p for l-arginine is equivalent to that of Mlp24p, and the affinities for l-serine and l-alanine are one order of magnitude higher than those for Mlp24p 21 . These results demonstrate that Mlp37 is a chemoreceptor for taurine, l-serine, l-alanine and l-arginine.   26 . This structure belongs to a family of bacterial sensor domains 27,28 . It provides two potential ligand-binding pockets per monomer (the membrane distal and proximal ones are hereafter referred to as pockets I and II, respectively). The 3C8C structure was solved in a complex with l-alanine, in which alanine was bound to pocket I. Among the known ligands for Mlp37, only taurine is not an l-amino acid. Although the structure of taurine is quite distinct from that of amino acids, its dissociation constant is comparable to those for l-serine and l-alanine. To elucidate the ligand recognition mechanism of Mlp37, we  Table S2). The taurine complex, like the l-alanine complex, forms a dimer with a neighbouring molecule in the asymmetric unit. In contrast, the l-serine complex forms a dimer with a molecule related by crystallographic two-fold symmetry (Supplementary Fig. S5c-e). In spite of these differences in molecular packing, the dimer structures are basically identical (Supplementary Fig. S5a-e). α 2 and the C-terminal region of α 1 stabilise the dimer by forming an up-and-down four-helix bundle structure with its dimer mate ( Fig. 4a,b). Taurine and l-serine are bound only in pocket I in the crystal structures. The amino acid backbone atoms of bound l-serine are located at essentially the same positions as those of the bound l-alanine molecule in the 3C8C structure (Fig. 4c,d). The amino group of l-serine is in contact with Tyr-170, Asp-201 and Asp-172, which are triangularly arranged around it. The carboxyl group of l-serine interacts mainly with the guanidino group of Arg-152. The indole NH of Trp-154 and the main-chain amino group of Ser-173 also contribute to the binding of the carboxyl group of the l-serine ligand. These interactions are exactly conserved in the l-alanine complex (Fig. 4c,d). The side-chain hydroxyl group of l-serine is recognised by Asp-172 and the indole NH of Trp-141 (Fig. 4d).
The binding of taurine is remarkably similar to that of l-serine (Fig. 4d,e and Supplementary Fig. S5f). The amino group of taurine lies at the equivalent position of that of l-serine and interacts with Tyr-170, Asp-201 and Asp-172. Two oxygen atoms of the sulfonate group are located in the corresponding positions to the carboxyl oxygen atoms of l-serine, but they are out from the plane formed by the guanidino group of Arg-152. Thus, the indole NH of Trp-147 seems to contribute to the interaction in conjunction with the guanidino group of Arg-152, the indole NH of Trp-154 and the main-chain amino group of Ser-173. The remaining oxygen of the sulfonate is located near the hydroxyl oxygen of l-serine, and it interacts with the indole NH of Trp-147 and a water molecule bound to Asp-172, the indole NH of Trp-141, and the main-chain amino group of Ala-174. These structural features are essentially the same in the two subunits of the dimeric Mlp37p taurine complex ( Supplementary  Fig. S5g). The residues in contact with taurine can be superimposed onto the corresponding residues of the l-serine complex ( Supplementary Fig. S5f), with root mean square deviations of about 0.3 Å (0.314 for A-subunit and 0.265 for B-subunit), indicating that taurine mimics l-serine in Mlp37. The entrance of Pocket I of one subunit in the l-alanine bound dimer is in a fully closed conformation that embraces the ligand (Supplementary Fig. S6). In contrast, that of the other subunit has an opening almost identical to those in l-serine and the taurine complexes. The distance between the indole nitrogen of Trp-141 and one of the carboxyl oxygen atoms of Asp-172 in the closed conformation is 3.2 Å, indicating that Trp-141 in the loop connecting β 2 and α 4 interacts directly with Asp-172 in the loop connecting β 4 and β 5. In contrast, these residues in the l-serine and taurine complexes are much further apart, because Trp-141 and Asp-172 interact with the hydroxyl group of the l-serine ligand and with a water molecule in the taurine complex. As a result, the entrances to pocket I in the l-serine and taurine complexes are slightly open. Such an opening would allow larger amino acid R groups of other amino acids to stick out from the pocket without significantly affecting interactions with the amino acid backbone atoms, a situation that could explain the ability of Mlp37 to sense a diversity of amino acids. The opening, however, is not wide enough to allow passage of an attractant molecule itself. Thus, ligand binding must involve some type of open-to-closed conformational change in pocket I.

Mutational analysis of attractant recognition.
To examine the in vivo roles of the residues in contact with the ligands (Trp-141, Trp-147, Arg-152, Trp-154, Tyr-170 Asp-172 and Asp-201), we constructed mutant receptors with individual alanine replacements at each residue. Ser-173 was not tested because ligand interaction involves its main chain. The mutant receptors were expressed in strain Vmlp201, which lacks mlp24 and mlp37 and therefore shows only residual tactic responses to amino acids. Although the expression levels of all of the mutant proteins were essentially the same as that of the wild-type protein ( Supplementary Fig. S4e), all showed defects in mediating taxis toward l-alanine, l-serine and taurine (Fig. 4f). The most profound defects were observed at the lowest attractant concentration tested (1 mM in the capillary) (Fig. 4f). Cells expressing the Y170A and D201A proteins showed impaired chemotaxis even at higher attractant concentrations (10 or 100 mM in the capillary), suggesting that recognition of the amino group by these residues is essential for ligand binding. It should be noted that cells respond to attractants diffusing out from the mouth of the capillary; therefore, the attractant concentration the cells encounter when migrating up the gradient are much lower than those in the capillary. Cells expressing other mutant proteins showed nearly wild-type performance at high attractant concentrations (10 or 100 mM in the capillary), implying that these mutants have reduced affinities for the attractants. Each mutation had similar effects on responses to the three attractants tested, as was expected from the similarities in the crystal structures. These results reinforce the importance of these residues in recognition of the attractants, l-alanine, l-serine and taurine.
The crystal structures suggest that pocket II is not involved in attractant sensing, as was shown for Mlp24 21 and Bacillus subtilis chemoreceptors 29,30 . To confirm this hypothesis, we substituted alanine for two of the well-conserved potential ligand-binding residues in pocket II, Y221A and H234A that are well conserved among the related amino acid chemoreceptors of different species. In contrast to the pocket I mutations, the pocket II mutations had mild to negligible effects ( Supplementary Fig. S7a,b), demonstrating that recognition of taurine and amino acids involves pocket I, but not pocket II.

Observation of Mlp37 with a fluorescently labelled ligand. The small openings in the pocket I bind-
ing sites for serine and taurine prompted us to test whether localisation of Mlp37 can be visualised in vivo with a fluorescently labelled amino acid: l-serine 5(6)-carboxyfluorescein ester ( Supplementary Fig. S7d, hereafter referred to as Ser-FAM), in which carboxyfluorescein is linked to the hydroxyl group of l-serine through an ester bond. When treated with Ser-FAM, fluorescent spots were observed at the poles of ∆mlp24 ∆mlp37 (Vmlp201) cells expressing Mlp37 from pMlp37, but not for cells carrying the empty vector (Fig. 5a,b). Pre-incubation with a 10-fold molar excess of l-serine or taurine before Ser-FAM labelling dramatically decreased the occurrence of polar fluorescent spots. By contrast, pre-incubation with l-glutamate, one of the weakest attractants, did not affect Ser-FAM labelling ( Fig. 5a,b). These results suggest that l-serine and taurine compete with Ser-FAM for binding to Mlp37. To confirm that the polar fluorescent spots correspond to Ser-FAM bound to Mlp37, we constructed a plasmid encoding Mlp37-TagRFP (named pKRB116). This fluorescent fusion protein expressed in strain Vmlp201 (∆mlp24 ∆mlp37) supported essentially wild-type chemotaxis toward serine ( Supplementary  Fig. S7b) and showed polar localisation (Fig. 5c). When treated with Ser-FAM, cells expressing Mlp37-TagRFP (Vmlp201/pKRB116) showed Ser-FAM spots that coincided with Mlp37-TagRFP (Fig. 5c). These results not only confirm the mechanism of ligand recognition by Mlp37 but also provide, for the first time, a tool to visualise ligand binding to a bacterial chemoreceptor in vivo.

Discussion
This study demonstrated that the classical biotype of V. cholerae shows attractant chemotaxis to taurine. This finding is consistent with the previous report that the El Tor biotype of V. cholerae is attracted by bile 15 , as taurine is a major component of bile. Taurine taxis can also account for the previous observation that bile enhances the motility of V. cholerae in semisolid agar 12 .
Several lines of evidence support the conclusion that the MCP-like protein Mlp37 mediates taxis to taurine and various amino acids. First, the deletion of mlp37 severely affected taxis to taurine and several amino acids and those defects were complemented by a plasmid expressing the mlp37 gene. Second, methylation of Mlp37 was enhanced in the presence of taurine and various amino acids. Third, ITC measurements with a periplasmic fragment of Mlp37 (Mlp37p) demonstrated direct binding of Mlp37p to taurine as well as several amino acids.
The crystal structures of Mlp37p in a complex with attractant ligands argue strongly that pocket I, the membrane-distal of the tandem PAS-like domains in Mlp37p, undergoes a substantial (open-to-closed-type) conformational change upon ligand binding. The l-serine complex, in which the hydroxyl group of the ligand points toward the small opening of pocket I, reveals the molecular basis for the diversity of amino acids that can be sensed by Mlp37. An enlarged opening could accommodate the longer R groups of l-arginine, l-lysine and l-glutamine. Remarkably, taurine and l-serine binding involve the same set of Mlp37 residues with almost identical three-dimensional arrangements. These observations readily explain why the dissociation constant of taurine is close to that of l-serine. The taurine complex structure also suggests why taurocholic acid and taurodeoxycholic acid act as weak attractants, whereas cholic acid and deoxycholic acid do not. The amino group of taurine lies near the entrance of the binding pocket, and the entrance is slightly open (Supplementary Fig. S6). In taurocholic acid and taurodeoxycholic acid the acidic groups are linked to the amino group of taurine. If the pocket entrance opens a little more, it could accommodate the tauroconjugates by binding the taurine moiety in the pocket while leaving the cholic and deoxycholic moieties outside of the pocket. However, the binding affinity may be greatly reduced because some of the interactions with the amino group of taurine would be lost in this configuration.
The slightly open conformation of pocket I in the serine complex enabled us to visualise ligand binding in vivo with a fluorescently labelled amino acid. Binding of Ser-FAM to Mlp37, which can be outcompeted by excess amounts of serine or taurine, validates the structural information. It also provides a novel tool to study the cell biology of bacterial chemotaxis.
The ligand complex structures and the mutational analyses demonstrate that only one of the two potential ligand-binding pockets (the membrane-distal pocket I) is involved in ligand binding by Mlp37. This observation is consistent with previous reports on the related amino acid chemoreceptors, McpB of B. subtilis 29 and Mlp24 of V. cholerae 21 . Some histidine kinases with two tandem PAS-like domains have also been reported to bind their ligands through pocket I 31,32 . There has been no evidence that pocket II in any MCP or histidine kinase with tandem PAS-like domains binds small ligands except that the membrane-proximal PAS-like domain of McpC interacts with lipid-anchored ligand-binding proteins 30 .
What is the physiological relevance of taurine taxis? Taurine is abundant in the intestines of humans and other vertebrates, including fish. Marine invertebrates have high concentrations of taurine that serves as an osmolyte that regulates tissue osmolarity 33 . Vibrio species are very often associated with fish and aquatic invertebrates 34 . Chemoattraction to taurine could therefore enhance fitness of V. cholerae when it is associated with various hosts, particularly marine organisms. The fact that cultivation of the wild-type strain at 37 °C enhanced taurine chemotaxis dramatically (and l-serine chemotaxis to a lesser extent) ( Supplementary Fig. S1j,k), however, argues strongly that taurine chemotaxis is advantageous primarily in the intestines of a mammalian host rather than in marine/estuarine animals. Because bile is excreted from the gallbladder to the small intestine, taxis to taurine could also play a role in pathogenicity, as has been shown for chemotaxis to amino acids 21 . It is possibe that a taurine gradient allows V. cholerae to remain in the small intestine, which is their preferred site of colonisation in humans, for a longer time. It has been argued that bile serves as a repellent of V. cholerae and that this behaviour would help the bacteria to escape from the lumen by penetrating through the protective glycocalyx of the mucous that overlays the epithelium 3,9 . Chemotaxis toward higher concentrations of taurine would be disadvantageous in this respect. However, the mucus layer of the small intestine contains various amino acids 15 , which should attract V. cholerae cells and facilitate their migration through the mucosa toward the epithelium. Upon penetration into the mucus layer or adherence to epithelial cells, an unidentified sensor could detect a set of amino acids (asparagine, arginine, glutamate and serine) to turn on the ctxAB operon that encodes cholera toxin, as has been discussed previously 21,35 .
Our finding that V. cholerae can respond to taurine as well as various amino acids raises the intriguing possibility that the pathogen senses other host factors to migrate toward favourable environments as it passes through the gastrointestinal tract. Indeed, V. cholerae has more than forty MLPs, most of them without known functions. Comprehensive studies on the chemotaxis behaviour of V. cholerae within the host would extend our understanding of the mechanisms that underlie infection by, and the virulence of, this serious world-wide pathogen.

Methods
Bacterial strains. The classical biotype strain O395N1 is wild type for chemotaxis (Che + ) and its derivative VcheA2 lacks cheA2 19 is defective in general chemotaxis (Che -). Strains Vmlp24 (∆mlp24) and Vmlp201 (∆mlp24 ∆mlp37) are derivatives of O395N1 lacking mlp24 and/or mlp37 21 . Vmlp37 (∆mlp37) was constructed similarly in this study. The E. coli strain RP437 36 is Che + . The V. parahaemolyticus strain LM1017 37 and the V. alginolyticus strain YM4 38 are wild type for polar flagellation and chemotaxis but defective in lateral flagellation (Pof + Laf − Che + ). The E. coli strain HCB436 39 lacks the methylesterase CheB and the methyltransferase CheR as well as the chemoreceptors.
Construction of plasmids. Plasmid pMlp37 was constructed as follows. From genomic DNA of strain O395N1, the region encoding Mlp37 missing the N-terminal extension (see Results for details) was amplified by PCR to introduce restriction sites (EcoRI and SphI) at the 5′ and 3′ ends, respectively. The resulting DNA fragment was digested with these restriction enzymes and cloned into similarly digested pAH901 21 , yielding pMlp37, which encodes the FLAG-tagged Mlp37. Site-directed mutagenesis of mlp37 was carried out with QuickChange II Site-directed Mutagenesis Kit (Agilent Technologies/Stratagene, CA), using pMlp37 as a template.
The DNA fragment encoding the entire periplasmic domain (residues 58-303) of Mlp37 was amplified and subcloned into the expression vector pGEX-6P-2 so that the Mlp37-encoding sequence was fused in-frame to the 3′ of the GST-encoding sequence to yield pGEX-Mlp37p.
For construction of a plasmid encoding Mlp37-TagRFP, the TagRFP coding region was amplified by standard PCR using plasmid pTagRFP-C (Evrogen) as a template with primers designed for cloning and introducing a 3× Gly linker at the N-terminus of TagRFP. The amplified fragment was cloned into plasmid pTWV228 (Takara Bio) to yield pKRB112 expressing free TagRFP with the linker. The mlp37 gene was amplified using pMlp37 as a template and was cloned into plasmid pKRB112 to yield pKRB116 expressing the Mlp37-TagRFP fusion. Expression of the resulting proteins was verified by immunoblot using anti-TagRFP antibody (Evrogen).
All the cloned and mutated genes were verified by DNA sequencing.
Capillary assay. Chemotactic ability was examined by a capillary assay as described previously 21  Isothermal titration calorimetry (ITC). Purification of GST-fused Mlp24p and Mlp37p (encoded in plasmid pGEX-Mlp24p and pGEX-Mlp37p) and titrations of the periplasmic fragments with ligands using a VP-ITC micro-calorimeter (MicroCal Inc., Northampton, MA) were carried out essentially as described previously 21 Crystallisation, X-ray data collection and structure determination of the Mlp37p complexes with taurine and serine. Crystallisation of the Mlp37p complexes with l-serine and taurine was performed by the hanging-drop vapour-diffusion technique. Crystallisation drops were prepared by mixing Mlp37p solution containing 10 mM l-serine or 10 mM taurine with an equal volume of a reservoir solution. Initial screening was carried out using the following screening kit: Wizard I and II, Cryo I and II (Emerald Biostructures) and Crystal Screen I and II (Hampton Research), and then the conditions were optimised. Crystals appeared within 3 days. The final crystallisation conditions are summarised in Table S2. The taurine complex crystal belongs to an orthorhombic space group of P2 1  . The data were processed with MOSFLM 41 and scaled with SCALA 42 . Initial phase was calculated by molecular-replacement using the Phenix programme suite 43 with the Mlp37p-alanine complex structure (PDB ID: 3C8C) as a search model. The atomic model was constructed with Coot 44 and refined with Phenix to 1.8 Å for the l-serine complex and to 1.95 Å for the taurine complex. During the refinement process, iterative manual modifications were performed. The final refinement R factor and the free R factor of the l-serine complex were 18.7% and 21.4%, respectively. The Ramachandran plot indicated that 95.9% and 4.1% residues were located in the most favourable and allowed regions, respectively. The refinement R factor and the free R factor of the taurine complex were converged to 19.0% and 23.7%, respectively. The Ramachandran plot indicated that 92.9% and 7.1% residues were located in the most favourable and allowed regions, respectively. Data collection and refinement statistics are summarised in Supplementary Table S2.
Fluorescence microscopy. Cells were grown at 30 °C in tryptone glycerol (TG) broth [1% tryptone, 0.5% NaCl, 0.5% (w/v) glycerol] supplemented with 50 μ g/ml ampicillin. Overnight cultures were diluted 50-fold into fresh TG medium and incubated for 4 h at 37 °C. Cells were harvested by centrifugation at room temperature and resuspended in 100 μ l of TMN buffer [50 mM Tris-HCl (pH-7.4), 5 mM glucose, 100 mM NaCl] and 100 μ M Ser-FAM [l-serine 5(6)-carboxyfluorescein ester] (Eurofins Genomics Inc., Tokyo) was added. If necessary, non-labelled l-serine or taurine (final concentration: 1 mM) was added to TMN buffer. Cells were then incubated for 30 min at 37 °C, and washed once with TMN buffer. An aliquot of the cell suspension was spotted onto a MAS-coated glass slide (Matsunami Glass Inc., Osaka). Cells were then observed under a fluorescence microscope (Olympus IX71) equipped with a 100× oil-immersion objective lens. GFP and TagRFP were visualised using fluorescence mirror units, U-MNIBA3 and U-MWIG3 (Olympus), respectively. All images were recorded and processed by using a cooled charge-coupled-device camera ORCA-ERII (Hamamatsu Photonics) and the software Metamorph ver. 7.6 (Universal Imaging).