Cloning and functional characterization of two bacterial members of the NAT/NCS2 family in Escherichia coli

Figures & data

Figure 1. ClustalW alignment of the coding sequences of YgfO (AAC75920; 466 codons), YicE (AAC76678; 463 codons), YgfU (Q46821; 482 codons), YgfQ (AAT48153; 455 codons), YcdG (AAC74091; 464 codons), YbbY (AAC73615; 435 codons), YjcD (AAC77034; 449 codons), YicO (AAC76687; 470 codons) and YieG (AAC76737; 445 codons) with functionally known proteins of NAT/NCS2 family (http://saier-144–164.ucsd.edu/tcdb/; for Lpe1, see Argyrou et al. [2001]; for Xut1, see Goudela et al. [2005]) showing the NAT/NCS2 signature-motif region. Principal substrates for functionally characterized homologues (including YgfO and YicE from this work) are given in parentheses. Positions of conserved [Q/E/P], N, G, T, and [R/K/G] residues are highlighted (see text for details).

Figure 2. Xanthine uptake activities of YgfO and YicE. E. coli T184 harbouring pT7–5/ygfO (upper panel) or pT7–5/yicE (lower panel) was grown aerobically at 37°C in complete medium to mid-logarithmic phase, induced with IPTG (0.5 mM) for 2 h, and assayed for transport of [³H]xanthine (external concentration 1 µM), as described in Methods. Assays were performed in the absence (closed triangles) or presence of CCCP, 5 µM (open triangles) or 30 µM (closed rectangles); in all cases, control assays performed with T184 harbouring pT7–5 alone yielded values indistinguishable from the ones of YgfO or YicE treated with CCCP, 30 µM (closed rectangles).

Figure 3. Substrate transport specificity of YgfO and YicE. E. coli T184 harbouring pT7–5/ygfO (upper panels) or pT7–5/yicE (lower panels) was grown aerobically at 37°C in complete medium to mid-logarithmic phase, induced with IPTG (0.5 mM) for 2 h, and assayed for transport of [³H]xanthine (rhombuses; panels on the left, X; panels on the right), [¹⁴C]uric acid (triangles; panels on the left, UA; panels on the right), [³H]hypoxanthine (HX; panels on the right) or [³H]uracil (URA; panels on the right), at the indicated external concentrations, as described in Methods. Initial uptake rates were measured at 5 and 10 sec, and maximal uptake values were taken from measurements over a 1 to 20-min incubation period. Control values obtained from T184 harboring vector pT7–5 alone (maximal uptake averaging 0.01, 0.02, 0.01, or 0.01 nmol/mg and 0.2, 1.2, 0.3, or 0.5 nmol/mg, for 1 µM and 100 µM of xanthine, hypoxanthine, uracil, or uric acid, respectively) were subtracted from the sample measurements in all cases. Data with standard deviation bars represent the means of three independent determinations.

Figure 4. Ligand recognition profiles of YgfO and YicE. E. coli T184 harbouring pT7–5/ygfO (upper panel) or pT7–5/yicE (lower panel) was grown, induced and assayed for transport of [³H]xanthine (1 µM), as described in the legend to Figure 2. Prior to initiation of the assay, cells were equilibrated with the indicated non-radiolabelled nucleobases (1 mM) for 5 min. Uptake rates were measured at 5 and 10 sec and control values obtained with T184 harboring pT7–5 alone were subtracted from the sample measurements in all cases. Results are presented as the percentage of transport activity retained relative to the activity of untreated positive controls, pre-equilibrated with solvent vehicle alone. Values represent the means of three determinations with standard deviations shown. Nucleobases and analogues used were: X, xanthine; U, uric acid; H, hypoxanthine; Ur, uracil; A, adenine; T, thymine; C, cytosine; G, guanine; Th, theophylline (1,3-dimethylxanthine); Cf, caffeine (1,3,7-trimethylxanthine); Ap, allopurinol; Op, oxypurinol; 1, 1-methylxanthine; 3, 3-mthylxanthine; 2, 2-thioxanthine; 6, 6-thioxanthine; 7, 7-methylxanthine; 8, 8-methylxanthine; 9, 9-methylxanthine; 8z, 8-azaxanthine; 9d, 9-deazaxanthine; 7d, 7-deazaxanthine; F, 5-fluoro-uracil; Al, allantoin; Im, imidazol.

Figure 5. Transport analysis and expression levels of YgfO-BAD and YicE-BAD. E. coli T184 harbouring pT7–5/ygfO, pT7–5/ygfO-BAD or pT7–5 alone (panels A and B) or pT7–5/yicE, pT7–5/yicE-BAD or pT7–5 alone (panel C) was grown, induced and assayed for transport of [³H]xanthine (1 µM), in the absence (A, C) or presence (B) of the indicated non-radiolabelled nucleobases (1 mM), as described in the legend to Figures 2 and 4. In parallel experiments, membrane fractions of induced T184 harbouring pT7–5/melY-BAD (as a control; Frillingos & Kaback, [2001]) (lane 1) or pT7–5/yicE-BAD (lane 2) or pT7–5/ygfO-BAD (lanes 3 and 4) were prepared and subjected to immunoblot analysis (100 µg of protein per lane) (panel D), as described in Methods. Proteins on the blot were probed both with anti-LacY-epitope antibody and with HRP-conjugated avidin, as indicated. Migration positions of prestained molecular weight standards (Bio-Rad, low range) are shown on the left (panel D). Nucleobases and analogues used in panel B for analysis of YgfO-BAD (dark-coloured bars) and YgfO (light-coloured bars) were: X, xanthine; U, uric acid; H, hypoxanthine; Ur, uracil; A, adenine; T, thymine; C, cytosine; G, guanine; Ap, allopurinol; Op, oxypurinol; 1, 1-methylxanthine; 3, 3-mthylxanthine; 2, 2-thioxanthine; 6, 6-thiolxanthine; 7, 7-methylxanthine; 8, 8-methylxanthine; 9, 9-methylxanthine; 8z, 8-azaxanthine.

Figure 6. IPTG-inducible expression and activity of YgfO-BAD. E. coli T184 harbouring pT7–5/ygfO-BAD or vector pT7–5 alone was grown aerobically at 37°C in complete medium, to mid-logarithmic phase, and induced with IPTG (0.5 mM) for the indicated periods of time. Part of the harvested cells was used to prepare membrane fractions and subjected to immunoblot analysis (100 µg protein per lane) using the anti-LacY-epitope antibody (upper panel), and part of them was resuspended in reaction buffer (35 µg of protein per 50 µL) to assay transport of [³H]xanthine (1 µM) (lower panel), as indicated. Prestained molecular weight standards (Bio-Rad, low range) are shown on the right of the upper panel.

Table I.  K_m and V_max values of YgfO and YicE for xanthine uptake¹.

Permease	Km(µM)	Vmax (nmol min^−1 mg ^−1 protein)	Vmax/Km (µL min^−1 mg ^−1)
YgfO	4.2±0.7	6.36±0.52	1514
YgfO-BAD	4.6±0.3	6.34±0.56	1378
YicE	2.9±0.5	0.59±0.14	203
YicE-BAD	3.8±0.6	0.85±0.06	224

¹E. coli T184 expressing the corresponding constructs were assayed for initial rates of xanthine uptake at 5 and 10 sec, in the concentration range of 0.1–100 µM, as described in the Methods section; negative control values obtained from T184 harboring vector pT7–5 alone (see Figure 2) were subtracted from the sample measurements in all cases. Kinetic parameters were determined from non-linear regression fitting to the Michaelis-Menten equation using Prism4; values represent the means of three independent determinations with standard deviations shown.

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