Multiple routes for non-physiological l-threonine uptake in Escherichia coli K-12

Abstract

In this study, we identified eight multicopy suppressors (yhjE, sdaC, ydgI, alaE, ychE, yqeG, proP, and yjeM) and three distinct classes of chromosomal mutations (lrp, marC, and cycA) capable of complementing the growth defect caused by threonine uptake deficiency in the sstT tdcC livKHMGF brnQ thrP strain. YhjE, SdaC, YdgI, AlaE, mutant MarC, and CycA exhibited measurable threonine-specific uptake activity in the in vitro assay. Phenotypic assays revealed that YhjE and SdaC were the main entry points for threonine in a strain lacking major threonine-specific permeases. A derivative of the threonine-auxotrophic sstT tdcC livKHMGF brnQ thrP mutant, harboring deletions of eight multicopy suppressors, exhibited significantly reduced fitness at subsaturating threonine concentrations and improved fitness at toxic threonine concentrations, indicating a defect in membrane permeability. These results may help guide the effective construction of threonine-producing strains, extend knowledge on the substrate preferences of SdaC, AlaE, and ProP, and provide clues for further studies on the exact substrate range of YhjE, YdgI, YjeM, YchE, MarC, and YqeG whose physiologically relevant functions have not yet been established.

Keywords: Escherichia coli; amino acid transporter; l-threonine uptake; membrane proteins; transmembrane transport.

Figure 1

Screening and analysis of the multicopy suppressors of threonine uptake defect. Threonine transport activity was measured as described under “Threonine uptake assay” in the Materials and Methods. “Thr” indicates l-threonine. (A) Phenotypic assay of the B1895 threonine-auxotrophic strain and its derivatives carrying pBR-ychE, pBR-yjeM, pBR-yhjE, pBR-alaE, pBR-proP, pBR-yqeG, pBR-ydgI, and pBR-sdaC plasmids, and the empty pBR322 vector on minimal plates with varying threonine concentrations. (B) Comparison of the phenotypes of B1895 and its derivatives lacking incremental combinations of the multicopy suppressors on minimal plates with varying threonine concentrations. (C) Comparison of threonine transport activity in the B2394 strain lacking the SstT, TdcC, ThrP, BrnQ, and LIV-I carriers with its derivatives overexpressing SdaC, YchE, YjeM, YhjE, AlaE, ProP, YqeG, and YdgI due to the presence of the corresponding plasmids. Measurements were performed using 800 μM l-threonine, and the reaction time was 1 min. The values shown are the average of three independent biological replicates. Error bars indicate standard deviation. p-values were calculated using two-tailed Student’s t-test with unequal variances. (D) Comparison of the growth fitness of B1895 and its derivative, B2818, lacking eight multicopy suppressors on minimal plates with toxic threonine concentrations.

Figure 2

Screening and analysis of chromosomal mutations that suppress threonine uptake defect. Threonine transport activity was measured as described under “Threonine uptake assay” in the Materials and Methods. “Thr” indicated l-threonine. (A) Phenotypic assay for the B1950 threonine-auxotrophic strain and its derivatives harboring mutations in marC, cycA, and lrp loci on minimal plates with varying threonine concentrations. (B) Comparison of threonine transport activity in the B1950 strain its derivatives harboring mutations in marC, cycA, and lrp loci. Measurements were performed using 2.5 mM l-threonine, and the reaction time was 1 min. The values shown are the average of three independent biological replicates. Error bars indicate standard deviation. p-values were calculated using two-tailed Student’s t-test with unequal variances. (C,D) MarC-YchE and CycA-ThrP alignments performed using the Clustal Omega algorithm (Sievers and Higgins, 2014) and UniProt web service (Bateman et al., 2025). The gray and red fillings indicate identical amino acid residues and the amino acid substitutions detected in the mutant derivatives of B1950 that can grow at a non-permissive threonine concentration, respectively. (E–H) Ternary structures of CycA and MarC predicted by AlphaFold2 (Jumper et al., 2021). The red filling indicates mutated residues. (E) CycA, side view. (F) CycA, top view. (G) MarC, side view. (H) MarC, top view.

Figure 3

Analysis of the lrp^T134A mutation phenotype. The threonine uptake measurement and in vivo luminescence assays were performed as described under “Threonine uptake assay” and “Measurement of in vivo luminescence and bacterial growth,” respectively, in the Materials and Method. “Thr” indicated l-threonine. (A) Phenotypic assay for the B2055 threonine-auxotrophic strain carrying the lrp^T134A allele and its derivatives harboring yhjE, proP, alaE, and yqeG deletions on minimal plates with varying threonine concentrations. (B) Quantification of yhjE promoter activity via bioluminescence measurement in strains carrying P_yhjE-luxCDABE transcriptional fusion and either the lrp^wt, lrp^T134A, or ∆lrp allele. The values shown are the average of three independent biological replicates. Horizontal error bars and filled area indicate standard deviation for OD₆₀₀ and RLU/OD₆₀₀, respectively. (C) Evaluation of AlaE activity in the B2055 strain carrying the lrp^T134A allele. Measurements were performed using 800 μM l-threonine, and the reaction time was 1 min. The values shown are the average of three independent biological replicates. Error bars indicate standard deviation. p-values were calculated using two-tailed Student’s t-test with unequal variances. (D) Comparison of the growth fitness of B1895 and its derivatives overexpressing either AlaE or SdaC on minimal plates supplemented with toxic threonine concentrations.

Figure 4

Phylogenic tree of the transport systems involved in threonine uptake. The tree with the highest log likelihood (−13131.09) is shown. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. All positions with less than 50% site coverage were eliminated. The indicated phylogenetic affiliations of transporters were determined according to the Transporter Classification Database (Saier et al., 2014).