The following strains of E. coli were used in this study: MG1655 (K-12 strain), BL21 (B strain, ATCC #BAA-1025), C122 (C strain, NCTC122, Public Health England), Crooks (ATCC #8739), and W (ATCC #9637). All strains were grown in lysogeny broth Miller (LBM) or on LBM agar, supplemented with carbenicillin (Sigma-Aldrich #C9231) and/or spectinomycin (Sigma-Aldrich #S4014).
A phylogenetic tree of 16 diverse E. coli strains with Escherichia fergusonii as an outgroup was created using the Phylogeny.fr analysis pipeline. Sequences were collected from NCBI GenBank: BL21 (TaKaRa) (NZ_CP010816.1), Crooks ATCC 8739 (NC_010468.1), K-12 substr. W3110 (NC_007779.1), K-12 substr. MG1655 (NC_000913.3), Nissle 1917 (NZ_CP007799.1), W strain ATCC9637 (NC_017635.1), C strain C122 (NZ_LT906474.1), O6:K15:H31 536 (NC_008253.1), 55989 (NC_011748.1), O127:H6 E2348/69 (NC_011601.1), IAI39 (NC_011750.1), O157:H7 str. Sakai (NC_002695.1), UMN026 (NC_011751.1), CFT073 (NC_004431.1), SMS-3-5 (NC_010498.1), Escherichia fergusonii ATCC 35469 (NC_011740.1). MLST analysis of sequences was performed by MLST2.0 run by the Center for Genomic Epidemiology, DTU (https://cge.cbs.dtu.dk/services/MLST/) using the MLST E. coli#1 configuration (adk, fumC, gyrB, icd, mdh, purA, recA genes) and default settings. MLST allele sequences for each of the 16 strains were downloaded, concatenated, and submitted to results from the Phylogeny.fr Phylogeny Analysis pipeline (http://www.phylogeny.fr/). In this pipeline, a MUSCLE multiple sequence alignment of concatenated MLST alleles was performed. The alignment was then processed by Gblocks and phylogeny analysis performed by PhyML with tree drawing by TreeDyn.
Reporter plasmids were designed to contain superfolder GFP (sfGFP) with different start codons. A 1971bp gBlock (Integrated DNA Technologies, Inc) was designed and synthesized that contained the metY gene from MG1655 (GenBank: U00096.3) with an anticodon mutation (CAU > CUA). A pULTRA-CNF vector (Addgene #48215) with a Clo DF13 origin of replication and a spectinomycin-resistance cassette was used as the plasmid backbone. The amber initiator plasmid was assembled via in vitro homologous assembly. Cultures for plasmid construction were grown in LBM supplemented with spectinomycin. Plasmids were isolated from transformants, sequence verified, and stored in elution buffer (10 mM Tris-Cl, pH 8.5).
Culture growth conditions for assay measurements
Transformants from glycerol stocks were streaked on LBM plates with appropriate antibiotics: spectinomycin (100 µg/mL) for amber initiator plasmid and/or carbenicillin (100 µg/mL) for reporter plasmids and grown overnight at 37°C. Individual colonies were inoculated in 2 mL of LBM containing appropriate antibiotics and grown overnight at 37°C shaking at 200 rpm. After overnight growth, each culture was diluted 1:100 into 400 µL LBM in a 96-well deep well plate containing either 1 mM IPTG to induce, or 2% glucose (w/v) to repress, metY(CUA) expression.
Measurements of fluorescence intensity from the amber initiator plasmid system was performed as before with the following modifications: Absorbance was measured at 600 nm (OD600) to estimate culture density, followed by fluorescence (excitation = 485 nm, emission = 520 nm, bandwidth = 9 nm) measured at a single gain setting (high gain sensitivity = 111), on a PHERAstar FSX (BMG Labtech) plate reader.
Strains were grown overnight as described above. Cell densities were measured and cells were passaged to a starting OD600 of 0.1 into fresh 200 µL LBM with appropriate antibiotics and either 1 mM IPTG or 2% glucose (w/v). Cultures were grown in a flat-bottom 96-well plate sealed with gas-permeable seal (Sigma-Aldrich # Z763624) at 37°C (300 rpm) while culture light scattering (OD600) over time was measured on a SPECTROstar NANO (BMG Labtech) plate reader at 5 min intervals for 10 h. Our analysis method was adapted from previous work and used to compare ratios of growth rate and maximal cell density for individual strains.
The growth rate and maximal cell density (max OD600) were calculated using the R package Growthcurver v.0.2.1. Ratios of growth rates and maximal cell densities were determined for strains with the amber initiation plasmid under induced growth condition (1 mM IPTG) versus repressed growth condition (2% glucose (w/v)).
Whole Genome Alignment
The following nucleotide sequences from GenBank were analyzed using Mauve whole genome alignment performed with default parameters: K-12 substr. MG1655 (NC_000913.3), BL21 (NZ_CP010816.1), C strain C122 (NZ_LT906474.1), W strain ATCC9637 (NC_017635.1), and Crooks ATCC 8739 (NC_010468.1).
metY Multiple Sequence Alignment
Gene sequences were extracted from GenBank genome sequences and aligned using MUSCLE algorithm using default parameters.