Translation initiation in Escherichia coli relies on recruitment of initiation factors 1-3 (IF1-IF3), the 30S ribosomal subunit, and the initiator tRNA (tRNAfMet) to a messenger RNA (mRNA). Several excellent reviews with more detailed information on the process of bacterial translation initiation have been published.

E. coli encodes two sequence variants of the initiator tRNA, called tRNAfMet1 and tRNAfMet2. The difference between these sequence variants is one nucleotide at position 46 where tRNAfMet1 is a G (modified to 7mG in tRNA) and tRNAfMet2 is an A. Both the tRNAfMet1 (metZWV genes) and tRNAfMet2 (metY gene) variants are present in the E. coli K-12 strains, but other strains of E. coli lack the tRNAfMet2 and instead have a fourth copy of tRNAfMet1 in the metY loci in their genomes (Fig. 1B). tRNAfMet2 has been introduced previously into E. coli B strains with no apparent deleterious effect.

The amber initiator $\textrm{tRNA}{fMet2 \atop CUA}$ (Fig. 1C) was created by Varshney and RajBhandary from a modified E. coli K-12 metY gene encoding a tRNA that can initiate translation at the ‘amber’ stop codon (UAG) instead of the usual AUG start codon. A major activity of biological engineers is the construction of genetic parts and devices that tell the cell when to turn on a gene, resulting in protein production. Predicting whether engineered genetic constructs will work across different organisms is non-trivial and often results in non-functional or incorrectly functioning systems. Differences in basal levels of amber suppression within common K-12 strains is already known. Expressing the amber initiator tRNA shows great promise as a way of programming orthogonal translation initiation at UAG sites in engineered genetic constructs, but it is unknown whether $\textrm{tRNA}{fMet2 \atop CUA}$ will function outside of the K-12 lineage of E. coli.

To enable the adoption of this powerful amber initiator tRNA system to drive programmable gene expression, we wanted to know how transportable it is across E. coli strains? In particular, how would the amber initiator functions in laboratory E. coli strains that normally do not contain the tRNAfMet2 sequence variant?

We began our investigation by building a paired set of plasmids to both express the amber initiator tRNA as well as measure the translation initiation efficiency at AUG and UAG codons. The amber initiator plasmid features a recoded metY gene (metY(CUA)) that expresses $\textrm{tRNA}{fMet2 \atop CUA}$. The amber initiator tRNA features an altered anticodon sequence that is the reverse complement of the desired UAG start codon (Fig. 1C). The plasmid contains an IPTG-inducible tac promoter and the Clo DF13 (medium copy) origin of replication expected to function across diverse E. coli strains (Fig. S3). We designed three reporter plasmids to measure translation initiation events caused by expression of $\textrm{tRNA}{fMet2 \atop CUA}$ from the amber initiation plasmid. The reporter plasmids use super-folder green fluorescent protein (sfGFP) with three start codon variants: UAG to measure translation initiation efficiency from the amber initiator tRNA, AUG as a positive control, and GCC as a negative control (Fig. S3). The sfGFP reporter is driven by a constitutive T5 promoter and has the ColE1 origin of replication, which is compatible with the Clo DF13 origin, so both plasmids can be simultaneously maintained within one E. coli cell.

We next transformed the amber initiator and reporter plasmids into five common generally recognized as safe (GRAS) laboratory strains of E. coli from a diverse set: MG1655 (K-12 strain), BL21 (B strain), C122 (C strain), ATCC 9637 (W strain), and ATCC 8739 (Crooks strain). All five E. coli strains are designated risk group 1 but arise from distinct lineages (Fig. 1A).

After transforming both amber initiator plasmid and reporter plasmids into all strains, we measured bulk fluorescence under repressing and inducing conditions (Fig. 1D). We observed similar trends in all tested strains. As expected, the reporter strain with sfGFP(GCC) start codon did not appreciably change expression due to $\textrm{tRNA}{fMet2 \atop CUA}$ expression. In contrast, the sfGFP(UAG) reporter had fluorescence levels similar to sfGFP(GCC) when under $\textrm{tRNA}{fMet2 \atop CUA}$ repressed conditions, but increased markedly when $\textrm{tRNA}{fMet2 \atop CUA}$ expression was induced (Fig. 1D). On average across the strains we saw an 88-fold increase in fluorescence upon $\textrm{tRNA}{fMet2 \atop CUA}$ expression with MG1655 showing the smallest increase and C122 showing the largest increase. As noted previously, the combination of lack of expression when repressed, along with a large increase after tRNA induction, could be useful for conditionally expressing toxic proteins due to lack of translational leakage from the UAG stop codon in the absence of the $\textrm{tRNA}{fMet2 \atop CUA}$.

Surprisingly, $\textrm{tRNA}{fMet2 \atop CUA}$ induction also caused increased fluorescence from the normal sfGFP(AUG) start codon reporter in all strains. We would not have expected the increase because it has been well established that the wild-type tRNAfMet2 initiator does not initiate translation from UAG stop codons. We would therefore not expect the reciprocal codon-anticodon pairing between the AUG start codon and the amber initiator $\textrm{tRNA}{fMet2 \atop CUA}$ to result in productive translation initiation. We next expressed wild-type metY from the pULTRA plasmid and found no fluorescence increase from the UAG reporter as expected, and a similar magnitude increase in fluorescence from the AUG reporter as was seen with the amber initiator. We speculate that $\textrm{tRNA}{fMet2 \atop CUA}$ expression has caused an increase in the cellular concentration of ribosomes and this has resulted in increased translation from the sfGFP(AUG) reporter. Support for this idea comes from a recent paper showing evidence that expressing initiator tRNA in E. coli increases mature ribosome levels and accelerates rRNA processing and our recent work showing the amber initiator can increase ribosome protein abundance in cells. We can speculate that expressing $\textrm{tRNA}{fMet2 \atop CUA}$ has a similar effect on ribosomal maturation and rRNA processing in the cell, considering the presence of identical identity elements (aside from the anticodon sequence) in both initiator tRNAs.

We next compared the effects of expressing the amber initiator on relative increases of sfGFP(AUG) and sfGFP(UAG) reporter expression to determine the suitability of the strains using the amber initiator (Fig. 1E). The results show the strains are all relatively similar, with E. coli C122 being the best and Crooks being the worst for selectively expressing UAG start codons over AUG initiation.

To determine the fitness effects of expressing the amber initiator $\textrm{tRNA}{fMet2 \atop CUA}$ on the different E. coli strains, we measured growth rate and maximal optical density differences when the $\textrm{tRNA}{fMet2 \atop CUA}$ transcription was induced versus repressed (Fig. 1F). We observed that the Crooks strain had both its growth rate and maximum optical density moderately reduced when the $\textrm{tRNA}{fMet2 \atop CUA}$ was induced, while the W and K-12 strains had a major reduction in growth rate but not maximum optical density (Fig. 1F). The B and C strain growth characteristics were unaffected by $\textrm{tRNA}{fMet2 \atop CUA}$ induction. The patterns of fitness changes following $\textrm{tRNA}{fMet2 \atop CUA}$ induction seemed to mirror the strain’s relative abilities to produce UAG-initiated reporter protein (Fig. 1E). That is, the B and C strains produced the most UAG reporter fluorescence compared to AUG reporter fluorescence, while the Crooks and W strains produced the least (Fig. 1E). The exception to this pattern is the K-12 strain, which produced the third highest amount of UAG reporter per AUG reporter fluorescence despite the growth rate being reduced by the most (~50%) (Fig. 1E and 1F). The reason for the difference in K-12 may be due to interference between the amber $\textrm{tRNA}{fMet2 \atop CUA}$ and the genomic copy of wild-type tRNAfMet2 in this strain, which is missing in the other strains examined in this study (Fig. 1B). Further work will need to be done to uncover the reasons for these strain-specific fitness differences.

In this work, we have shown that the amber initiator $\textrm{tRNA}{fMet2 \atop CUA}$ can be expressed in a fully functional form in five different strains of generally regarded as safe laboratory E. coli. Furthermore, the genomic absence of the tRNAfMet2 (metY) gene variant does not seem to have a major bearing on the effectiveness of the amber initiator $\textrm{tRNA}{fMet2 \atop CUA}$ function within those strains (B, C, W, and Crooks) (Fig. 1B). Lastly, this work points towards possible uses for the amber initiator tRNA to enable expression of toxic proteins in strains of E. coli, such as BL21, that are often used for the production of protein for structural biology, investigation of protein function, and for therapeutics.

Bacterial strains

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).

Phylogenetic Tree

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 (). 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.

Plasmid construction

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.

Fluorescence measurements

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.

Fitness analysis

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.

PRJ was supported by the Molecular Sciences Department, Faculty of Science & Engineering, and the Deputy Vice-Chancellor (Research) of Macquarie University. RMV is a recipient of the Macquarie University Research Excellence PhD scholarship (MQRES). PFY was supported by a summer research scholarship from the Molecular Sciences Department of Macquarie University.

This study was completed in accordance with Macquarie University biosafety committee approval, application ID 5201600567.