An orthogonal amber initiator tRNA functions similarly across diverse Escherichia coli laboratory strains

Abstract

Translation initiation is a sequential process involving interactions between the 30S small ribosomal subunit, initiation factors and initiator tRNA. The Escherichia coli K-12 strain is unique in the Escherichia because it has two different initiator tRNA sequences, tRNAfMet1 encoded by the metZWVgenes and tRNAfMet2 encoded by the metY gene. A mutant of the metY gene was previously made where the anticodon sequence, responsible for specifying the start codon where translation initiation begins, was changed so that it bound to the amber stop codon UAG instead of the usual AUG start codon. This amber initiator tRNA has already been shown to be functional in the K-12 strain, but it is unclear whether it would function in other strains normally lacking the tRNA variant. In this work, we transformed E. coli K-12, and four other generally regarded as safe (GRAS) laboratory strains, with a plasmid expressing the amber initiator tRNA and evaluated its functionality and growth effects on the bacteria. We performed these tests because, despite these strains all belonging to E. coli phylogenetic group A, it is well known that there is significant variation between even closely related E. coli strains in their metabolism, transcriptional response to exogenous DNA expression and rates of amber stop codon suppression. We found that the amber initiator functions similarly across the five strains, effectively initiating translation at the orthogonal UAG start codon and that it had modest growth-slowing effects in the Crooks, W, and K-12 strains. The five tested E. coli strains in this work (K-12, B, C, W, and Crooks) are important workhorses of academic and industrial research and development. The path is now clear to deploy the amber initiator tRNA into these five strains to precisely control gene expression. Figure 1. An orthogonal amber initiator tRNA functions similarly across diverse Escherichia coli laboratory strains. (A) Phylogenetic tree of 16 diverse E. coli strains. The tree has drawn from MUSCLE multiple sequence alignment of concatenated multisequence locus typing (MSLT) gene sequences (adk, fumC, gyrB, icd, mdh, purA, recA). Alignment processed by Gblocks and phylogeny analysis by PhyML. The tree was drawn by TreeDyn. Bootstrapping values (red) displayed on tree generated by MrBayes v3.2 and represent the confidence level of the displayed branching topology, with 1 being the highest level of confidence. The scale bar represents the number of nucleotide substitutions per site. Phylogeny.fr used for analysis pipeline. Five E. coli laboratory strains analyzed in this work shown by bold green text. See figure S1 for Mauve whole genome alignment between E. coli strains in green. (B) metZWV and metY loci in E. coli strains. Initiator tRNAfMet1 and tRNAfMet2 differ in sequence by a single nucleotide at position 46 in the variable loop, where tRNAfMet1 has 7mG and tRNAfMet2 has an A. The dashed lines represent similar genomic loci in each strain while boxes schematically represent the variation of tRNAfMet1 and tRNAfMet2 placement within each locus. See figure S2 for metY multiple sequence alignment. (C) Initiator tRNA structure showing anticodon pairing with the mRNA start codon. Left inset, wild-type initiator tRNAfMet2 pairing with canonical AUG start codon. Right inset, amber initiator tRNA(CUA) pairing with amber stop codon UAG. (D) Amber initiator tRNA(CUA) can initiate translation from UAG start codons in five common laboratory E. coli lineages. Normalized expression levels from sfGFP reporter beginning with one of three start codons (AUG, UAG, or GCC) with wild-type tRNAfMet2 or amber initiator tRNA(CUA) expression either repressed or induced. Each bar displays the average of three biological replicate measurements. Error bars represent one standard deviation. See figure S3 for amber initiator expression plasmid and fluorescent reporter plasmid maps. (E) tRNA(CUA) expression results in higher UAG versus AUG start codon initiation in K-12, C and B strains versus W and Crooks strains. Each data point displays the average of three biological replicate measurements. Error bars represent one standard deviation. A diagonal line indicates the equal amount of normalized fluorescence from UAG and AUG-initiating reporters. (F) tRNA(CUA) expression results in decreased fitness in Crooks, W, and K-12 strains. All strains harbor the pULTRA::tac-metY(CUA) plasmid. Ratios on the Yand Xaxis were calculated by dividing growth rate and max OD600 from cultures induced with 1 mM IPTG to those repressed with 2% glucose. Each data point is the average of three biological replicates. Error bars represent one standard deviation.