<i>Escherichia coli</i> Reporter Strains Allow for the In Vivo Evaluation of Recombinant Elongation Factor Protein (EF-P)

Abstract

Background: Elongation factor protein (EF-P) in bacteria helps ribosomes to incorporate contiguous proline residues (xPro) into proteins. In this way, EF-P rescues ribosomes from stalling at these xPro motifs. Whereas EF-P deficiency is lethal for some species, others show reduced virulence or generally lower growth rates, such as Escherichia coli (E. coli). EF-P needs to be post-translationally modified to gain full functionality. Methods: We constructed E. coli K-12 mutant strains with deletion of the serA gene leading to an auxotrophy for L-serine. Then, we engineered a 6xPro motif in the recombinant serA gene, which was then chromosomally inserted under its native promoter. Furthermore, mutant strains which were deleted for efp and/or epmA (encoding the EF-P modification protein EpmA) were engineered. Results: Δefp, ΔepmA, and Δefp/ΔepmA double mutants showed already significantly reduced growth rates in minimal media. ΔserA derivatives of these strains were complemented by the wt serA gene but not by 6xPro-serA. ΔserA mutants with intact efp were complemented by all serA-constructs. Chromosomal expression of the recombinant efp gene from E. coli or from the pathogen, Staphylococcus aureus (S. aureus), restored growth, even without epmA expression. Conclusions: We provide a novel synthetic reporter system for in vivo evaluation of EF-P deficiency. In addition, we demonstrated that both EF-P-E. coli and EF-P-S. aureus restored the growth of a 6xPro-serA: Δefp, ΔepmA strain, which is evidence that modification of EF-P might be dispensable for rescuing of ribosomes stalled during translation of proline repeats.

Keywords

• elongation factor protein EF-P
• polyproline stretch
• ribosome stalling
• <i>serA</i> gene
• efp
• epmA