E. coli strains and plasmids used in this study are listed in table S1. Because of its high toxicity, hipA was cloned into expression plasmids together with a Shine-Dalgarno sequence in which the consensus sequence of the Shine-Dalgarno, the spacer between the Shine-Dalgarno and the start codon, or the start codon was changed to decrease the translation efficiency and the toxicity of HipA (49). In table S1, sd8 indicates a consensus sequence AAGGAA with a spacer of eight nucleotides to the ATG start codon. Oligonucleotides used are listed in table S2.

pNDM220::hipA and pNDM220::hipA7. The hipA gene was amplified from pEG5 and hipA7 from pEG9 with primers OMS43 and OMS44. The polymerase chain reaction (PCR) products were digested with Eco RI and Bam HI and ligated with pNDM220 digested with the same enzymes. The resulting plasmids contain the hipA or hipA7 gene with a mitigated Shine-Dalgarno (sd8ATG) sequence downstream of the Plac promoter.

pMG25::hipA and pMG25::hipA7. The hipA gene was amplified from pEG5 and hipA7 from pEG9 with primers OMS41 and OMS42. The PCR products were digested with Eco RI and Bam HI and ligated with pMG25 digested with the same enzymes. The resulting plasmids contain the hipA or hipA7 gene with a mitigated Shine-Dalgarno (sd8ATG) sequence downstream of the Plac promoter.

pBAD33::6his hipA and pBAD33::6his hipA7. The hipA gene was amplified from pEG5 and hipA7 from pEG9 with primers OEG110 and OEG111. The PCR products were digested with Xba I and Sph I and ligated with pBAD33 digested with the same enzymes. The resulting plasmids contain the hipA or hipA7 gene with a mitigated Shine-Dalgarno (sd8ATG) sequence downstream of the PBAD promoter and the sequence that encodes six histidine residues at protein N terminus.

pBAD33::hipA S150A, pBAD33::hipA S150D, pBAD33::hipA S359A, and pBAD33::hipA S359D. Those mutants were amplified using a two-step PCR technique that consists of PCR amplification of each fragment upstream and downstream of the point mutation and then a third PCR mixing of both fragments to obtain hipA variant with the external primers OEG57 and OEG111. The hipA gene was amplified from pEG5 with primer pairs OEG57-OEG28 and OEG29-OEG111 to construct the S150A mutant, primer pairs OEG57-OEG242 and OEG241-OEG111 to construct the S150D mutant, primer pairs OEG57-OEG244 and OEG243-OEG111 to construct the S359A mutant, and primer pairs OEG57-OEG246 and OEG245-OEG111 to construct the S359D mutant. The final PCR products were digested with Xba I and Sph I and ligated with pBAD33 digested with the same enzymes. The resulting plasmids contain the hipA mutant gene with a mitigated Shine-Dalgarno (sd8ATG) sequence downstream of the PBAD promoter.

pET28a::gltX and pET28a::rplK. The gltX and rplK genes were amplified from MG1655 E. coli strain with primer pairs OMS14-OMS15 and OMS3-OMS4, respectively. The PCR products were digested with Nde I and Xho I and ligated with pET28a digested with the same enzymes. The resulting plasmids contain the gltX or rplK gene, together with the sequence upstream of the gltX or rplK gene that, at protein N terminus, encodes for MGSS, six histidine residues, and SSGLVPRGSHM.

pET28a::seqA. The seqA gene was amplified from MG1655 with primers OMS26 and OMS27. The PCR product was digested with Nco I and Xho I and ligated with pET28a digested with the same enzymes. The resulting plasmid contains the seqA gene together with the sequence upstream of the seqA gene that encodes for the MA sequence at protein N terminus and the sequence downstream of the seqA gene that encodes for the LE sequence and six histidine residues at protein C terminus.

The MG1655 rplK S102A and rplK S102D strains were constructed by replacing a chromosomal rplK gene that encodes for RplK with the rplK gene that encodes for phosphoablative mutant RplK-S102A or phosphomimetic mutant RplK-S102D. To construct the mutants, two or three point mutations were introduced into the wild-type rplK gene (gcg codon for rplK S102A and gat for rplK S102D instead of tcc codon) at the position that encodes for Ser102. Those mutants were amplified using a two-step PCR technique, as described above. The external primers used to obtain rplK variant were OMS20 and OMS21. rplK S102A was constructed using OMS20-OMS11 and OMS10-OMS21, and for rplK S102D variant, we used OMS20-OMS13 and OMS12-OMS21 (table S2). The gene constructs containing nusG, rplK S102A/rplK S102D, and rplA genes were cloned into the pKOV plasmid using Not I and Xba I restriction enzymes with primers OMS20 and OMS21, and allelic replacement was performed as previously described (50). The pKOV plasmid was a gift from G. Church (Addgene plasmid #25769). Colonies were screened for mutations using temperature switch PCR genotyping, as previously described (51) and confirmed by DNA sequencing.

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