Regulation of the macrolide resistance ABC-F translation factor MsrD

Abstract

Antibiotic resistance ABC-Fs (ARE ABC-Fs) are translation factors that provide resistance against clinically important ribosome-targeting antibiotics which are proliferating among pathogens. Here, we combine genetic and structural approaches to determine the regulation of streptococcal ARE ABC-F gene msrD in response to macrolide exposure. We show that binding of cladinose-containing macrolides to the ribosome prompts insertion of the leader peptide MsrDL into a crevice of the ribosomal exit tunnel, which is conserved throughout bacteria and eukaryotes. This leads to a local rearrangement of the 23 S rRNA that prevents peptide bond formation and accommodation of release factors. The stalled ribosome obstructs the formation of a Rho-independent terminator structure that prevents msrD transcriptional attenuation. Erythromycin induction of msrD expression via MsrDL, is suppressed by ectopic expression of mrsD, but not by mutants which do not provide antibiotic resistance, showing correlation between MsrD function in antibiotic resistance and its action on this stalled complex.

Structure Elucidation and Interaction Dynamics of MefA-MsrD Efflux Proteins in Streptococcus pneumoniae: Impact on Macrolide Susceptibility

“…The mefA / E and msrD genes are typically present in an operon located in the MEGA. The operon is translated as a polycistronic mRNA (in the presence of erythromycin) from a single promoter that is situated around 350 bp upstream of the mefA gene . The mef ( A ) gene is often carried into phage φ1207.3.…”

Base composition at the start of the coding sequence controls the balance between translation initiation and mRNA degradation inE. coli

“…Plasmids were constructed by PCR amplification of the whole plasmids with the CloneAmp Hifi polymerase (Takara) and assembly of the insert part with the NEBuilder HiFi DNA Assembly kit. The yfp variants plasmids were constructed by amplification of the pMMB67EH (72) backbone with primers and amplification of the yfp gene (75,76) with primers that contain or not the modification of the first codon of the yfp or that add the different TBT sequences, the primers are described in Table 1. The same approach was used to insert the TBT sequence in the pET21 expressed test genes ycaQ, RSP_2139, SCO1897 and SRU_1983 that we have used in our previous study (13).…”

Global regulation via modulation of ribosome pausing by the ABC-F protein EttA

“…The pMMB plasmids used for target validation in the WT, Δ ettA , and C ettA strains, were derived from the low-copy plasmid pMMBpLlacO-1-67EH - . It allows controlled gene expression from the IPTG inducible PLlacO-1 promoter 33 with a transcription start that permits to retain the native 5’UTR of the inserted gene. All of EttA target genes, (except the mgtL_mgtA 1-5aa insert sequence, which was synthesized by the company TWIST bioscience) were PCR amplified from genomic DNA of the MG1655 strain with primers hybridizing at the transcription initiation site and to the stop codon of the ORF.…”

“…With the EF-P/eIF-5A protein that has been identified to rescue ribosomes stalled during the synthesis of polyproline-containing proteins [29][30][31] , ABC-F are the only factors known to bind to the ribosomal E site. Some ABC-F have a clear physiological function such as the antibiotic resistance ABC-F (ARE ABC-F) factors 9,10,12,[14][15][16][17][18][19][20]32,33 which provide resistance toward antibiotics that target the PTC and the nascent peptide exit tunnel (NPET) of the ribosome 9,10,12,16,17,19,20 . However, for most of the family, their physiological function remains unclear.…”

“…Some ABC-F have a clear physiological function such as the antibiotic resistance ABC-F (ARE ABC-F) factors 9,10,12,14–20,32,33 which provide resistance toward antibiotics that target the PTC and the nascent peptide exit tunnel (NPET) of the ribosome 9,10,12,16,17,19,20 . However, for most of the family, their physiological function remains unclear.…”