Fig. 2: Context-dependent ribosomal stalling induced by linezolid is countered by PoxtA.

a A heatmap of log₂-relative change in the 5Pseq coverage of wild-type E. faecalis upon linezolid treatment. The distance (in nucleotides, nt) from the 5ʹ of the sequenced mRNA fragments to synonymous codons encoding a specified amino acid is indicated on the x axis. The 0 nt position corresponds to the first nucleotide of the codon. Dark blue signifies decreased 5PSeq coverage upon linezolid treatment, yellow signifies increased coverage upon linezolid treatment. A specific increase in the 5Pseq coverage associated with synonymous codons located at –8 nucleotides is indicative of a ribosomal stall with the corresponding amino acid in the −1 position of the nascent chain, as illustrated schematically in panels b and c (ref. ³³). b, c Metagene analysis of 5Pseq libraries aligned to alanine codons. Linezolid-induced ribosomal stalling results in a specific increase in 5Pseq read counts 8 nt upstream of alanine codons, indicating a ribosomal stall with alanine residues in the −1 position (see schematics). Samples were prepared with E. faecalis harbouring the empty pCIE_spec vector (a, b) or upon poxtA expression (c, blue line), with and without linezolid treatment. Exponentially growing cells were treated with 4 μg/mL linezolid (final concentration) 10 min before harvest. All analyses were performed on pooled datasets from three biological replicates.