Translation control by altered start codon usage as a means of modulating the general stress response and virulence...

In the food-borne pathogen Listeria monocytogenes, SigB is the central regulator of general stress response (GSR) and it mediates host entry by promoting acid resistance and epithelial cell attachment. However, mutations can readily arise to disable regulators of SigB (Rsb proteins), which suggests a considerable genetic plasticity in the GSR. To further investigate this, we defined the complete genome sequence of a clinical isolate and elucidated how sequential mutations within sigB operon (rsbX N77K and rsbU Q317*) impacted fitness through modulation of SigB activity. To investigate the plasticity of the GSR, we followed its genetic adaptation to lethal acidic challenge (mimicking the selective pressure encountered during entry into the host). Acid resistance developed rapidly and all 6 acid resistant derivatives (ARDs) selected for analysis had acquired mutations in rsbW, which encodes an antagonist of SigB that suppresses SigB activity during non-stress conditions. These mutations resulted in non-canonical start codons (rsbWATG to rsbWATA or rsbWATT) or premature translation termination (rsbW-) and all were found to result in increased SigB activity. A translational reporter assay demonstrated distinct differences in translation efficiency between three start codons: ATG > ATA > ATT, suggesting that a perturbation of RsbW:SigB stoichiometry alters SigB activity. We then analysed start codon usage for all conserved genes in 60,692 L. monocytogenes genomes. This analysis revealed flexible usage of start codons associated with genetic clades in 39 conserved genes, 13 of which are involved in virulence and stress response. Further, we show that flexible use of canonical start codons (ATG and GTG) can also mediate different levels of expression of virulence and stress response genes. Taken together, we show the genetic plasticity of GSR regulation in a model pathogen, and highlight the importance of translational control as a means of fine-tuning gene expression during short-term adaptation and long-term evolution for optimal fitness.