The stringent response does not influence ribosome pausing in Bacillus subtilis

The stringent response represses translation and is activated when cells enter the stationary phase and intracellular amino acid levels drop. Bacillus subtilis, a well-known industrial enzyme production organism, secretes most enzymes during the stationary phase. However, translation pausing can affect protein folding and production. Here, we examined whether the stringent response affects ribosome pausing in B. subtilis. To investigate this, genome-wide ribosome profiling was performed using a stringent response mutant that overexpressed the α-amylase AmyM. Blocking the stringent response did increase overall protein synthesis, however AmyM production was reduced. Ribosome profiling revealed that this was not caused by a reduction in translation. In fact, absence of the stringent response did not markedly influence ribosome pausing. Late into the stationary phase an increased ribosome pausing at tryptophan codons was observed, suggesting a depletion of tryptophan. A strong suppression of tryptophan biosynthesis and acquisition genes, under control of the trp RNA-binding attenuation protein TRAP, likely accounts for this. Why this occurs is unclear since TRAP does not belong to the stringent response regulon of B. subtilis. Finally, the genome-wide ribosome profiles revealed several operon genes with unusually low translation activities, illustrating the importance of translation initiation as a regulatory element of expression.