The stringent response does not influence ribosome pausing in Bacillus subtilis [RNA-Seq]

The stringent response represses translation and is activated when amino acid levels drop, for example during the stationary phase. Bacillus subtilis, a well-known industrial enzyme production organism, secretes most enzymes during the stationary phase. We were curious whether the stringent response affects ribosome pausing, and whether its absence improves protein production yields in the stationary phase. To investigate this, genome-wide ribosome profiling was used using a stringent response mutant that overexpressed the α-amylase AmyM. Although, blocking the stringent response did increases overall protein synthesis, the secretion of AmyM was actually reduced. The ribosome profiling data revealed that this was not caused by a reduction in translation. In fact, absence of the stringent response did not seem to markedly influence ribosome pausing. However, late in stationary phase an increased ribosome pausing at tryptophane codons emerges, suggesting a depletion of tryptophane. A strong suppression of tryptophane biosynthesis and acquisition genes, under control of the trp RNA-binding attenuation protein TRAP, likely accounts for this, although TRAP does not belong to the stringent response regulon of B. subtilis. Finally, the ribosome profiles revealed several genes with unusually low translation activities, illustrating the importance of translation initiation as a regulatory element of expression. A high-copy expression plasmid was used to overexpress the heterologous α-amylase AmyM from G. stearothermophilus. Throughout this study this strain is referred to as the wild type (WT) strain. To eliminate the stringent response, we took this strain and deleted rel, sasA and sasB. This mutant is referred to as the ppGpp0 strain. Both strains were grown in rich medium for 16 h at 30 °C with continuous shaking.