Effect of the stringent response on Bacillus subtilis ribosome profiles and protein production

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 absence of the stringent response improves protein production in the stationary phase, and whether the stringent response affects ribosome pausing, as this may influence protein folding. To investigate this, genome-wide ribosome profiling was used using a stringent response mutant that overexpressed the amylase AmyM. Firstly, we confirmed that blocking the stringent response increases overall protein synthesis. However, this lead to a reduction in AmyM production. Ribosome profiling revealed that this was not caused by a reduction in translation. In fact, the absence of the stringent response did not seem to influence ribosome pausing, in general. However, late in the stationary phase we noticed an increase in ribosome pausing at tryptophane codons, suggesting a depletion of tryptophane. A strong suppression of tryptophane biosynthesis and acquisition genes, under control of the trp RNA binding attenuation protein TRAP, might account for this. TRAP does not belong to the known stringent response regulon of Bacillus subtilis, and in fact many genes of this regulon did not show a different expression in our stringent response mutant. Finally, the ribosome profile data revealed several genes with unusually low translation activities, underlining the importance of translation initiation as a regulatory element of expression.