The alarmone pGpp inhibits translation through GTP depletion I

Bacteria produce the alarmone nucleotides ppGpp and pppGpp during stress to affect replication, transcription, and metabolism. ppGpp and pppGpp also attenuate translation by competitively binding translational GTPases to conserve resources during stress. Recently, pGpp was identified as a third alarmone, and important pathogens like Clostridioides difficile exclusively produce pGpp in response to stress. Despite its abundance as an alarmone, the precise role of pGpp in mediating stress responses is poorly understood. Here, we show that, while pGpp is a weaker inhibitor of protein synthesis than ppGpp and pppGpp in vitro, pGpp production in the model Gram-positive bacterium Bacillus subtilis leads to faster translation inhibition in vivo. pGpp production leads to fewer ribosomes engaged in translation and more hibernating ribosome dimers than (p)ppGpp production, suggesting that translation initiation is strongly inhibited. Additionally, pGpp production depletes cellular GTP more rapidly than (p)ppGpp production, which we show is sufficient for translation inhibition. Faster GTP depletion during pGpp production is also accompanied by more robust transcriptome remodeling. This work expands the model by which alarmones inhibit translation to include GTP depletion and demonstrates how production of different alarmone species exert varying effects on physiology. Overall design: RNA-seq comparing Bacillus subtilis pGpp inducible mutants (168 trpC2 dsasBdsasA amyE::Pxyl-sasA drel::kan, KAE60) and (p)ppGpp inducible mutants (168 trpC2 dsasBdsasAdnahA amyE::Pxyl-sasA drel::kan, KAE74). Two biological replicates of each strain were grown with repeated sampling pre-alarmone induction, 10 minutes post-, and 20 minutes post-induction.