The alarmone (p)ppGpp serves as the signalling molecule for the bacterial universal stringent response and plays a crucial role in bacterial virulence, persistence, and stress adaptation. Consequently, there is a significant focus on developing new drugs that target and modulate the levels of (p)ppGpp as a potential strategy for controlling bacterial infections. However, despite the availability of various methods for detecting (p)ppGpp, a simple and straightforward detection method is needed. In this study, we demonstrated that malachite green, a well-established compound used for phosphate detection, can directly detect (p)ppGpp and its analogues esp., pGpp. By utilizing malachite green, we identified three new inhibitors of the hydrolase activity of SpoT, one of the two RelA-SpoT homolog (RSH) proteins responsible for making and hydrolyzing (p)ppGpp in Escherichia coli. These findings highlight the convenience and practicality of malachite green, which can be widely employed in high-throughput studies to investigate (pp)pGpp in vitro and discover novel regulators of RSH proteins.

• Keywords
• SpoT, malachite green, pGpp, ppGpp, pppGpp, thermorubin,
• Publication type
• Journal Article MeSH

Bacillus subtilis cells are well suited to study how bacteria sense and adapt to proteotoxic stress such as heat, since temperature fluctuations are a major challenge to soil-dwelling bacteria. Here, we show that the alarmones (p)ppGpp, well known second messengers of nutrient starvation, are also involved in the heat stress response as well as the development of thermo-resistance. Upon heat-shock, intracellular levels of (p)ppGpp rise in a rapid but transient manner. The heat-induced (p)ppGpp is primarily produced by the ribosome-associated alarmone synthetase Rel, while the small alarmone synthetases RelP and RelQ seem not to be involved. Furthermore, our study shows that the generated (p)ppGpp pulse primarily acts at the level of translation, and only specific genes are regulated at the transcriptional level. These include the down-regulation of some translation-related genes and the up-regulation of hpf, encoding the ribosome-protecting hibernation-promoting factor. In addition, the alarmones appear to interact with the activity of the stress transcription factor Spx during heat stress. Taken together, our study suggests that (p)ppGpp modulates the translational capacity at elevated temperatures and thereby allows B. subtilis cells to respond to proteotoxic stress, not only by raising the cellular repair capacity, but also by decreasing translation to concurrently reduce the protein load on the cellular protein quality control system.

• MeSH
• Bacillus subtilis genetics   MeSH
• Bacterial Proteins genetics   MeSH
• Ligases genetics   MeSH
• Heat-Shock Response genetics   MeSH
• Gene Expression Regulation, Bacterial genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Bacterial Proteins MeSH
• guanosine 3',5'-polyphosphate synthetases MeSH Browser
• Ligases MeSH