Jonathan Guilherme Lucas dos Santos¹, Youngki You², Ernesto Satoshi Nakayasu², Frederico José Gueiros Filho ¹

The stringent response is a conserved bacterial stress adaptation triggered by nutrient limitation and other environmental cues, essential for survival. Its effectors are the second messengers guanosine tetraphosphate (ppGpp) and pentaphosphate (pppGpp), collectively called (p)ppGpp or \"alarmones\". Their accumulation reprograms metabolism, slowing anabolism and growth to promote survival. This occurs through effects on: (1) transcription; (2) ribosome biogenesis (via rRNA repression and GTPase inhibition); (3) translation (by blocking GTPases at all stages); and (4) DNA replication. The RelA/SpoT Homologue (RSH) superfamily regulates (p)ppGpp levels and includes two classes: (1) long RSHs, with both synthesis and hydrolysis domains (mono- or bifunctional); and (2) short RSHs, containing only a synthesis (SAS) or hydrolysis (SAH) domain. Bacillus subtilis encodes one bifunctional long RSH (Rel_Bs) and two short synthetases (RelP and RelQ). (p)ppGpp is essential for B. subtilis survival during fatty acid (FA) starvation. Studies suggest RSHs are regulated by protein-protein interactions, metabolite binding to conserved C-terminal domains, and, more recently, by post-translational modifications (PTMs) that fine-tune activity during stress. However, how Rel_Bs is regulated under FA starvation remains unclear. To explore this, we proposed a proteomics-driven approach to identify Rel_Bs PTMs and partners during FA starvation. We engineered a B. subtilis strain expressing functional 3xFLAG-tagged Rel_Bs and optimized culture conditions to induce FA starvation using cerulenin treatment. Cultures were grown under starved and control conditions, harvested at mid-log phase, lysed by mechanical disruption with protease inhibitors, and Rel_Bs was purified using agarose beads conjugated with anti-FLAG antibody. Preliminary MS/MS analysis revealed Rel_Bs glutarylation in control conditions, absent under starvation,  suggesting stress-induced PTM changes. This finding will be confirmed using newly prepared samples, and if the results are reproduced, functional assays with PTM-mimicking or -blocking mutants will be done to evaluate their role in Rel_Bs activation. SDS-PAGE of immunoprecipitated Rel_Bs showed additional bands under FA starvation, suggesting specific co-immunoprecipitation events under this condition. Proteomic analysis found 22 proteins enriched and 11 depleted in starved samples. Also, 15 proteins were unique to control, and 39 to FA-starved conditions. These data may guide the selection of candidate proteins for genetic and phenotypic assays to assess impact on Rel_Bs function. To complement the search for starvation-dependent Rel_Bs interactors, we will use TurboID proximity-dependent biotinylation, followed by streptavidin-based enrichment and mass spectrometry. TurboID assay conditions are being optimized with proper controls to distinguish real interactors from background. By uncovering condition-specific interactors and PTMs involved in Rel_Bs regulation under FA starvation, this study will provide critical insights into the molecular mechanisms governing the stringent response of Gram-positive bacteria. The insights gained may also support the development of strategies to disrupt stringent response pathways as a means to impair bacterial survival under nutrient-limited or stress conditions.

Agradecimentos: Thanks to the collaborators involved in this work, the Institute of Chemistry – University of São Paulo (IQ-USP), the laboratory team, and my advisor for guidance and support. Financial support from FAPESP is also gratefully acknowledged.