Accurately modeling the structures of proteins and their complexes using artificial intelligence is revolutionizing molecular biology. Experimental data enable a candidate‐based approach to systematically model novel protein assemblies. Here, we use a combination of in‐cell crosslinking mass spectrometry and co‐fractionation mass spectrometry (CoFrac‐MS) to identify protein–protein interactions in the model Gram‐positive bacterium Bacillus subtilis . We show that crosslinking interactions prior to cell lysis reveals protein interactions that are often lost upon cell lysis. We predict the structures of these protein interactions and others in the Subti Wiki database with AlphaFold‐Multimer and, after controlling for the false‐positive rate of the predictions, we propose novel structural models of 153 dimeric and 14 trimeric protein assemblies. Crosslinking MS data independently validates the AlphaFold predictions and scoring. We report and validate novel interactors of central cellular machineries that include the ribosome, RNA polymerase, and pyruvate dehydrogenase, assigning function to several uncharacterized proteins. Our approach uncovers protein–protein interactions inside intact cells, provides structural insight into their interaction interfaces, and is applicable to genetically intractable organisms, including pathogenic bacteria.

Summary The second messenger cyclic di‐AMP (c‐di‐AMP) is essential for growth of many bacteria because it controls osmolyte homeostasis. c‐di‐AMP can regulate the synthesis of potassium uptake systems in some bacteria and it also directly inhibits and activates potassium import and export systems, respectively. Therefore, c‐di‐AMP production and degradation have to be tightly regulated depending on the environmental osmolarity. The Gram‐positive pathogen Listeria monocytogenes relies on the membrane‐bound diadenylate cyclase CdaA for c‐di‐AMP production and degrades the nucleotide with two phosphodiesterases. While the enzymes producing and degrading the dinucleotide have been reasonably well examined, the regulation of c‐di‐AMP production is not well understood yet. Here we demonstrate that the extracytoplasmic regulator CdaR interacts with CdaA via its transmembrane helix to modulate c‐di‐AMP production. Moreover, we show that the phosphoglucosamine mutase GlmM forms a complex with CdaA and inhibits the diadenylate cyclase activity in vitro. We also found that GlmM inhibits c‐di‐AMP production in L. monocytogenes when the bacteria encounter osmotic stress. Thus, GlmM is the major factor controlling the activity of CdaA in vivo. GlmM can be assigned to the class of moonlighting proteins because it is active in metabolism and adjusts the cellular turgor depending on environmental osmolarity.

Accurately modeling the structures of proteins and their complexes using artificial intelligence is revolutionizing molecular biology. Experimental data enable a candidate-based approach to systematically model novel protein assemblies. Here, we use a combination of in-cell crosslinking mass spectrometry, co-fractionation mass spectrometry and the SubtiWiki database to identify protein-protein interactions in the model Gram-positive bacterium Bacillus subtilis. Pairing this with structure prediction by AIphaFold-Multimer, we identify novel interactors of central machineries that include the ribosome, RNA polymerase and pyruvate dehydrogenase, as well as interactions involving uncharacterized proteins, which we functionally validate. After controlling for the false-positive rate of the AlphaFold approach, we propose novel structural models of 153 dimeric and 14 trimeric protein assemblies. We show that crosslinking MS data can independently validate AlphaFold predictions in situ. Our approach uncovers protein-protein interactions inside cells, provides structural insight into their interaction interface, and is applicable to genetically intractable organisms, including pathogenic bacteria.

Differentiation, growth, and virulence of the vascular plant pathogen Verticillium dahliae depend on a network of interconnected cellular signaling cascades. The transcription factor Hac1 of the endoplasmic reticulum-associated unfolded protein response (UPR) is required for initial root colonization, fungal growth, and vascular propagation by conidiation. Hac1 is essential for the formation of microsclerotia as long-time survival resting structures in the field. Single endoplasmic reticulum-associated enzymes for linoleic acid production as precursors for oxylipin signal molecules support fungal growth but not pathogenicity. Microsclerotia development, growth, and virulence further require the pheromone response mitogen-activated protein kinase (MAPK) pathway, but without the Ham5 scaffold function. The MAPK phosphatase Rok1 limits resting structure development of V.dahliae, but promotes growth, conidiation, and virulence. The interplay between UPR and MAPK signaling cascades includes several potential targets for fungal growth control for supporting disease management of the vascular pathogen V.dahliae.

29 Development and virulence of the vascular plant pathogen Verticillium dahliae 30 are connected and depend on a complex interplay between the unfolded protein 31 response, a Ham5 independent pheromone MAP kinase module and formation 32 of precursors for oxylipin signal molecules. 33  Genes coding for the unfolded protein response regulator Hac1, the Ham5 34 MAPK scaffold protein, and the oleate Δ12-fatty acid desaturase Ode1 were 35 deleted and their functions in growth, differentiation, and virulence on plants 36 were studied using genetic, cell biology, and plant infection experiments. 37  The unfolded protein response transcription factor Hac1 is required for initial 38 root colonization, fungal conidiation and propagation inside the host and is 39 essential for resting structure formation. Microsclerotia development, growth 40 and virulence require the pheromone response MAPK pathway, but without the 41 Ham5 scaffold function. Single ER-associated enzymes for linoleic acid 42 production make important contributions to fungal growth but have only a minor 43 impact on the pathogenicity of V. dahliae. 44  Fungal growth, sporulation, dormant structure formation and plant infection 45 require a network of the Hac1-regulated unfolded protein response, a scaffold-46 independent pheromone response MAPK pathway and formation of precursors 47 for signalling. This network includes interesting targets for disease management 48 of the vascular pathogen V. dahliae. 49 50