Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis, remains one of the most prevalent human pathogens and a major cause of mortality worldwide. Metabolic network is a central mediator and defining feature of the pathogenicity of Mtb. Increasing evidence suggests that lysine succinylation dynamically regulates enzymes in carbon metabolism in both bacteria and human cells; however, its extent and function in Mtb remain unexplored. Here, we performed a global succinylome analysis of the virulent Mtb strain H37Rv by using high accuracy nano-LC-MS/MS in combination with the enrichment of succinylated peptides from digested cell lysates and subsequent peptide identification. In total, 1545 lysine succinylation sites on 626 proteins were identified in this pathogen. The identified succinylated proteins are involved in various biological processes and a large proportion of the succinylation sites are present on proteins in the central metabolism pathway. Site-specific mutations showed that succinylation is a negative regulatory modification on the enzymatic activity of acetylCoA synthetase. Molecular dynamics simulations demonstrated that succinylation affects the conformational stability of acetyl-CoA synthetase, which is critical for its enzymatic activity. Further functional studies showed that CobB, a sirtuin-like deacetylase in Mtb, functions as a desuccinylase of acetyl-CoA synthetase in in vitro assays. Together, our findings reveal widespread roles for lysine succinylation in regulating metabolism and diverse processes in Mtb. Our data provide a rich resource for functional analyses of lysine succinylation and facilitate the dissection of metabolic networks in this life-threatening pathogen.

Cyanobacteria are the oldest known life form inhabiting Earth and the only prokaryotes capable of performing oxygenic photosynthesis. Synechocystis sp. PCC 6803 (Synechocystis) is a model cyanobacterium used extensively in research on photosynthesis and environmental adaptation. Posttranslational protein modification by lysine acetylation plays a critical regulatory role in both eukaryotes and prokaryotes; however, its extent and function in cyanobacteria remain unexplored. Herein, we performed a global acetylome analysis on Synechocystis through peptide prefractionation, antibody enrichment, and high accuracy LC−MS/MS analysis; identified 776 acetylation sites on 513 acetylated proteins; and functionally categorized them into an interaction map showing their involvement in various biological processes. Consistent with previous reports, a large fraction of the acetylation sites are present on proteins involved in cellular metabolism. Interestingly, for the first time, many proteins involved in photosynthesis, including the subunits of phycocyanin (CpcA, CpcB, CpcC, and CpcG) and allophycocyanin (ApcA, ApcB, ApcD, ApcE, and ApcF), were found to be lysine acetylated, suggesting that lysine acetylation may play regulatory roles in the photosynthesis process. Six identified acetylated proteins associated with photosynthesis and carbon metabolism were further validated by immunoprecipitation and Western blotting. Our data provide the first global survey of lysine acetylation in cyanobacteria and reveal previously unappreciated roles of lysine acetylation in the regulation of photosynthesis. The provided data set may serve as an important resource for the functional analysis of lysine acetylation in cyanobacteria and facilitate the elucidation of the entire metabolic networks and photosynthesis process in this model cyanobacterium.

a Bcl2-associated athanogene 3 (BAG3), a member of the BAG family of co-chaperones, plays a critical role in regulating apoptosis, development, cell motility, autophagy, and tumor metastasis and in mediating cell adaptive responses to stressful stimuli. BAG3 carries a BAG domain, a WW domain, and a proline-rich repeat (PXXP), all of which mediate binding to different partners. To elucidate BAG3's interaction network at the molecular level, we employed quantitative immunoprecipitation combined with knockdown and human proteome microarrays to comprehensively profile the BAG3 interactome in humans. We identified a total of 382 BAG3-interacting proteins with diverse functions, including transferase activity, nucleic acid binding, transcription factors, proteases, and chaperones, suggesting that BAG3 is a critical regulator of diverse cellular functions. In addition, we characterized interactions between BAG3 and some of its newly identified partners in greater detail. In particular, bioinformatic analysis revealed that the BAG3 interactome is strongly enriched in proteins functioning within the proteasome-ubiquitination process and that compose the proteasome complex itself, suggesting that a critical biological function of BAG3 is associated with the proteasome. Functional studies demonstrated that BAG3 indeed interacts with the proteasome and modulates its activity, sustaining cell survival and underlying resistance to therapy through the down-modulation of apoptosis. Taken as a whole, this study expands our knowledge of the BAG3 interactome, provides a valuable resource for understanding how BAG3 affects different cellular functions, and demonstrates that biologically relevant data can be harvested using this kind of integrated approach. Molecular

Bacteria are capable of sensing and responding to environmental changes by several signal transduction systems with different mechanisms. Much attention is paid to model organisms with complex signaling networks to understand their composition and function, but how a complicated network evolved from a simple one or vice versa lacks systematic studies.

The evolution of macromolecular complex is a fundamental biological question, which is related to the origin of life and also guides our practice in synthetic biology. The chemosensory system is one of the complex structures that evolved very early in bacteria and displays enormous diversity and complexity in terms of composition and array structure in modern species. However, how the diversity and complexity of the chemosensory system evolved remains unclear. Here, using the Campylobacterota phylum with a robust “eco-evo” framework, we investigated the co-evolution of the chemosensory system and one of its important signaling outputs, flagellar machinery. Our analyses show that substantial flagellar gene alterations will lead to switch of its primary chemosensory class from one to another, or result in a hybrid of two classes. Unexpectedly, we discovered that the high-torque generating flagellar motor structure of Campylobacter jejuni and Helicobacter pylori likely evolved in the last common ancestor of the Campylobacterota phylum. Later lineages that experienced significant flagellar alterations lost some key components of complex scaffolding structures, thus derived simpler structures than their ancestor. Overall, this study revealed the co-evolutionary path of the chemosensory system and flagellar system, and highlights that the evolution of flagellar structural complexity requires more investigation in the Bacteria domain based on a resolved phylogenetic framework, with no assumptions on the evolutionary direction.

Summary Campylobacter jejuni is the leading cause of foodborne gastrointestinal illness worldwide, and chemotaxis plays an important role in its host colonization and pathogenesis. Although many studies on chemotaxis have focused on the physical organization and signaling mechanism of the system’s protein complex, much less is known about the transcriptional regulation of its components. Here, we describe two novel regulators, CJJ81176_0275 and CJJ81176_0276 (designated as CheP and CheQ), which specifically activate the transcription of the chemotaxis core genes cheV, cheA and cheW in C. jejuni and they are also essential for chemotactic responses. CheP has a single HD‐related output domain (HDOD) domain and can promote CheQ binding to the cheVAW operon promoter through a protein‐protein interaction. Mutagenesis analyses identified key residues critical for CheP function and/or interaction with CheQ. Further structural characterization of CheQ revealed a novel fold with strong positive surface charges that allow for its DNA binding. These findings reveal the gene regulatory mechanism of the chemotaxis system in an important bacterial pathogen and provide potential anti‐virulence targets for campylobacteriosis treatment. In addition, ChePQ is an example of how proteins with the widespread but functionally obscure HDOD can coordinate with a signal output DNA‐binding protein/domain to regulate the expression of important signaling pathways.

Campylobacter jejuni is a food-borne zoonotic pathogen of worldwide concern and the leading cause of bacterial diarrheal disease. In contrast to other enteric pathogens, C. jejuni has strict growth and nutritional requirements but lacks many virulence factors that have evolved for pathogenesis or interactions with the host. It is unclear how this bacterium has adapted to an enteric lifestyle. Here, we discovered that the CheO protein (CJJ81176_1265) is required for C. jejuni colonization of mice gut through its role in chemotactic control of flagellar rotation in oxygen-limiting environments. CheO interacts with the chemotaxis signaling proteins CheA and CheZ, and also with the flagellar rotor components FliM and FliY. Under microaerobic conditions, CheO localizes at the cellular poles where the chemosensory array and flagellar machinery are located in C. jejuni and its polar localization depends on chemosensory array formation. Several chemoreceptors that mediate energy taxis coordinately determine the bipolar distribution of CheO. Suppressor screening for a ΔcheO mutant identified that a single residue variation in FliM can alleviate the phenotype caused by the absence of CheO, confirming its regulatory role in the flagellar rotor switch. CheO homologs are only found in species of the Campylobacterota phylum, mostly species of host-associated genera Campylobacter, Helicobacter and Wolinella. The CheO results provide insights into the complexity of chemotaxis signal transduction in C. jejuni and closely related species. Importantly, the recruitment of CheO into chemosensory array to promote chemotactic behavior under hypoxia represents a new adaptation strategy of C. jejuni to human and animal intestines.

Microbes rely on signal transduction systems to sense and respond to environmental changes for survival and reproduction. It is generally known that niche adaptation plays an important role in shaping the signaling repertoire. However, the evolution of bacterial signaling capacity lacks systematic studies with a temporal direction. Particularly, it is unclear how complexity evolved from simplicity or vice versa for signaling networks. Here we examine the evolutionary processes of major signal transduction systems in Campylobacterota (formerly Epsilonproteobacteria), a phylum with sufficient evolutionary depth and ecological diversity. Evolution of signaling systems within Campylobacterota shows two opposite trends. During niche expansion, signaling complexity increases with gene expansions through horizontal gene transfer (HGT), gene duplication, fusion and fission, which create opportunities for genetic innovation and pathway integration. In contrast, as the lineages adapt to a specialized niche, complexity decreases with massive gene losses that lead to the decline or disappearance of pathways mediated by multiple transmitters. Overall, signaling capacity and complexity arise and drop together in Campylobacterota, determined by sensory demand, genetic resources and co-evolution within the genomic context. These findings reflects plausible evolutionary principles for other cellular networks and genome evolution of the Bacteria domain.