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DNA binding protein from starved cells (Dps) is a miniature ferritin complex, which plays a vital role in protecting bacterial DNA during starvation to maintain the integrity of bacteria under hostile conditions. Several approaches, including cryo-electron tomography, have been previously implemented by other research groups to decipher the structure of the Dps protein bound to DNA. However, none of the structures of the Dps-DNA complex was resolved to high resolution to identify the DNA binding residues. Like other bacteria, Mycobacterium smegmatis also expresses Dps2 (called MsDps2), which binds DNA to protect it under oxidative stress conditions. In this study, we implemented various biochemical and biophysical studies to characterize the DNA protein interactions of Dps2 protein from Mycobacterium smegmatis. We employed single-particle cryo-EM-based structural analysis of MsDps2-DNA complexes and identified that the region close to the N-terminus confers the DNA binding property. Based on cryo-EM data, we also pinpointed several arginine residues, proximal to the DNA binding region, responsible for DNA binding. We also performed mutations of these residues, which dramatically reduced the MsDps2-DNA interaction. In addition, we proposed a model that elucidates the mechanism of DNA compaction, which adapts a lattice-like structure. We performed single-molecule imaging of MsDps2-DNA interactions that corroborate well with our structural studies. Taken together, our results delineate the specific MsDps2 residues that play an important role in DNA binding and compaction, providing new insights into Mycobacterial DNA compaction mechanisms under stress conditions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOXs) have a major role in the physiology of eukaryotic cells by mediating reactive oxygen species production. Evolutionarily distant proteins with the NOX catalytic core have been found in bacteria, including Streptococcus pneumoniae NOX (SpNOX), which is proposed as a model for studying NOXs because of its high activity and stability in detergent micelles. We present here cryo-electron microscopy structures of substrate-free and nicotinamide adenine dinucleotide (NADH)-bound SpNOX and of NADPH-bound wild-type and F397A SpNOX under turnover conditions. These high-resolution structures provide insights into the electron-transfer pathway and reveal a hydride-transfer mechanism regulated by the displacement of F397. We conducted structure-guided mutagenesis and biochemical analyses that explain the absence of substrate specificity toward NADPH and suggest the mechanism behind constitutive activity. Our study presents the structural basis underlying SpNOX enzymatic activity and sheds light on its potential in vivo function., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

The marine bacterium Vibrio alginolyticus possesses a polar flagellum driven by a sodium ion flow. The main components of the flagellar motor are the stator and rotor. The C-ring and MS-ring, which are composed of FliG and FliF, respectively, are parts of the rotor. Here, we purified an MS-ring composed of FliF-FliG fusion proteins and solved the near-atomic resolution structure of the S-ring-the upper part of the MS-ring-using cryo-electron microscopy. This is the first report of an S-ring structure from Vibrio , whereas, previously, only those from Salmonella have been reported. The Vibrio S-ring structure reveals novel features compared with that of Salmonella , such as tilt angle differences of the RBM3 domain and the β-collar region, which contribute to the vertical arrangement of the upper part of the β-collar region despite the diversity in the RBM3 domain angles. Additionally, there is a decrease of the inter-subunit interaction between RBM3 domains, which influences the efficiency of the MS-ring formation in different bacterial species. Furthermore, although the inner-surface electrostatic properties of Vibrio and Salmonella S-rings are altered, the residues potentially interacting with other flagellar components, such as FliE and FlgB, are well structurally conserved in the Vibrio S-ring. These comparisons clarified the conserved and non-conserved structural features of the MS-ring across different species.IMPORTANCEUnderstanding the structure and function of the flagellar motor in bacterial species is essential for uncovering the mechanisms underlying bacterial motility and pathogenesis. Our study revealed the structure of the Vibrio S-ring, a part of its polar flagellar motor, and highlighted its unique features compared with the well-studied Salmonella S-ring. The observed differences in the inter-subunit interactions and in the tilt angles between the Vibrio and Salmonella S-rings highlighted the species-specific variations and the mechanism for the optimization of MS-ring formation in the flagellar assembly. By concentrating on the region where the S-ring and the rod proteins interact, we uncovered conserved residues essential for the interaction. Our research contributes to the advancement of bacterial flagellar biology., Competing Interests: The authors declare no conflict of interest.

Mycobacteria are known for their complex cell wall, which comprises layers of peptidoglycan, polysaccharides and unusual fatty acids known as mycolic acids that form their unique outer membrane. Polyketide synthase 13 (Pks13) of Mycobacterium tuberculosis , the bacterial organism causing tuberculosis, catalyses the last step of mycolic acid synthesis prior to export to and assembly in the cell wall. Due to its essentiality, Pks13 is a target for several novel anti-tubercular inhibitors, but its 3D structure and catalytic reaction mechanism remain to be fully elucidated. Here, we report the molecular structure of the catalytic core domains of M. tuberculosis Pks13 (Mt-Pks13), determined by transmission cryo-electron microscopy (cryoEM) to a resolution of 3.4 Å. We observed a homodimeric assembly comprising the ketoacyl synthase (KS) domain at the centre, mediating dimerization, and the acyltransferase (AT) domains protruding in opposite directions from the central KS domain dimer. In addition to the KS-AT di-domains, the cryoEM map includes features not covered by the di-domain structural model that we predicted to contain a dimeric domain similar to dehydratases, yet likely lacking catalytic function. Analytical ultracentrifugation data indicate a pH-dependent equilibrium between monomeric and dimeric assembly states, while comparison with the previously determined structures of M. smegmatis Pks13 indicates architectural flexibility. Combining the experimentally determined structure with modelling in AlphaFold2 suggests a structural scaffold with a relatively stable dimeric core, which combines with considerable conformational flexibility to facilitate the successive steps of the Claisen-type condensation reaction catalysed by Pks13.

Protein filaments are ubiquitous in nature and have diverse biological functions. Cryo-electron microscopy (cryo-EM) enables the determination of atomic structures, even from native samples, and is capable of identifying previously unknown filament species through high-resolution cryo-EM maps. In this study, we determine the structure of an unreported filament species from a cryo-EM dataset collected from Bacillus amyloiquefaciens biofilms. These filaments are composed of GerQ, a spore coat protein known to be involved in Bacillus spore germination. GerQ assembles into a structurally stable architecture consisting of rings containing nine subunits, which stacks to form filaments. Molecular dockings and model predictions suggest that this nine-subunit structure is suitable for binding CwlJ, a protein recruited by GerQ and essential for Ca 2+ -DPA induced spore germination. While the assembly state of GerQ within the spores and the direct interaction between GerQ and CwlJ have yet to be validated through further experiments, our findings provide valuable insights into the self-assembly of GerQ and enhance our understanding of its role in spore germination., (© 2024. The Author(s).)

Drug-resistant Tuberculosis (TB) is a global public health problem. Resistance to rifampicin, the most effective drug for TB treatment, is a major growing concern. The etiological agent, Mycobacterium tuberculosis ( Mtb ), has a cluster of ATP-binding cassette (ABC) transporters which are responsible for drug resistance through active export. Here, we describe studies characterizing Mtb Rv1217c-1218c as an ABC transporter that can mediate mycobacterial resistance to rifampicin and have determined the cryo-electron microscopy structures of Rv1217c-1218c. The structures show Rv1217c-1218c has a type V exporter fold. In the absence of ATP, Rv1217c-1218c forms a periplasmic gate by two juxtaposed-membrane helices from each transmembrane domain (TMD), while the nucleotide-binding domains (NBDs) form a partially closed dimer which is held together by four salt-bridges. Adenylyl-imidodiphosphate (AMPPNP) binding induces a structural change where the NBDs become further closed to each other, which downstream translates to a closed conformation for the TMDs. AMPPNP binding results in the collapse of the outer leaflet cavity and the opening of the periplasmic gate, which was proposed to play a role in substrate export. The rifampicin-bound structure shows a hydrophobic and periplasm-facing cavity is involved in rifampicin binding. Phospholipid molecules are observed in all determined structures and form an integral part of the Rv1217c-1218c transporter system. Our results provide a structural basis for a mycobacterial ABC exporter that mediates rifampicin resistance, which can lead to different insights into combating rifampicin resistance., Competing Interests: Competing interests statement:The authors declare no competing interest.

Oleate hydratase (OhyA) is a bacterial peripheral membrane protein that catalyzes FAD-dependent water addition to membrane bilayer-embedded unsaturated fatty acids. The opportunistic pathogen Staphylococcus aureus uses OhyA to counteract the innate immune system and support colonization. Many Gram-positive and Gram-negative bacteria in the microbiome also encode OhyA. OhyA is a dimeric flavoenzyme whose carboxy terminus is identified as the membrane binding domain; however, understanding how OhyA binds to cellular membranes is not complete until the membrane-bound structure has been elucidated. All available OhyA structures depict the solution state of the protein outside its functional environment. Here, we employ liposomes to solve the cryo-electron microscopy structure of the functional unit: the OhyA•membrane complex. The protein maintains its structure upon membrane binding and slightly alters the curvature of the liposome surface. OhyA preferentially associates with 20-30 nm liposomes with multiple copies of OhyA dimers assembling on the liposome surface resulting in the formation of higher-order oligomers. Dimer assembly is cooperative and extends along a formed ridge of the liposome. We also solved an OhyA dimer of dimers structure that recapitulates the intermolecular interactions that stabilize the dimer assembly on the membrane bilayer as well as the crystal contacts in the lattice of the OhyA crystal structure. Our work enables visualization of the molecular trajectory of membrane binding for this important interfacial enzyme., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Although eukaryotic Argonautes have a pivotal role in post-transcriptional gene regulation through nucleic acid cleavage, some short prokaryotic Argonaute variants (pAgos) rely on auxiliary nuclease factors for efficient foreign DNA degradation 1 . Here we reveal the activation pathway of the DNA defence module DdmDE system, which rapidly eliminates small, multicopy plasmids from the Vibrio cholerae seventh pandemic strain (7PET) 2 . Through a combination of cryo-electron microscopy, biochemistry and in vivo plasmid clearance assays, we demonstrate that DdmE is a catalytically inactive, DNA-guided, DNA-targeting pAgo with a distinctive insertion domain. We observe that the helicase-nuclease DdmD transitions from an autoinhibited, dimeric complex to a monomeric state upon loading of single-stranded DNA targets. Furthermore, the complete structure of the DdmDE-guide-target handover complex provides a comprehensive view into how DNA recognition triggers processive plasmid destruction. Our work establishes a mechanistic foundation for how pAgos utilize ancillary factors to achieve plasmid clearance, and provides insights into anti-plasmid immunity in bacteria., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Many enzymes can self-assemble into higher-order structures with helical symmetry. A particularly noteworthy example is that of nitrilases, enzymes in which oligomerization of dimers into spiral homo-oligomers is a requirement for their enzymatic function. Nitrilases are widespread in nature where they catalyze the hydrolysis of nitriles into the corresponding carboxylic acid and ammonia. Here, we present the Cryo-EM structure, at 3 Å resolution, of a C-terminal truncate nitrilase from Rhodococcus sp. V51B that assembles in helical filaments. The model comprises a complete turn of the helical arrangement with a substrate-intermediate bound to the catalytic cysteine. The structure was solved having added the substrate to the protein. The length and stability of filaments was made more substantial in the presence of the aromatic substrate, benzonitrile, but not for aliphatic nitriles or dinitriles. The overall structure maintains the topology of the nitrilase family, and the filament is formed by the association of dimers in a chain-like mechanism that stabilizes the spiral. The active site is completely buried inside each monomer, while the substrate binding pocket was observed within the oligomerization interfaces. The present structure is in a closed configuration, judging by the position of the lid, suggesting that the intermediate is one of the covalent adducts. The proximity of the active site to the dimerization and oligomerization interfaces, allows the dimer to sense structural changes once the benzonitrile was bound, and translated to the rest of the filament, stabilizing the helical structure., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Streptomyces are a genus of ubiquitous soil bacteria from which the majority of clinically utilized antibiotics derive 1 . The production of these antibacterial molecules reflects the relentless competition Streptomyces engage in with other bacteria, including other Streptomyces species 1,2 . Here we show that in addition to small-molecule antibiotics, Streptomyces produce and secrete antibacterial protein complexes that feature a large, degenerate repeat-containing polymorphic toxin protein. A cryo-electron microscopy structure of these particles reveals an extended stalk topped by a ringed crown comprising the toxin repeats scaffolding five lectin-tipped spokes, which led us to name them umbrella particles. Streptomyces coelicolor encodes three umbrella particles with distinct toxin and lectin composition. Notably, supernatant containing these toxins specifically and potently inhibits the growth of select Streptomyces species from among a diverse collection of bacteria screened. For one target, Streptomyces griseus, inhibition relies on a single toxin and that intoxication manifests as rapid cessation of vegetative hyphal growth. Our data show that Streptomyces umbrella particles mediate competition among vegetative mycelia of related species, a function distinct from small-molecule antibiotics, which are produced at the onset of reproductive growth and act broadly 3,4 . Sequence analyses suggest that this role of umbrella particles extends beyond Streptomyces, as we identified umbrella loci in nearly 1,000 species across Actinobacteria., (© 2024. The Author(s).)

Prokaryotes have evolved intricate innate immune systems against phage infection 1-7 . Gabija is a highly widespread prokaryotic defence system that consists of two components, GajA and GajB 8 . GajA functions as a DNA endonuclease that is inactive in the presence of ATP 9 . Here, to explore how the Gabija system is activated for anti-phage defence, we report its cryo-electron microscopy structures in five states, including apo GajA, GajA in complex with DNA, GajA bound by ATP, apo GajA-GajB, and GajA-GajB in complex with ATP and Mg 2+ . GajA is a rhombus-shaped tetramer with its ATPase domain clustered at the centre and the topoisomerase-primase (Toprim) domain located peripherally. ATP binding at the ATPase domain stabilizes the insertion region within the ATPase domain, keeping the Toprim domain in a closed state. Upon ATP depletion by phages, the Toprim domain opens to bind and cleave the DNA substrate. GajB, which docks on GajA, is activated by the cleaved DNA, ultimately leading to prokaryotic cell death. Our study presents a mechanistic landscape of Gabija activation., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Membrane proteins constitute about 20% of the human proteome and play crucial roles in cellular functions. However, a complete understanding of their structure and function is limited by their hydrophobic nature, which poses significant challenges in purification and stabilization. Detergents, essential in the isolation process, risk destabilizing or altering the proteins' native conformations, thus affecting stability and functionality. This study leverages single-particle cryo-electron microscopy to elucidate the structural nuances of membrane proteins, focusing on the SLAC1 bacterial homolog from Haemophilus influenzae ( Hi TehA) purified with diverse detergents, including n-dodecyl β-D-maltopyranoside (DDM), glycodiosgenin (GDN), β-D-octyl-glucoside (OG), and lauryl maltose neopentyl glycol (LMNG). This research not only contributes to the understanding of membrane protein structures but also addresses detergent effects on protein purification. By showcasing that the overall structural integrity of the channel is preserved, our study underscores the intricate interplay between proteins and detergents, offering insightful implications for drug design and membrane biology.

To survive bacteriophage (phage) infections, bacteria developed numerous anti-phage defence systems 1-7 . Some of them (for example, type III CRISPR-Cas, CBASS, Pycsar and Thoeris) consist of two modules: a sensor responsible for infection recognition and an effector that stops viral replication by destroying key cellular components 8-12 . In the Thoeris system, a Toll/interleukin-1 receptor (TIR)-domain protein, ThsB, acts as a sensor that synthesizes an isomer of cyclic ADP ribose, 1''-3' glycocyclic ADP ribose (gcADPR), which is bound in the Smf/DprA-LOG (SLOG) domain of the ThsA effector and activates the silent information regulator 2 (SIR2)-domain-mediated hydrolysis of a key cell metabolite, NAD + (refs.  12-14 ). Although the structure of ThsA has been solved 15 , the ThsA activation mechanism remained incompletely understood. Here we show that 1''-3' gcADPR, synthesized in vitro by the dimeric ThsB' protein, binds to the ThsA SLOG domain, thereby activating ThsA by triggering helical filament assembly of ThsA tetramers. The cryogenic electron microscopy (cryo-EM) structure of activated ThsA revealed that filament assembly stabilizes the active conformation of the ThsA SIR2 domain, enabling rapid NAD + depletion. Furthermore, we demonstrate that filament formation enables a switch-like response of ThsA to the 1''-3' gcADPR signal., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

To conserve energy during starvation and stress, many organisms use hibernation factor proteins to inhibit protein synthesis and protect their ribosomes from damage 1,2 . In bacteria, two families of hibernation factors have been described, but the low conservation of these proteins and the huge diversity of species, habitats and environmental stressors have confounded their discovery 3-6 . Here, by combining cryogenic electron microscopy, genetics and biochemistry, we identify Balon, a new hibernation factor in the cold-adapted bacterium Psychrobacter urativorans. We show that Balon is a distant homologue of the archaeo-eukaryotic translation factor aeRF1 and is found in 20% of representative bacteria. During cold shock or stationary phase, Balon occupies the ribosomal A site in both vacant and actively translating ribosomes in complex with EF-Tu, highlighting an unexpected role for EF-Tu in the cellular stress response. Unlike typical A-site substrates, Balon binds to ribosomes in an mRNA-independent manner, initiating a new mode of ribosome hibernation that can commence while ribosomes are still engaged in protein synthesis. Our work suggests that Balon-EF-Tu-regulated ribosome hibernation is a ubiquitous bacterial stress-response mechanism, and we demonstrate that putative Balon homologues in Mycobacteria bind to ribosomes in a similar fashion. This finding calls for a revision of the current model of ribosome hibernation inferred from common model organisms and holds numerous implications for how we understand and study ribosome hibernation., (© 2024. The Author(s).)

Bacteria encode hundreds of diverse defence systems that protect them from viral infection and inhibit phage propagation 1-5 . Gabija is one of the most prevalent anti-phage defence systems, occurring in more than 15% of all sequenced bacterial and archaeal genomes 1,6,7 , but the molecular basis of how Gabija defends cells from viral infection remains poorly understood. Here we use X-ray crystallography and cryo-electron microscopy (cryo-EM) to define how Gabija proteins assemble into a supramolecular complex of around 500 kDa that degrades phage DNA. Gabija protein A (GajA) is a DNA endonuclease that tetramerizes to form the core of the anti-phage defence complex. Two sets of Gabija protein B (GajB) dimers dock at opposite sides of the complex and create a 4:4 GajA-GajB assembly (hereafter, GajAB) that is essential for phage resistance in vivo. We show that a phage-encoded protein, Gabija anti-defence 1 (Gad1), directly binds to the Gabija GajAB complex and inactivates defence. A cryo-EM structure of the virally inhibited state shows that Gad1 forms an octameric web that encases the GajAB complex and inhibits DNA recognition and cleavage. Our results reveal the structural basis of assembly of the Gabija anti-phage defence complex and define a unique mechanism of viral immune evasion., (© 2023. The Author(s).)