A novel bacterial sulfite dehydrogenase that requires three c-type cytochromes for electron transfer.

c-type Cytochromes (c-Cyts), primarily as electron carriers and oxidoreductases, play a key role in energy transduction processes in virtually all living organisms. Many bacteria, such as Shewanella oneidensis, are particularly rich in c-Cyts, supporting respiratory versatility not seen in eukaryotes. Unfortunately, a large number of c-Cyts are underexplored, and their biological functions remain unknown. In this study, we identify SorCABD of S. oneidensis as a novel sulfite dehydrogenase (SDH), which catalyzes the oxidation of sulfite to sulfate. In addition to catalytic subunit SorA, this enzymatic complex includes three c-Cyt subunits, which all together carry out electron transfer. The electrons extracted from sulfite oxidation are ultimately delivered to oxygen, leading to oxygen reduction, a process relying on terminal oxidase cyt cbb3. Genomic analysis suggests that the homologs of this SDH are present in a small number of bacterial genera, Shewanella and Vibrio in particular. Because these bacteria are generally capable of reducing sulfite under anaerobic conditions, the co-existence of a sulfite oxidation system implies that they may play especially important roles in the transformation of sulfur species in natural environments.Importancec-type Cytochromes (c-Cyts) endow bacteria with high flexibility in their oxidative/respiratory systems, allowing them to extracellularly transform diverse inorganic and organic compounds for survival and growth. However, a large portion of the bacterial c-Cyts remain functionally unknown. Here, we identify three c-Cyts that work together as essential electron transfer partners for the catalytic subunit of a novel SDH in sulfite oxidation in Shewanella oneidensis. This characteristic makes S. oneidensis the first organism known to be capable of oxidizing and reducing sulfite. The findings suggest that Shewanella, along with a small number of other aquatic bacteria, would serve as a particular driving force in the biogeochemical sulfur cycle in nature.

Bacterial TANGO2 homologs are heme-trafficking proteins that facilitate biosynthesis of cytochromes c.

Heme, an essential molecule for virtually all living organisms, acts primarily as a cofactor in a large number of proteins. However, how heme is mobilized from the site of synthesis to the locations where hemoproteins are assembled remains largely unknown in cells, especially bacterial ones. In this study, with Shewanella oneidensis as the model, we identified HtpA (SO0126) as a heme-trafficking protein and homolog of TANGO2 proteins found in eukaryotes. We showed that HtpA homologs are widely distributed in all domains of living organisms and have undergone parallel evolution. In its absence, the cytochrome (cyt) c content and catalase activity decreased significantly. We further showed that both HtpA and representative TANGO2 proteins bind heme with 1:1 stoichiometry and a relatively low dissociation constant. Protein interaction analyses substantiated that HtpA directly interacts with the cytochrome c maturation system. Our findings shed light on cross-membrane transport of heme in bacteria and extend the understanding of TANGO2 proteins. IMPORTANCE The intracellular trafficking of heme, an essential cofactor for hemoproteins, remains underexplored even in eukaryotes, let alone bacteria. Here we developed a high-throughput method by which HtpA, a homolog of eukaryotic TANGO2 proteins, was identified to be a heme-binding protein that enhances cytochrome c biosynthesis and catalase activity in Shewanella oneidensis. HtpA interacts with the cytochrome c biosynthesis system directly, supporting that this protein, like TANGO2, functions in intracellular heme trafficking. HtpA homologs are widely distributed, but a large majority of them were found to be non-exchangeable, likely a result of parallel evolution. By substantiating the heme-trafficking nature of HtpA and its eukaryotic homologs, our findings provide general insight into the heme-trafficking process and highlight the functional conservation along evolution in all living organisms.

Generation and Physiology of Hydrogen Sulfide and Reactive Sulfur Species in Bacteria.

Sulfur is not only one of the most abundant elements on the Earth, but it is also essential to all living organisms. As life likely began and evolved in a hydrogen sulfide (H2S)-rich environment, sulfur metabolism represents an early form of energy generation via various reactions in prokaryotes and has driven the sulfur biogeochemical cycle since. It has long been known that H2S is toxic to cells at high concentrations, but now this gaseous molecule, at the physiological level, is recognized as a signaling molecule and a regulator of critical biological processes. Recently, many metabolites of H2S, collectively called reactive sulfur species (RSS), have been gradually appreciated as having similar or divergent regulatory roles compared with H2S in living organisms, especially mammals. In prokaryotes, even in bacteria, investigations into generation and physiology of RSS remain preliminary and an understanding of the relevant biological processes is still in its infancy. Despite this, recent and exciting advances in the fields are many. Here, we discuss abiotic and biotic generation of H2S/RSS, sulfur-transforming enzymes and their functioning mechanisms, and their physiological roles as well as the sensing and regulation of H2S/RSS.

A Common Target of Nitrite and Nitric Oxide for Respiration Inhibition in Bacteria.

Nitrite and nitric oxide (NO) are well-known bacteriostatic agents with similar biochemical properties. However, many studies have demonstrated that inhibition of bacterial growth by nitrite is independent of NO. Here, with Shewanella oneidensis as the research model because of its unusually high cytochrome (cyt) c content, we identify a common mechanism by which nitrite and NO compromise cyt c biosynthesis in bacteria, and thereby inhibit respiration. This is achieved by eliminating the inference of the cyclic adenosine monophosphate-catabolite repression protein (cAMP-Crp), a primary regulatory system that controls the cyt c content and whose activity is subjected to the repression of nitrite. Both nitrite and NO impair the CcmE of multiple bacteria, an essential heme chaperone of the System I cyt c biosynthesis apparatus. Given that bacterial targets of nitrite and NO differ enormously and vary even in the same genus, these observations underscore the importance of cyt c biosynthesis for the antimicrobial actions of nitrite and NO.

The stringent starvation protein SspA modulates peptidoglycan synthesis by regulating the expression of peptidoglycan synthases.

The peptidoglycan (PG) layer of bacterial cells is essential for maintaining the cell shape and survival of cells; therefore, the synthesis of PG needs to be spatiotemporally controlled. While it is well established that PG synthesis is mediated posttranslationally through interactions between PG synthases and their cognate partners, much less is known about the transcriptional regulation of genes encoding these synthases. Based on a previous finding that the Gram-negative bacterium Shewanella oneidensis lacking the prominent PG synthase exhibits impaired cell wall integrity, we performed genetic selections to isolate the suppressors. We discovered that disrupting the sspA gene encoding stringent starvation protein A (SspA) is sufficient to suppress compromised PG. SspA serves as a transcriptional repressor that regulates the expression of the two types of PG synthases, class A penicillin-binding proteins and SEDS/bPBP protein complexes. SspA is an RNA polymerase-associated protein, and its regulation involves interactions with the σ70 -RNAP complex and an antagonistic effect of H-NS, a global nucleoid-associated protein. We also present evidence that the regulation of PG synthases by SspA is conserved in Escherichia coli, adding a new dimension to the current understanding of PG synthesis and its regulation.

Nitric Oxide, Nitric Oxide Formers and Their Physiological Impacts in Bacteria.

Nitric oxide (NO) is an active and critical nitrogen oxide in the microbe-driven nitrogen biogeochemical cycle, and is of great interest to medicine and the biological sciences. As a gas molecule prior to oxygen, NO respiration represents an early form of energy generation via various reactions in prokaryotes. Major enzymes for endogenous NO formation known to date include two types of nitrite reductases in denitrification, hydroxylamine oxidoreductase in ammonia oxidation, and NO synthases (NOSs). While the former two play critical roles in shaping electron transport pathways in bacteria, NOSs are intracellular enzymes catalyzing metabolism of certain amino acids and have been extensively studied in mammals. NO interacts with numerous cellular targets, most of which are redox-active proteins. Doing so, NO plays harmful and beneficial roles by affecting diverse biological processes within bacterial physiology. Here, we discuss recent advances in the field, including NO-forming enzymes, the molecular mechanisms by which these enzymes function, physiological roles of bacterial NOSs, and regulation of NO homeostasis in bacteria.

Pleiotropic Effects of Hfq on the Cytochrome c Content and Pyomelanin Production in Shewanella oneidensis.

Shewanella oneidensis is the best understood model microorganism for the study of diverse cytochromes (cytos) c that support its unparallel respiratory versatility. Although RNA chaperone Hfq has been implicated in regulation of cyto c production, little is known about the biological pathways that it affects in this bacterium. In this study, from a spontaneous mutant that secretes pyomelanin and has a lowered cyto c content, we identified Hfq to be the regulator that critically associates with both phenotypes in S. oneidensis. We found that expression of the key genes in biosynthesis and degradation of heme is differentially affected by Hfq at under- and overproduced levels, and through modulating heme levels, Hfq influences the cyto c content. Although Hfq in excess results in overproduction of the enzymes responsible for both generation and removal of homogentisic acid (HGA), the precursor of pyomelanin, it is compromised activity of HmgA that leads to excretion and polymerization of HGA to form pyomelanin. We further show that Hfq mediates HmgA activity by lowering intracellular iron content because HmgA is an iron-dependent enzyme. Overall, our work highlights the significance of Hfq-mediated posttranscriptional regulation in the physiology of S. oneidensis, unraveling unexpected mechanisms by which Hfq affects cyto c biosynthesis and pyomelanin production. IMPORTANCE In bacteria, Hfq has been implicated in regulation of diverse biological processes posttranslationally. In S. oneidensis, Hfq affects the content of cytos c that serve as the basis of its respiratory versatility and potential application in bioenergy and bioremediation. In this study, we found that Hfq differentially regulates heme biosynthesis and degradation, leading to altered cyto c contents. Hfq in excess causes a synthetic effect on HmgA, an enzyme responsible for pyomelanin formation. Overall, the data presented manifest that the biological processes in a given bacterium regulated by Hfq are highly complex, amounting to required coordination among multiple physiological aspects to allow cells to respond to environmental changes promptly.

Biotransformation of 2D Nanomaterials through Stimulated Bacterial Respiration-Produced Extracellular Reactive Oxygen Species: A Common but Overlooked Process.

The biotransformation of 2D nanomaterials is still poorly understood, although their environmental fates are becoming an increasing concern with their broad applications. Here, we found that Ti3C2Tx nanosheets, a typical 2D nanomaterial, could be oxidized by reactive oxygen species (ROS) produced by both Gram-negative (Escherichia coli and Shewanella oneidensis) and Gram-positive (Bacillus subtilis) bacteria, with the formation of titanium dioxide (TiO2) on the nanosheet surfaces and impairment of structural integrity. Specifically, Ti3C2Tx nanosheets stimulated bacterial respiration Complex I, leading to increased generation of extracellular O2•- and the formation of H2O2 and •OH via Fenton-like reactions, which intensified the oxidation of the nanosheets. Surface modifications with KOH and hydrazine (HMH), especially HMH, could limit bacterial oxidation of the nanosheets. These findings reveal a common but overlooked process in which oxygen-respiring bacteria are capable of oxidizing 2D nanosheets, providing new knowledge for environmental fate evaluation and future design of functional 2D nanomaterials.

Recent Advances in the Siderophore Biology of Shewanella.

Despite the abundance of iron in nature, iron acquisition is a challenge for life in general because the element mostly exists in the extremely insoluble ferric (Fe3+) form in oxic environments. To overcome this, microbes have evolved multiple iron uptake strategies, a common one of which is through the secretion of siderophores, which are iron-chelating metabolites generated endogenously. Siderophore-mediated iron transport, a standby when default iron transport routes are abolished under iron rich conditions, is essential under iron starvation conditions. While there has been a wealth of knowledge about the molecular basis of siderophore synthesis, uptake and regulation in model bacteria, we still know surprisingly little about siderophore biology in diverse environmental microbes. Shewanella represent a group of γ-proteobacteria capable of respiring a variety of organic and inorganic substrates, including iron ores. This respiratory process relies on a large number of iron proteins, c-type cytochromes in particular. Thus, iron plays an essential and special role in physiology of Shewanella. In addition, these bacteria use a single siderophore biosynthetic system to produce an array of macrocyclic dihydroxamate siderophores, some of which show particular biological activities. In this review, we first outline current understanding of siderophore synthesis, uptake and regulation in model bacteria, and subsequently discuss the siderophore biology in Shewanella.

NapB Restores cytochrome c biosynthesis in bacterial dsbD-deficient mutants.

Cytochromes c (cyts c), essential for respiration and photosynthesis in eukaryotes, confer bacteria respiratory versatility for survival and growth in natural environments. In bacteria having a cyt c maturation (CCM) system, DsbD is required to mediate electron transport from the cytoplasm to CcmG of the Ccm apparatus. Here with cyt c-rich Shewanella oneidensis as the research model, we identify NapB, a cyt c per se, that suppresses the CCM defect of a dsbD mutant during anaerobiosis, when NapB is produced at elevated levels, a result of activation by cAMP-Crp. Data are then presented to suggest that NapB reduces CcmG, leading to the suppression. We further show that NapB proteins capable of rescuing CCM in the dsbD mutant form a small distinct clade. The study sheds light on multifunctionality of cyts c, and more importantly, unravels a self-salvation strategy through which bacteria have evolved to better adjust to the natural world.

Complex Interplay of Heme-Copper Oxidases with Nitrite and Nitric Oxide.

Nitrite and nitric oxide (NO), two active and critical nitrogen oxides linking nitrate to dinitrogen gas in the broad nitrogen biogeochemical cycle, are capable of interacting with redox-sensitive proteins. The interactions of both with heme-copper oxidases (HCOs) serve as the foundation not only for the enzymatic interconversion of nitrogen oxides but also for the inhibitory activity. From extensive studies, we now know that NO interacts with HCOs in a rapid and reversible manner, either competing with oxygen or not. During interconversion, a partially reduced heme/copper center reduces the nitrite ion, producing NO with the heme serving as the reductant and the cupric ion providing a Lewis acid interaction with nitrite. The interaction may lead to the formation of either a relatively stable nitrosyl-derivative of the enzyme reduced or a more labile nitrite-derivative of the enzyme oxidized through two different pathways, resulting in enzyme inhibition. Although nitrite and NO show similar biochemical properties, a growing body of evidence suggests that they are largely treated as distinct molecules by bacterial cells. NO seemingly interacts with all hemoproteins indiscriminately, whereas nitrite shows high specificity to HCOs. Moreover, as biologically active molecules and signal molecules, nitrite and NO directly affect the activity of different enzymes and are perceived by completely different sensing systems, respectively, through which they are linked to different biological processes. Further attempts to reconcile this apparent contradiction could open up possible avenues for the application of these nitrogen oxides in a variety of fields, the pharmaceutical industry in particular.

Functional Irreplaceability of Escherichia coli and Shewanella oneidensis OxyRs Is Critically Determined by Intrinsic Differences in Oligomerization.

LysR-type transcriptional regulators (LTTRs), which function in diverse biological processes in prokaryotes, are composed of a conserved structure with an N-terminal DNA-binding domain (DBD) and a C-terminal signal-sensing regulatory domain (RD). LTTRs that sense and respond to the same signal are often functionally exchangeable in bacterial species across wide phyla, but this phenomenon has not been demonstrated for the H2O2-sensing and -responding OxyRs. Here, we systematically examined the biochemical and structural determinants differentiating activator-only OxyRs from dual-activity ones by comparing OxyRs from two Gammaproteobacteria, Escherichia coli and Shewanella oneidensis. Our data show that EcOxyR could function as neither an activator nor a repressor in S. oneidensis. Using SoOxyR-based OxyR chimeras and mutants, we demonstrated that residues 283 to 289, which form the first half of the last C-terminal α-helix (α10), are critical for the proper function of SoOxyR and cannot be replaced with the EcOxyR counterpart. Crystal structural analysis reveals that α10 is important for the oligomerization of SoOxyR, which, unlike EcOxyR, forms several high-order oligomers upon DNA binding. As the mechanisms of OxyR oligomerization vary substantially among bacterial species, our findings underscore the importance of subtle structural features in determining regulatory activities of structurally similar proteins descending from a common ancestor. IMPORTANCE Evolution may drive homologous proteins to be functionally nonexchangeable in different organisms. However, much is unknown about the mechanisms underlying this phenomenon beyond amino acid substitutions. Here, we systematically examined the biochemical and structural determinants differentiating functionally nonexchangeable OxyRs, H2O2-responding transcriptional regulators from two Gammaproteobacteria, Escherichia coli and Shewanella oneidensis. Using SoOxyR-based OxyR chimeras and mutants, we demonstrated that residues 283 to 289, which form the first half of the last C-terminal α-helix (α10), are critical for the proper function of SoOxyR and cannot be replaced with the EcOxyR counterpart. Crystal structural analysis reveals that this last helix is critical for formation of high-order oligomers upon DNA binding, a phenomenon not observed with EcOxyR. Our findings provide a new dimension to differences in sequence and structural features among bacterial species in determining regulatory activities of homologous regulators.

Promoting Extracellular Electron Transfer of Shewanella oneidensis MR-1 by Optimizing the Periplasmic Cytochrome c Network.

The low efficiency of extracellular electron transfer (EET) is a major bottleneck for Shewanella oneidensis MR-1 acting as an electroactive biocatalyst in bioelectrochemical systems. Although it is well established that a periplasmic c-type cytochrome (c-Cyt) network plays a critical role in regulating EET efficiency, the understanding of the network in terms of structure and electron transfer activity is obscure and partial. In this work, we attempted to systematically investigate the impacts of the network components on EET in their absence and overproduction individually in microbial fuel cell (MFC). We found that overexpression of c-Cyt CctA leads to accelerated electron transfer between CymA and the Mtr system, which function as the primary quinol oxidase and the outer-membrane (OM) electron hub in EET. In contrast, NapB, FccA, and TsdB in excess severely impaired EET, reducing EET capacity in MFC by more than 50%. Based on the results from both strategies, a series of engineered strains lacking FccA, NapB, and TsdB in combination while overproducing CctA were tested for a maximally optimized c-Cyt network. A strain depleted of all NapB, FccA, and TsdB with CctA overproduction achieved the highest maximum power density in MFCs (436.5 mW/m2), ∼3.62-fold higher than that of wild type (WT). By revealing that optimization of periplasmic c-Cyt composition is a practical strategy for improving EET efficiency, our work underscores the importance in understanding physiological and electrochemical characteristics of c-Cyts involved in EET.

Thiosulfate oxidation in sulfur-reducing Shewanella oneidensis and its unexpected influences on the cytochrome c content.

Thiosulfate, an important form of sulfur compounds, can serve as both electron donor and acceptor in various microorganisms. In Shewanella oneidensis, a bacterium renowned for respiratory versatility, thiosulfate reduction has long been recognized but whether it can catalyse thiosulfate oxidation remains elusive. In this study, we discovered that S. oneidensis is capable of thiosulfate oxidation, a process specifically catalysed by two periplasmic cytochrome c (cyt c) proteins, TsdA and TsdB, which act as the catalytic subunit and the electron transfer subunit respectively. In the presence of oxygen, oxidation of thiosulfate has priority over reduction. Intriguingly, thiosulfate oxidation negatively regulates the cyt c content in S. oneidensis cells, largely by reducing intracellular levels of cAMP, which as the cofactor modulates activity of global regulator Crp required for transcription of many cyt c genes. This unexpected finding provides an additional dimension to interplays between the respiration regulator and the respiratory pathways in S. oneidensis. Moreover, the data presented here identified S. oneidensis as the first bacterium known to date owning both functional thiosulfate reductase and dehydrogenase, and importantly, genomics analyses suggested that the number of bacterial species possessing this feature is rather limited.

Shewanella oneidensis arcA Mutation Impairs Aerobic Growth Mainly by Compromising Translation.

Arc (anoxic redox control), one of the most intensely investigated two-component regulatory systems in γ-proteobacteria, plays a major role in mediating the metabolic transition from aerobiosis to anaerobiosis. In Shewanella oneidensis, a research model for respiratory versatility, Arc is crucial for aerobic growth. However, how this occurs remains largely unknown. In this study, we demonstrated that the loss of the response regulator ArcA distorts the correlation between transcription and translation by inhibiting the ribosome biosynthesis. This effect largely underlies the growth defect because it concurs with the effect of chloramphenicol, which impairs translation. Reduced transcription of ArcA-dependent ribosomal protein S1 appears to have a significant impact on ribosome assembly. We further show that the lowered translation efficiency is not accountable for the envelope defect, another major defect resulting from the ArcA loss. Overall, our results suggest that although the arcA mutation impairs growth through multi-fold complex impacts in physiology, the reduced translation efficacy appears to be a major cause for the phenotype, demonstrating that Arc is a primary system that coordinates proteomic resources with metabolism in S. oneidensis.

Lipopolysaccharide Transport System Links Physiological Roles of σE and ArcA in the Cell Envelope Biogenesis in Shewanella oneidensis.

The bacterial cell envelope is not only a protective structure that surrounds the cytoplasm but also the place where a myriad of biological processes take place. This multilayered complex is particularly important for electroactive bacteria such as Shewanella oneidensis, as it generally hosts branched electron transport chains and numerous reductases for extracellular respiration. However, little is known about how the integrity of the cell envelope is established and maintained in these bacteria. By tracing the synthetic lethal effect of Arc two-component system and σE in S. oneidensis, in this study, we identified the lipopolysaccharide transport (Lpt) system as the determining factor. Both Arc and σE, by regulating transcription of lptFG and lptD, respectively, are required for the Lpt system to function properly. The ArcA loss results in an LptFG shortage that triggers activation of σE and leads to LptD overproduction. LptFG and LptD at abnormal levels cause a defect in the lipopolysaccharide (LPS) transport, leading to cell death unless σE-dependent envelope stress response is in place. Overall, our report reveals for the first time that Arc works together with σE to maintain the integrity of the S. oneidensis cell envelope by participating in the regulation of the LPS transport system. IMPORTANCE Arc is a well-characterized global regulatory system that modulates cellular respiration by responding to changes in the redox status in bacterial cells. In addition to regulating expression of respiratory enzymes, Shewanella oneidensis Arc also plays a critical role in cell envelope integrity. The absence of Arc and master envelope stress response (ESR) regulator σE causes a synthetic lethal phenotype. Our research shows that the Arc loss downregulates lptFG expression, leading to cell envelope defects that require σE-mediated ESR for viability. The complex mechanisms revealed here underscore the importance of the interplay between global regulators in bacterial adaption to their natural inhabits.

Physiological Roles of Nitrite and Nitric Oxide in Bacteria: Similar Consequences from Distinct Cell Targets, Protection, and Sensing Systems.

Nitrite and nitric oxide (NO) are two active nitrogen oxides that display similar biochemical properties, especially when interacting with redox-sensitive proteins (i.e., hemoproteins), an observation serving as the foundation of the notion that the antibacterial effect of nitrite is largely attributed to NO formation. However, a growing body of evidence suggests that they are largely treated as distinct molecules by bacterial cells. Although both nitrite and NO are formed and decomposed by enzymes participating in the transformation of these nitrogen species, NO can also be generated via amino acid metabolism by bacterial NO synthetase and scavenged by flavohemoglobin. NO seemingly interacts with all hemoproteins indiscriminately, whereas nitrite shows high specificity to heme-copper oxidases. Consequently, the homeostasis of redox-sensitive proteins may be responsible for the substantial difference in NO-targets identified to date among different bacteria. In addition, most protective systems against NO damage have no significant role in alleviating inhibitory effects of nitrite. Furthermore, when functioning as signal molecules, nitrite and NO are perceived by completely different sensing systems, through which they are linked to different biological processes.

Distinct H2O2-Scavenging System in Yersinia pseudotuberculosis: KatG and AhpC Act Together to Scavenge Endogenous Hydrogen Peroxide.

To colonize in the digestive tract of animals and humans, Yersinia pseudotuberculosis has to deal with reactive oxygen species (ROS) produced by host cells and microbiota. However, an understanding of the ROS-scavenging systems and their regulation in this bacterium remains largely elusive. In this study, we identified OxyR as the master transcriptional regulator mediating cellular responses to hydrogen peroxide (H2O2) in Y. pseudotuberculosis through genomics and transcriptomics analyses. OxyR activates transcription of diverse genes, especially the core members of its regulon, including those encoding catalases, peroxidases, and thiol reductases. The data also suggest that sulfur species and manganese may play a particular role in the oxidative stress response of Y. pseudotuberculosis. Among the three H2O2-scavenging systems in Y. pseudotuberculosis, catalase/peroxidase KatE functions as the primary scavenger for high levels of H2O2; NADH peroxidase alkyl hydroperoxide reductase (AhpR) and catalase KatG together are responsible for removing low levels of H2O2. The simultaneous loss of both AhpC (the peroxidatic component of AhpR) and KatG results in activation of OxyR. Moreover, we found that AhpC, unlike its well-characterized Escherichia coli counterpart, has little effect on protecting cells against toxicity of organic peroxides. These findings provide not only novel insights into the structural and functional diversity of bacterial H2O2-scavenging systems but also a basic understanding of how Y. pseudotuberculosis copes with oxidative stress.

Structural basis of assembly and torque transmission of the bacterial flagellar motor.

The bacterial flagellar motor is a supramolecular protein machine that drives rotation of the flagellum for motility, which is essential for bacterial survival in different environments and a key determinant of pathogenicity. The detailed structure of the flagellar motor remains unknown. Here we present an atomic-resolution cryoelectron microscopy (cryo-EM) structure of the bacterial flagellar motor complexed with the hook, consisting of 175 subunits with a molecular mass of approximately 6.3 MDa. The structure reveals that 10 peptides protruding from the MS ring with the FlgB and FliE subunits mediate torque transmission from the MS ring to the rod and overcome the symmetry mismatch between the rotational and helical structures in the motor. The LP ring contacts the distal rod and applies electrostatic forces to support its rotation and torque transmission to the hook. This work provides detailed molecular insights into the structure, assembly, and torque transmission mechanisms of the flagellar motor.