Three classes of ion-driven protein motors have been identified to date: ATP synthase, the bacterial flagellar motor, and a proton-driven motor that powers gliding motility and the Type 9 protein secretion system (T9SS) in Bacteroidetes bacteria. Here, we present cryo-EM structures of the gliding motility/T9SS motors GldLM from Flavobacterium johnsoniae and PorLM from Porphyromonas gingivalis . The motor is an asymmetric inner membrane protein complex in which the single transmembrane helices of two periplasm-spanning GldM/PorM proteins are positioned inside a ring of five GldL/PorL proteins. Mutagenesis and single-molecule tracking identifies protonatable amino acid residues in the transmembrane domain of the complex that are important for motor function. Our data provide evidence for a mechanism in which proton flow results in rotation of the periplasm-spanning GldM/PorM dimer inside the intra-membrane GldL/PorL ring to drive processes at the bacterial outer membrane.

How complex, multi-component macromolecular machines evolved remains poorly understood. Here we reveal the evolutionary origins of the chemosensory machinery that controls flagellar motility in Escherichia coli. We first identify ancestral forms still present in Vibrio cholerae, Pseudomonas aeruginosa, Shewanella oneidensis and Methylomicrobium alcaliphilum, characterizing their structures by electron cryotomography and finding evidence that they function in a stress response pathway. Using bioinformatics, we trace the evolution of the system through γ-Proteobacteria, pinpointing key evolutionary events that led to the machine now seen in E. coli. Our results suggest that two ancient chemosensory systems with different inputs and outputs (F6 and F7) existed contemporaneously, with one (F7) ultimately taking over the inputs and outputs of the other (F6), which was subsequently lost.

Data information: s, S-layer; m, membrane; c, conical structure; a, archaella; r, ring. Scale bars, 100 nm; scale bar in (A) applies to (1-3); scale bar in (B) applies to (B-E). EMBO reportsª 2017 The Authors EMBO reportsStructure of an archaellar motor and cone Ariane Briegel et al EV1 Figure EV2. Double-cone structure observed in Thermococcus kodakaraensis.A tomographic slice through a side view shows two associated conical structures (c1 and c2), both associated with archaella (a). s, S-layer; m, membrane. Scale bar, 100 nm. A, B Tomographic slices through two cells, highlighting the association between the cone and the archaella. C, D 3D segmentations of the cells in (A) and (B), respectively, with cones in blue and archaella in red, embedded in tomographic slices. E Tomographic slices of individual archaella show the varying orientations of archaella with respect to the cell envelope, as well as apparent connections to the cone.Data information: Scale bars, 100 nm in (A) and (B), 50 nm in (E) (applies to all panels in E); segmentations not to scale. Figure EV4. Individual particles from the subtomogram average show heterogeneity in the L3 density and angle of cone density. EMBO reportsª 2017 The Authors EMBO reports Structure of an archaellar motor and cone Ariane Briegel et al EV3The L3 density appears as either two dots of similar (first two panels) or different intensity (third panel), a single dot (fourth panel), or a dot and an extended line (fifth panel). m, membrane; c, conical structure. Scale bar, 10 nm (applies to all panels).ª 2017 The Authors EMBO reportsAriane Briegel et al Structure of an archaellar motor and cone EMBO reports EV4

Nearly all motile bacterial cells use a highly sensitive and adaptable sensory system to detect changes in nutrient concentrations in the environment and guide their movements toward attractants and away from repellents. The best-studied bacterial chemoreceptor arrays are membrane-bound. Many motile bacteria contain one or more additional, sometimes purely cytoplasmic, chemoreceptor systems. Vibrio cholerae contains three chemotaxis clusters (I, II, and III). Here, using electron cryotomography, we explore V. cholerae's cytoplasmic chemoreceptor array and establish that it is formed by proteins from cluster I. We further identify a chemoreceptor with an unusual domain architecture, DosM, which is essential for formation of the cytoplasmic arrays. DosM contains two signaling domains and spans the two-layered cytoplasmic arrays. Finally, we present evidence suggesting that this type of receptor is important for the structural stability of the cytoplasmic array.chemotaxis | chemoreceptor arrays | Vibrio cholerae | electron cryotomography | microscopy M ost motile bacteria move toward favorable environments through a process called chemotaxis. The molecular basis of this behavior is best understood in Escherichia coli, where transmembrane methyl-accepting chemotaxis proteins (MCPs, or chemoreceptors) form large arrays at the cell pole. The chemoreceptors bind attractants or repellents in the periplasm (1-3) and relay signals to histidine kinases (CheA) in the cytoplasm (4). When activated, CheA first autophosphorylates and then transfers the phosphoryl group to the response regulators CheY and CheB. Phosphorylated CheY binds to the flagellar motor, changing the direction of flagellar rotation. This allows the cells to switch from swimming forward smoothly (so-called "runs") to tumbling randomly. Changes in the duration and frequency of run and tumble phases drive a biased random walk that moves the cells toward favorable environments (5). The other response regulator, CheB, is a methylesterase, which, in conjunction with the constitutively active methyltransferase CheR, tunes the sensitivity of the system by changing the methylation state of the chemoreceptors (6-8).Although there is only one chemotaxis system in E. coli, most chemotactic bacterial and archaeal species have multiple systems (9). For instance, Rhodobacter sphaeroides has three chemotaxis systems encoded in three distinct clusters of genes, one of which (CheOp2) encodes two receptors without predicted transmembrane domains, TlpT and TlpC (10). Analysis of fluorescently tagged TlpT and TlpC revealed that they localize in foci around midcell. The foci split before cell division and are segregated to the midcell position within the future daughter cells. The chromosome-associated ParA-like ATPase PpfA controls the localization and segregation of the foci through an interaction with the N terminus of TlpT in a ParB-like manner (11).Using electron cryotomography (ECT), we discovered that these cytoplasmic foci in R. sphaerodes are ordered arrays of chemore...

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