The direction of rotation of the Escherichia coli flagellum is controlled by an assembly called the switch complex formed from multiple subunits of the proteins FliG, FliM, and FliN. Structurally, the switch complex corresponds to a drum-shaped feature at the bottom of the basal body, termed the C-ring. Stimulus-regulated reversals in flagellar motor rotation are the basis for directed movement such as chemotaxis. In E. coli, the motors turn counterclockwise (CCW) in their default state, allowing the several filaments on a cell to join together in a bundle and propel the cell smoothly forward. In response to the chemotaxis signaling molecule phospho-CheY (CheY P ), the motors can switch to clockwise (CW) rotation, causing dissociation of the filament bundle and reorientation of the cell. CheY P has previously been shown to bind to a conserved segment near the N terminus of FliM. Here, we show that this interaction serves to capture CheY P and that the switch to CW rotation involves the subsequent interaction of CheY P with FliN. FliN is located at the bottom of the C-ring, in close association with the C-terminal domain of FliM (FliM C ), and the switch to CW rotation has been shown to involve relative movement of FliN and FliM C . Using a recently developed structural model for the FliN/FliM C array, and the CheY P -binding site here identified on FliN, we propose a mechanism by which CheY P binding could induce the conformational switch to CW rotation.switching | cell motility | signal transduction | molecular motors M any motile bacteria control the direction of their swimming by regulating the sense of flagellar rotation in response to sensory cues. In the well-studied enteric species Escherichia coli and Salmonella (Salmonella enterica serovar typhimurium), counterclockwise (CCW) rotation allows the several flagellar filaments on a cell to join in a bundle to propel the cell smoothly, whereas clockwise (CW) rotation of one or more flagella disrupts the bundle and causes the cell to tumble. In the absence of external stimuli, a cell executes smooth runs of about a second punctuated by brief tumbles that send it in a new, essentially random, direction; by delaying the switch to CW rotation in response to attractant stimuli, cells prolong runs that happen to be in a favorable direction and so bias their movement toward nutrients, temperatures, or other factors conducive to survival (1, 2).

The Middle East respiratory syndrome coronavirus (MERS-CoV) recently spread from an animal reservoir to infect humans, causing sporadic severe and frequently fatal respiratory disease. Appropriate public health and control measures will require discovery of the zoonotic MERS coronavirus reservoirs. The relevant animal hosts are liable to be those that offer optimal MERS virus cell entry. Cell entry begins with virus spike (S) protein binding to DPP4 receptors. We constructed chimeric DPP4 receptors that have the virus-binding domains of indigenous Middle Eastern animals and assessed the activities of these receptors in supporting S protein binding and virus entry. Human, camel, and horse receptors were potent and nearly equally effective MERS virus receptors, while goat and bat receptors were considerably less effective. These patterns reflected S protein affinities for the receptors. However, even the low-affinity receptors could hypersensitize cells to infection when an S-cleaving protease(s) was present, indicating that affinity thresholds for virus entry must be considered in the context of host-cell proteolytic environments. These findings suggest that virus receptors and S protein-cleaving proteases combine in a variety of animals to offer efficient virus entry and that several Middle Eastern animals are potential reservoirs for transmitting MERS-CoV to humans. IMPORTANCE MERS is a frequently fatal disease that is caused by a zoonotic CoV. The animals transmitting MERS-CoV to humans are not yet known. Infection by MERS-CoV requires receptors and proteases on host cells. We compared the receptors of humans and Middle Eastern animals and found that human, camel, and horse receptors sensitized cells to MERS-CoV infection more robustlythan goat and bat receptors. Infection susceptibility correlated with affinities of the receptors for viral spike proteins. We also found that the presence of a cell surface lung protease greatly increases susceptibility to MERS-CoV, particularly in conjunction with low-affinity receptors. This cataloguing of human and animal host cell factors allows one to make inferences on the distribution of MERS-CoV in nature.

SUMMARY Recent studies have shown that conjugation systems of Gram-negative bacteria are composed of distinct inner and outer membrane core complexes (IMCs and OMCCs, respectively). Here, we functionally characterized the OMCC, focusing first on a cap domain that forms a channel across the outer membrane. Strikingly, the OMCC caps of the Escherichia coli pKM101 Tra and Agrobacterium tumefaciens VirB/VirD4 systems are completely dispensable for substrate transfer, but required for formation of conjugative pili. The pKM101 OMCC cap and extended pilus also are dispensable for activation of a Pseudomonas aeruginosa type VI secretion system (T6SS). Chimeric conjugation systems composed of the IMCpKM101 joined to OMCCs from the A. tumefaciens VirB/VirD4, E. coli R388 Trw, and Bordetella pertussis Ptl systems support conjugative DNA transfer in E. coli and trigger P. aeruginosa T6SS killing, but not pilus production. The A. tumefaciens VirB/VirD4 OMCC, solved by transmission electron microscopy, adopts a cage structure similar to the pKM101 OMCC. Our findings establish that OMCCs are highly structurally and functionally conserved - but also intrinsically conformationally flexible - scaffolds for translocation channels. Furthermore, the OMCC cap and a pilus tip protein coregulate pilus extension but are not required for channel assembly or function.

The bitopic membrane protein VirB10 of the Agrobacterium VirB/VirD4 type IV secretion system (T4SS) undergoes a structural transition in response to sensing of ATP binding or hydrolysis by the channel ATPases VirD4 and VirB11. This transition, detectable as a change in protease susceptibility, is required for DNA substrate passage through the translocation channel. Here, we present evidence that DNA substrate engagement with VirD4 and VirB11 also is required for activation of VirB10. Several DNA substrates (oncogenic T-DNA and plasmids RSF1010 and pCloDF13) induced the VirB10 conformational change, each by mechanisms requiring relaxase processing at cognate oriT sequences. VirD2 relaxase deleted of its translocation signal or any of the characterized relaxases produced in the absence of cognate DNA substrates did not induce the structural transition. Translocated effector proteins, e.g., VirE2, VirE3, and VirF, also did not induce the transition. By mutational analyses, we supplied evidence that the N-terminal periplasmic loop of VirD4, in addition to its catalytic site, is essential for early-stage DNA substrate transfer and the VirB10 conformational change. Further studies of VirB11 mutants established that three T4SS-mediated processes, DNA transfer, protein transfer, and pilus production, can be uncoupled and that the latter two processes proceed independently of the VirB10 conformational change. Our findings support a general model whereby DNA ligand binding with VirD4 and VirB11 stimulates ATP binding/hydrolysis, which in turn activates VirB10 through a structural transition. This transition confers an open-channel configuration enabling passage of the DNA substrate to the cell surface.T he type IV secretion systems (T4SSs) mediate the transfer of DNA and protein substrates across the envelopes of many Gram-negative and -positive bacterial species (1). The conjugation systems comprise a large subfamily of the T4SSs. These medically important systems are responsible for widespread transmission of antibiotic resistance genes and virulence genes carried by conjugative plasmids and chromosomally integrated elements. Overall, conjugation can be depicted as three distinct biochemical reactions. The DNA transfer and replication (Dtr) proteins bind a cognate origin-of-transfer (oriT) sequence and initiate processing of the DNA substrate for transfer (2-4). Next, the Dtr-oriT complex, termed the relaxosome, engages with the type IV coupling protein (T4CP) (5). Finally, the T4CP delivers the DNA substrate to a transenvelope channel comprised of the mating pair formation (Mpf) proteins for translocation across the cell envelope (6, 7). Studies in recent years have begun to define mechanistic and structural details of conjugation systems. Additionally, an accumulating body of evidence suggests that a combination of intraand extracellular signals act to coordinate the conjugative DNA transfer reactions in space and time. The present investigations advance our understanding of a signaling mechanism required for DNA subs...

The bacterial flagellum contains a specialized secretion apparatus in its base that pumps certain protein subunits through the growing structure to their sites of installation beyond the membrane. A related apparatus functions in the injectisomes of gram-negative pathogens to export virulence factors into host cells. This mode of protein export is termed type-III secretion (T3S). Details of the T3S mechanism are unclear. It is energized by the proton gradient; here, a mutational approach was used to identify proton-binding groups that might function in transport. Conserved proton-binding residues in all the membrane components were tested. The results identify residues R147, R154, and D158 of FlhA as most critical. These lie in a small, well conserved cytoplasmic domain of FlhA, located between trans-membrane segments 4 and 5. Two-hybrid experiments demonstrate self-interaction of the domain, and targeted cross-linking indicates that it forms a multimeric array. A mutation that mimics protonation of the key acidic residue (D158N) was shown to trigger a global conformational change that affects the other, larger cytoplasmic domain that interacts with the export cargo. The results are discussed in the framework of a transport model based on proton-actuated movements in the cytoplasmic domains of FlhA.