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In Escherichia coli and Salmonella , the c-di-GMP effector YcgR inhibits flagellar motility by interacting directly with the motor to alter both its bias and speed. Here, we demonstrate that in both of these bacteria, YcgR acts sequentially, altering motor bias first and then decreasing motor speed. We show that when c-di-GMP levels are high, deletion of ycgR restores wild-type motor behavior in E. coli , indicating that YcgR is the only motor effector in this bacterium. Yet, motility and chemotaxis in soft agar do not return to normal, suggesting that there is a second mechanism that inhibits motility under these conditions. In Salmonella , c-di-GMP-induced synthesis of extracellular cellulose has been reported to entrap flagella and to be responsible for the YcgR-independent motility defect. We found that this is not the case in E. coli Instead, we found through reversion analysis that deletion of rssB , which codes for a response regulator/adaptor protein that normally directs ClpXP protease to target σ S for degradation, restored wild-type motility in the ycgR mutant. Our data suggest that high c-di-GMP levels may promote altered interactions between these proteins to downregulate flagellar gene expression. IMPORTANCE Flagellum-driven motility has been studied in E. coli and Salmonella for nearly half a century. Over 60 genes control flagellar assembly and function. The expression of these genes is regulated at multiple levels in response to a variety of environmental signals. Cues that elevate c-di-GMP levels, however, inhibit motility by direct binding of the effector YcgR to the flagellar motor. In this study conducted mainly in E. coli , we show that YcgR is the only effector of motor control and tease out the order of YcgR-mediated events. In addition, we find that the σ S regulator protein RssB contributes to negative regulation of flagellar gene expression when c-di-GMP levels are elevated., (Copyright © 2019 American Society for Microbiology.)

Objective: To evaluate and characterize tinnitus in elderly volunteers，try to find out the relevant factors that can affect the incidence of tinnitus. Method: The study included 150 elderly volunteers. All volunteers had taken the otology examination and pure tone audiometry. They were interviewed by the investigators who were trained together, using the same questionnaire. The characteristics of tinnitus and the relationship between all relevant factors and tinnitus were analyzed. Result: Average age was 71.4 years. There are now 77 patients with tinnitus(51.3%), of which 31 cases have sustained tinnitus for more than 3 months, accounting for 40.3% of existing tinnitus volunteers. There was negative correlation between tinnitus and age. There was positive correlation between tinnitus and hearing loss. Tinnitus and headache had no correlation( P >0.05). Conclusion: After reach a certain age(70 years old), the incidence of tinnitus decreased. Hearing loss might be the most dangerous factor.If the hearing loss was more serious, the incidence of the tinnitus became higher. Tinnitus in the elderly may be the result of a combination of factors., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Editorial Department of Journal of Clinical Otorhinolaryngology Head and Neck Surgery.)

Objective: To investigate the incidence of tinnitus and its influencing factors in patients with chronic suppurative otitis media, and to provide clinical data for the study of the pathogenesis of tinnitus. Method: The clinical data of 77 patients with chronic suppurative otitis media who underwent modified radical mastoidectomy and tympanoplasty were investigated. When tinnitus and otitis media happened in the same side,then the tinnitus is judged to be otitis media related. Patients were further divided into otitis media related tinnitus and the no tinnitus groups. The differences of tinnitus severity, sleep disturbance, migraine (migraine features), snoring and gastroesophageal reflux were compared between the two groups in tinnitus occurrence and classification. Result: The incidence of otitis media related tinnitus was 55.8%(43/77). Most of the tinnitus happened(33/43) later than the occurrence of otitis media for several years or even decades. There were 43 cases of tinnitus associated with otitis media, and 31 cases without tinnitus. Between the groups, significant differences were observed in migraine features, and the P value is 0.011, while no significant differences were noticed in the severity of hearing loss and sleep disorders, snoring, gastroesophageal reflux. Ranking of otitis media related tinnitus was positively related to the degree of hearing loss, especially the bone conduction threshold. With Spearman rank correlation test,P values of mean value(250Hz,500Hz,1kHz,2kHz,4kHz), middle frequency (1kHz, 2kHz)and high frequency (4kHz) of bone conduction threshold were 0.010,0.019 and 0.003, and the correlation coefficients were 0.391,0.356 and 0.443, respectively. Conclusion: The occurrence of tinnitus in patients with otitis media may not be consistent with the time of otitis media, and theoretically later than the occurrence of otitis media is more reasonable. Whether tinnitus occurs in patients with otitis media is not related to sleep disorders, the degree of air conduction and bone conduction hearing loss, but is related to migraine features. The severity of tinnitus associated with otitis media is associated with bone conduction hearing loss., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Editorial Department of Journal of Clinical Otorhinolaryngology Head and Neck Surgery.)

Bacteria use two-component signal transduction systems to elicit adaptive responses to environmental changes. The simplest of these systems comprises a transmembrane sensor with histidine kinase activity and its cytoplasmic response regulator partner. Stimulus-response studies of two-component signaling systems typically employ expression reporters, such as β-galactosidase, that operate with relatively slow kinetics and low precision. In this chapter, we illustrate a new strategy for directly measuring the signaling activities of two-component sensor kinases in vivo. Our method exploits recent work that defines the recognition determinants for sensor-response regulator signaling transactions, which enabled us to couple histidine kinases to a FRET-based assay that uses signaling components of the E. coli chemotaxis system. We demonstrate the approach with NarX, a nitrate/nitrite sensor kinase, but the method should be applicable to other two-component sensor kinases.

A sensory adaptation system that tunes chemoreceptor sensitivity enables motile Escherichia coli cells to track chemical gradients with high sensitivity over a wide dynamic range. Sensory adaptation involves feedback control of covalent receptor modifications by two enzymes: CheR, a methyltransferase, and CheB, a methylesterase. This study describes a CheR function that opposes the signaling consequences of its catalytic activity. In the presence of CheR, a variety of mutant serine chemoreceptors displayed up to 40-fold enhanced detection sensitivity to chemoeffector stimuli. This response enhancement effect did not require the known catalytic activity of CheR, but did involve a binding interaction between CheR and receptor molecules. Response enhancement was maximal at low CheR:receptor stoichiometry and quantitative analyses argued against a reversible binding interaction that simply shifts the ON-OFF equilibrium of receptor signaling complexes. Rather, a short-lived CheR binding interaction appears to promote a long-lasting change in receptor molecules, either a covalent modification or conformation that enhances their response to attractant ligands., Competing Interests: The authors declare no conflict of interest.

Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose.

Residues E402 and R404 of the Escherichia coli serine chemoreceptor, Tsr, appear to form a salt bridge that spans the interfaces between neighboring dimers in the Tsr trimer of dimers, a key structural component of receptor core signaling complexes. To assess their functional roles, we constructed full sets of single amino acid replacement mutants at E402 and R404 and characterized their signaling behaviors with a suite of in vivo assays. Our results indicate that the E402 and R404 residues of Tsr play their most critical signaling roles at their inner locations near the trimer axis where they likely participate in stabilizing the trimer-of-dimer packing and the kinase-ON state of core signaling complexes. Mutant receptors with a variety of side-chain replacements still accessed both the ON and OFF signaling states, suggesting that core signaling complexes produce kinase activity over a range of receptor conformations and dynamic motions. Similarly, the kinase-OFF state may not be a discrete conformation but rather a range of structures outside the range of those suitable for kinase activation. Consistent with this idea, some structural lesions at both E402 and R404 produced signaling behaviors that are not compatible with discrete two-state models of core complex signaling states. Those lesions might stabilize intermediate receptor conformations along the OFF-ON energy landscape. Amino acid replacements produced different constellations of signaling defects at each residue, indicating that they play distinct structure-function roles. R404, but not E402, was critical for high signal cooperativity in the receptor array., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

HAMP domains play key signaling roles in many bacterial receptor proteins. The four-helix HAMP bundle of the homodimeric Escherichia coli serine chemoreceptor (Tsr) interacts with an adjoining four-helix sensory adaptation bundle to regulate the histidine autokinase CheA bound to the cytoplasmic tip of the Tsr molecule. The adaptation helices undergo reversible covalent modifications that tune the stimulus-responsive range of the receptor: unmodified E residues promote kinase-off output, and methylated E residues or Q replacements at modification sites promote kinase-on output. We used mutationally imposed adaptational modification states and cells with various combinations of the sensory adaptation enzymes, CheR and CheB, to characterize the signaling properties of mutant Tsr receptors that had amino acid replacements in packing layer 3 of the HAMP bundle and followed in vivo CheA activity with an assay based on Förster resonance energy transfer. We found that an alanine or a serine replacement at HAMP residue I229 effectively locked Tsr output in a kinase-on state, abrogating chemotactic responses. A second amino acid replacement in the same HAMP packing layer alleviated the I229A and I229S signaling defects. Receptors with the suppressor changes alone mediated chemotaxis in adaptation-proficient cells but exhibited altered sensitivity to serine stimuli. Two of the suppressors (S255E and S255A) shifted Tsr output toward the kinase-off state, but two others (S255G and L256F) shifted output toward a kinase-on state. The alleviation of locked-on defects by on-shifted suppressors implies that Tsr-HAMP has several conformationally distinct kinase-active output states and that HAMP signaling might involve dynamic shifts over a range of bundle conformations., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The Tar chemoreceptor-CheA-CheW ternary complex of Escherichia coli is a transmembrane allosteric enzyme in which binding of ligands to the periplasmic domain modulates the activity of CheA kinase. Kinase activity is also affected by reversible methylation of four glutamyl residues in the cytoplasmic domain of the receptor. E. coli Tar contains 553 residues. Residues 549-553 comprise the NWETF pentapeptide that binds the CheR methyltransferase and CheB methylesterase. The crystal structure of the similar Tsr chemoreceptor predicts that residues 263-289 and 490-515 of Tar form the most membrane-proximal portion of the extended CD1-CD2 four-helix bundle of the cytoplasmic domain. The last methylation site, Glu-491, is in the C19 heptad, and the N22-19 and C22-19 heptads are present in all classes of bacterial transmembrane chemoreceptors. Residues 516-548 probably serve as a flexible tether for the NWETF pentapeptide. Here, we present a mutational analysis of residues 505-548. The more of this region that is deleted, the less sensitive Tar is to inhibition by aspartate. Tar deleted from residue 505 through the NWETF sequence stimulates CheA in vitro but is not inhibited by aspartate. Thus, interaction of the last two heptads (C21 and C22) of CD2 with the first two heptads (N22 and N21) of CD1 must be important for transmitting an inhibitory signal from the HAMP domain to the four-helix bundle. The R514A, K523A, R529A, R540A, and R542A substitutions, singly or together, increase the level of activation of CheA in vitro, whereas the R505A substitution decreases the level of CheA stimulation by 40% and lowers the aspartate K(i) 7-fold. The R505E substitution completely abolishes stimulation of CheA in vitro. Glu-505 may interact electrostatically with Asp-273 to destabilize the "on" signaling state by loosening the four-helix bundle.

Chemoreceptors in Escherichia coli are homodimeric transmembrane proteins that convert environmental stimuli into intracellular signals controlling flagellar motion. Chemoeffectors bind to the extracellular (periplasmic) domain of the receptors, whereas their cytoplasmic domain mediates signaling and adaptation. The second transmembrane helix (TM2) connects these two domains. TM2 contains an aliphatic core flanked by amphipathic aromatic residues that have specific affinity for polar-hydrophobic membrane interfaces. We previously showed that Trp-209, near the cytoplasmic end of TM2, helps maintain the normal baseline-signaling state of the aspartate chemoreceptor (Tar) and that Tyr-210 plays an auxiliary role in this control. We have now repositioned the Trp-209/Tyr-210 pair in single-residue increments about the cytoplasmic polar-hydrophobic interface. Changes from WY-2 to WY+1 modulate the baseline-signaling state of the receptor in predictable and incremental steps that can be compensated by adaptive methylation/demethylation. Greater displacements, as in WY-3, WY+2, and WY+3, bias the receptor to the off kinase-inhibiting state or the on kinase-stimulating state, respectively, to a degree that cannot be fully compensated by the adaptation system. Aromatic residues analogous to Trp-209/Tyr-210 are present in other chemoreceptors and many transmembrane sensor kinases, where they may serve a similar function.

Four chemoreceptors in Escherichia coli mediate responses to chemicals in the environment. The receptors self-associate and localize to the cell poles. This aggregation implies that interactions among receptors are important parameters of signal processing during chemotaxis. We examined this phenomenon using a receptor-coupled in vitro assay of CheA kinase activity. The ability of homogeneous populations of the serine receptor Tsr and the aspartate receptor Tar to stimulate CheA was directly proportional to the ratio of the receptor to total protein in cell membranes up to a fraction of 50%. Membranes containing mixed populations of Tar and Tsr supported an up to 4-fold greater stimulation of CheA than expected on the basis of the contributions of the individual receptors. Peak activity was seen at a Tar:Tsr ratio of 1:4. This synergy was observed only when the two proteins were expressed simultaneously, suggesting that, under our conditions, the fundamental "cooperative receptor unit" is relatively static, even in the absence of CheA and CheW. Finally, we observed that inhibition of receptor-stimulated CheA activity by serine or aspartate required significantly higher concentrations of ligand for membranes containing mixed Tsr and Tar populations than for membranes containing only Tsr (up to 10(2)-fold more serine) or Tar (up to 10(4)-fold more aspartate). Together with recent analyses of the interactions of Tsr and Tar in vivo, our results reveal the emergent properties of mixed receptor populations and emphasize their importance in the integrated signal processing that underlies bacterial chemotaxis.

The chemoreceptors of Escherichia coli are homodimeric membrane proteins that cluster in patches near the cell poles. They convert environmental stimuli into intracellular signals that control flagellar rotation. The functional domains of a receptor are physically separated by the cell membrane. Chemoeffectors bind to the extracellular (periplasmic) domain, and the cytoplasmic domain mediates signaling and adaptation. These two domains communicate through the second transmembrane helix (TM2) that connects them. In the high-abundance receptors Tar and Tsr, TM2 is flanked by tryptophan residues, which should localize preferentially to the interfacial zone between the polar and hydrophobic layers of the phospholipid bilayer. To investigate the functional significance of the Trp residues that flank TM2 of Tar, we used site-directed mutagenesis to generate the W192A and W209A substitutions. The W192A protein retains full activity in vivo and in vitro, but it increases the K(i) for aspartate in the in vitro assay 3-fold. The W209A replacement eliminates receptor-mediated stimulation of CheA in vitro, and it leads to an increased level of adaptive methylation in vivo. This phenotype in some respects mimics the changes seen upon binding aspartate. Since the W209A substitution may cause the C-terminus of TM2 to protrude farther into the cytoplasm, these results reinforce the hypothesis that aspartate binding causes a similar displacement. Moving Trp to each position from residue 206 to residue 212 generated a wide variety of Tar signaling states that are generally consistent with the predictions of the piston model of transmembrane signaling. None of these receptors was completely locked in one signaling mode, although most showed pronounced signaling biases. Our findings suggest that the Trp residues flanking TM2, especially Trp-209, are important in setting the baseline activity and ligand sensitivity of the Tar receptor. We also conclude that the Tyr-210 residue plays at least an auxiliary role in this control.