Search

Bacterial chemoreceptors form ternary signaling complexes with the histidine kinase CheA through the coupling protein CheW. Receptor complexes in turn cluster into cellular arrays that produce highly sensitive responses to chemical stimuli. In Escherichia coli, receptors of different types form mixed trimer-of-dimers signaling teams through the tips of their highly conserved cytoplasmic domains. To explore the possibility that the hairpin loop at the tip of the trimer contact region might promote interactions with CheA or CheW, we constructed and characterized mutant receptors with amino acid replacements at the two nearly invariant hairpin charged residues of Tsr: R388, the most tip-proximal trimer contact residue, and E391, the apex residue of the hairpin turn. Mutant receptors were subjected to in vivo tests for the assembly and function of trimers, ternary complexes, and clusters. All R388 replacements impaired or destroyed Tsr function, apparently through changes in trimer stability or geometry. Large-residue replacements locked R388 mutant ternary complexes in the kinase-off (F, H) or kinase-on (W, Y) signaling state, suggesting that R388 contributes to signaling-related conformational changes in the trimer. In contrast, most E391 mutants retained function and all formed ternary signaling complexes efficiently. Hydrophobic replacements of any size (G, A, P, V, I, L, F, W) caused a novel phenotype in which the mutant receptors produced rapid switching between kinase-on and -off states, indicating that hairpin tip flexibility plays an important role in signal state transitions. These findings demonstrate that the receptor determinants for CheA and CheW binding probably lie outside the hairpin tip of the receptor signaling domain.

HAMP domains are ~50-residue motifs, found in many bacterial signaling proteins, that consist of two amphiphilic helices joined by a nonhelical connector segment. The HAMP domain of Tsr, the serine chemoreceptor of Escherichia coli, receives transmembrane input signals from the periplasmic serine binding domain and in turn modulates output signals from the Tsr kinase control domain to elicit chemotactic responses. We created random amino acid replacements at each of the 14 connector residues of Tsr-HAMP to identify those that are critical for Tsr function. In all, we surveyed 179 connector missense mutants and identified three critical residues (G235, L237, and I241) at which most replacements destroyed Tsr function and another important residue (G245) at which most replacements impaired Tsr function. The region surrounding G245 tolerated 1-residue deletions and insertions of up to 10 glycines, suggesting a role as a relatively nonspecific, flexible linker. The critical connector residues are consistent with a structural model of the Tsr-HAMP domain based on the solution structure of an isolated thermophile HAMP domain (M. Hulko, F. Berndt, M. Gruber, J. U. Linder, V. Truffault, A. Schultz, J. Martin, J. E. Schultz, A. N. Lupas, and M. Coles, Cell 126:929-940, 2006) in which G235 defines a critical turn at the C terminus of the first helix and L237 and I241 pack against the helices, perhaps to stabilize alternative HAMP signaling conformations. Most I241 lesions locked Tsr signal output in the kinase-on mode, implying that this residue is responsible mainly for stabilizing the kinase-off signaling state. In contrast, lesions at L237 resulted in a variety of aberrant output patterns, suggesting a role in toggling output between signaling states.

The C-terminal P5 domain of the histidine kinase CheA is essential for coupling CheA autophosphorylation activity to chemoreceptor control through a binding interaction with the CheW protein. To locate P5 determinants critical for CheW binding and chemoreceptor control, we surveyed cysteine replacements at 39 residues predicted to be at or near the P5 surface in Escherichia coli CheA. Two-thirds of the Cys replacement proteins exhibited in vitro defects in CheW binding, either before or after modification with a bulky fluorescein group. The binding-defective sites were widely distributed on the P5 surface and were often interspersed with sites that caused no functional defects, implying that relatively minor structural perturbations, often far from the actual binding site, can influence its conformation or accessibility. The most likely CheW docking area included loop 2 in P5 folding subdomain 1. All but four of the binding-defective P5-Cys proteins were defective in receptor-mediated activation, suggesting that CheW binding, as measured in vitro, is necessary for assembly of ternary signaling complexes and/or subsequent CheA activation. Other Cys sites specifically affected receptor-mediated activation or deactivation of CheA, demonstrating that CheW binding is not sufficient for assembly and/or operation of receptor signaling complexes. Because P5 is quite similar to CheW, whose structure is known to be dynamic, we suggest that conformational flexibility and dynamic motions govern the signaling activities of the P5 domain. In addition, relative movements of the CheA domains may be involved in CheW binding, in ternary complex assembly, and in subsequent stimulus-induced conformational changes in receptor signaling complexes.

Enteric bacteria tumble, swim slowly, and are then paralyzed upon exposure to 390- to 530-nm light. Here, we analyze this complex response in Escherichia coli using standard fluorescence microscope optics for excitation at 440 [+ or -] 5 nm. The slow swimming and paralysis occurred only in dye-containing growth media or buffers. Excitation elicited complete paralysis within a second in 1 [micro]M proflavine dye, implying specific motor damage, but prolonged tumbling in buffer alone. The tumbling half-response times were subsecond for onset but more than a minute for recovery. The response required the chemotaxis signal protein CheY and receptor-dependent activation of its kinase CheA. The study of deletion mutants revealed a specific requirement for either the aerotaxis receptor Aer or the chemoreceptor Tar but not the Tar homolog Tsr. The action spectrum of the wild-type response was consistent with a flavin, but the chromophores remain to be identified. The motile response processed via Aer was sustained, with recovery to either step-up or -down taking more than a minute. The response processed via Tar was transient, recovering on second time scales comparable to chemotactic responses. The response duration and amplitude were dependent on relative expression of Aer, Tar, and Tsr. The main response features were reproduced when each receptor was expressed singly from a plasmid in a receptorless host strain. However, time-resolved motion analysis revealed subtle kinetic differences that reflect the role of receptor cluster interactions in kinase activation-deactivation dynamics.

Aer, a low-abundance signal transducer in Escherichia coli, mediates robust aerotactic behavior, possibly through interactions with methyl-accepting chemotaxis proteins (MCP). We obtained evidence for interactions between Aer and the high-abundance aspartate (Tar) and serine (Tsr) receptors. Aer molecules bearing a cysteine reporter diagnostic for trimer-of-dimer formation yielded cross-linking products upon treatment with a trifunctionai maleimide reagent. Aer also formed mixed cross-linking products with a similarly marked Tar reporter. An Aer trimer contact mutation known to abolish trimer formation by MCPs eliminated Aer trimer and mixed trimer formation. Trimer contact alterations known to cause epistatic behavior in MCPs also produced epistatic properties in Aer. Amino acid replacements in the Tar trimer contact region suppressed an epistatic Aer signaling defect, consistent with compensatory conformational changes between directly interacting proteins. In cells lacking MCPs, Aer function required high-level expression, comparable to the aggregate number of receptors in a wild-type cell. Aer proteins with clockwise (CW)-biased signal output cannot function under these conditions but do so in the presence of MCPs, presumably through formation of mixed signaling teams. The Tar signaling domain was sufficient for functional rescue. Moreover, CW-biased lesions did not impair aerotactic signaling in a hybrid Aer-Tar transducer capable of adjusting its steady-state signal output via methylation-dependent sensory adaptation. Thus, MCPs most likely assist mutant Aer proteins to signal productively by forming collaborative signaling teams. Aer evidently evolved to operate collaboratively with high-abundance receptors but can also function without MCP assistance, provided that it can establish a suitable prestimulus swimming pattern.

The Escherichia coli Aer protein contains an N-terminal PAS domain that binds flavin adenine dinucleotide (FAD), senses aerotactic stimuli, and communicates with the output signaling domain. To explore the roles of the intervening F1 and HAMP segments in Aer signaling, we isolated plasmid-borne aerotaxis-defective mutations in a host strain lacking all chemoreceptors of the methyl-accepting chemotaxis protein (MCP) family. Under these conditions, Aer alone established the cell's run/tumble swimming pattern and modulated that behavior in response to oxygen gradients. We found two classes of Aer mutants: null and clockwise (CW) biased. Most mutant proteins exhibited the null phenotype: failure to elicit CW flagellar rotation, no aerosensing behavior in MCP-containing hosts, and no apparent FAD-binding ability. However, null mutants had low Aer expression levels caused by rapid degradation of apparently nonnative subunits. Their functional defects probably reflect the absence of a protein product. In contrast, CW-biased mutant proteins exhibited normal expression levels, wild-type FAD binding, and robust aerosensing behavior in MCP-containing hosts. The CW lesions evidently shift unstimulated Aer output to the CW signaling state but do not block the Aer input-output pathway. The distribution and properties of null and CW-biased mutations suggest that the Aer PAS domain may engage in two different interactions with HAMP and the HAMP-proximal signaling domain: one needed for Aer maturation and another for promoting CW output from the Aer signaling domain. Most aerotaxis-defective null mutations in these regions seemed to affect maturation only, indicating that these two interactions involve structurally distinct determinants.

During chemotactic signaling by Escherichia coli, autophosphorylation of the histidine kinase CheA is coupled to chemoreceptor control by the CheW protein, which interacts with the C-terminal P5 domain of CheA. To identify P5 determinants important for CheW binding and receptor coupling control, we isolated and characterized a series of P5 missense mutants. The mutants fell into four phenotypic groups on the basis of in vivo behavioral and protein stability tests and in vitro assays with purified mutant proteins. Group 1 mutants exhibited autophosphorylation and receptor-coupling defects, and their CheA proteins were subject to relatively rapid degradation in vivo. Group 1 mutations were located at hydrophobic residues in P5 subdomain 2 and most likely caused folding defects. Group 2 mutants made stable CheA proteins with normal autophosphorylation ability but with defects in CheW binding and in receptor-mediated activation of CheA autophosphorylation. Their mutations affected residues in P5 subdomain 1 near the interface with the CheA dimerization (P3) and ATP-binding (P4) domains. Mutant proteins of group 3 were normal in all tests yet could not support chemotaxis, suggesting that P5 has one or more important but still unknown signaling functions. Group 4 mutant proteins were specifically defective in receptor-mediated deactivation control. The group 4 mutations were located in P5 subdomain 1 at the P3/PY interface. We conclude that P5 subdomain 1 is important for CheW binding and for receptor coupling control and that these processes may require substantial motions of the P5 domain relative to the neighboring P3 and P4 domains of CheA.

Aer is a membrane-associated protein that mediates aerotactic responses in Escherichia coli. Its C-terminal half closely resembles the signaling domains of methyl-accepting chemotaxis proteins (MCPs), which undergo reversible methylation at specific glutamic acid residues to adapt their signaling outputs to homogeneous chemical environments. MCP-mediated behaviors are dependent on two specific enzymes, CheR (methyltransferase) and CheB (methylesterase). The Aer signaling domain contains unorthodox methylation sites that do not conform to the consensus motif for CheR or CheB substrates, suggesting that Aer, unlike conventional MCPs, might be a methylation-independent transducer. Several lines of evidence supported this possibility. (i) The Aer protein was not detectably modified by either CheR or CheB. (ii) Amino acid replacements at the putative Aer methylation sites generally had no deleterious effect on Aer function. (iii) Aer promoted aerotactic migrations on semisolid media in strains that lacked all four of the E. coli MCPs. CheR and CheB function had no influence on the rate of aerotactic movements in those strains. Thus, Aer senses and signals efficiently in the absence of deamidation or methylation, methylation changes, methylation enzymes, and methyl-accepting chemotaxis proteins. We also found that chimeric transducers containing the PAS-HAMP sensing domain of Aer joined to the signaling domain and methylation sites of Tar, an orthodox MCP, exhibited both methylation-dependent and methylation-independent aerotactic behavior. The hybrid Aear transducers demonstrate that methylation independence does not emanate from the Aer signaling domain but rather may be due to transience of the cellular redox changes that are thought to trigger Aer-mediated behavioral responses.

CheA is a multidomain histidine kinase for chemotaxis in Escherichia coli. CheA autophosphorylates through interaction of its N-terminal phosphorylation site domain (P1) with its central dimerization (P3) and ATP-binding (P4) domains. This activity is modulated through the C-terminal P5 domain, which couples CheA to chemoreceptor control. CheA phosphoryl groups are donated to two response regulators, CheB and CheY, to control swimming behavior. The phosphorylated forms of CheB and CheY turn over rapidly, enabling receptor signaling complexes to elicit fast behavioral responses by regulating the production and transmission of phosphoryl groups from CheA. To promote rapid phosphotransfer reactions, CheA contains a phosphoacceptor-binding domain (P2) that serves to increase CheB and CheY concentrations in the vicinity of the adjacent P1 phosphodonor domain. To determine whether the P2 domain is crucial to CheA's signaling specificity, we constructed CheA[DELTA]P2 deletion mutants and examined their signaling properties in vitro and in vivo. We found that CheA[DELTAta]P2 autophosphorylated and responded to receptor control normally but had reduced rates of phosphotransfer to CheB and CheY. This defect lowered the frequency of tumbling episodes during swimming and impaired chemotactic ability. However, expression of additional P1 domains in the CheA[DELTA]P2 mutant raised tumbling frequency, presumably by buffering the irreversible loss of CheA[DELTA]P2-generated phosphoryl groups from CheB and CheY, and greatly improved its chemotactic ability. These findings suggest that P2 is not crucial for CheA signaling specificity and that the principal determinants that favor appropriate phosphoacceptor partners, or exclude inappropriate ones, most likely reside in the P1 domain.

The role of a fragment from Escherichia coli CheA kinase on the stimulatory chemoreceptor signaling was analyzed. The study demonstrated that a polypeptide is capable of corresponding to the T, M and C domains of CheA to block stimulatory receptor coupling in vivo and in vitro. Results showed that chemoreceptor coupled to CheA exhibit two discrete signaling states.

The aer gene of Escherichia coli encodes a 506-residue protein with an N terminus that is similar to the NifL aerosensor and a C terminus that is similar to the flagellar signalling domain of methyl-accepting chemoreceptors. Mutants without a functional Aer protein did not congregate around air bubbles or follow oxygen gradients in soft agar plates. Also, membranes with overexpressed Aer protein had high levels of noncovalently associated flavin adenine dinucleotide.

The effects of CheA (1-149) P1-containing polypeptide on the chemotactic abilities of Escherichia coli was analyzed to determine the mechanisms that mediate microbial chemotactic signalling pathway. The CheA (1-149) fragments disrupted chemotactic signaling pathway by diverting in vivo phosphate fluxes from normal signaling routes. The histidine kinase also exhibited transphosphorylation and phosphotransfer capabilities by interacting with CheB and CheY .

The lesions in CheA sub l (654 amino acids) and not the lack of CheA sub s were responsible for the the reduced chemotactic ability of the M981 mutant of Escherichia coli. Normal behaviour of M981 is noticed and its flagellar rotation pattern is not distinguishable from wild type exhibiting wild-type detection limit and peak positions in capillary chemotaxis assays. It is concluded that CheA sub makes a little contribution to chemotactic ability in the laboratory and its functional importance in other settings is not yet clear.

Isolation of soluble cytoplasmic domain segments from the serine chemoreceptor Tsr that enhances counterclockwise (CCW) and clockwise (CW) rotations reveals that a 180-residue fragment in the cytoplasmic domain of Tsr generates two active signals for CW and CCW. Interactions between CheA kinase and the full-length receptors mediate CCW signal release while CheW directly plays a role in CW signal release. The in vivo behavior of Tsr and the in vitro influence of the signaling fragments of Tsr on CheA functions are in agreement with the phosphorelay model for chemotactic signaling.

This article comments on a latest research finding in the area of bacterial chemotaxis and its regulation. Research indictes that the response regulator CheY, which in its phospho-CheY form binds to the FliM protein of the flagellar motors to mediate clockwise rotation. Data suggest CheY residue plays a role in the response regulator's phosphorylation activities.

The serine chemoreceptor of Escherichia coli contains four canonical methylation sites for sensory adaptation that lie near intersubunit helix interfaces of the Tsr homodimer. An unexplored fifth methylation site, E502, lies at an intrasubunit helix interface closest to the HAMP domain that controls input-output signaling in methyl-accepting chemotaxis proteins. We analyzed, with in vivo Förster resonance energy transfer (FRET) kinase assays, the serine thresholds and response cooperativities of Tsr receptors with different mutationally imposed modifications at sites 1 to 4 and/or at site 5. Tsr variants carrying E or Q at residue 502, in combination with unmodifiable D and N replacements at adaptation sites 1 to 4, underwent both methylation and demethylation/deamidation, although detection of the latter modifications required elevated intracellular levels of CheB. These Tsr variants could not mediate a chemotactic response to serine spatial gradients, demonstrating that adaptational modifications at E502 alone are not sufficient for Tsr function. Moreover, E502 is not critical for Tsr function, because only two amino acid replacements at this residue abrogated serine chemotaxis: Tsr-E502P had extreme kinase-off output and Tsr-E502I had extreme kinase-on output. These large threshold shifts are probably due to the unique HAMP-proximal location of methylation site 5. However, a methylation-mimicking glutamine at any Tsr modification site raised the serine response threshold, suggesting that all sites influence signaling by the same general mechanism, presumably through changes in packing stability of the methylation helix bundle. These findings are consistent with control of input-output signaling in Tsr through dynamic interplay of the structural stabilities of the HAMP and methylation bundles. [ABSTRACT FROM AUTHOR]

The four transmembrane chemoreceptors of Escherichia coil sense phenol as either an attractant (Tar) or a repellent (Tap, Trg, and Tsr). In this study, we investigated the Tar determinants that mediate its attractant response to phenol and the Tsr determinants that mediate its repellent response to phenol. Tar molecules with lesions in the aspartate-binding pocket of the periplasmic domain, with a foreign periplasmic domain (from Tsr or from several Pseudomonas chemoreceptors), or lacking nearly the entire periplasmic domain still mediated attractant responses to phenol. Similarly, Tar molecules with the cytoplasmic methylation and kinase control domains of Tsr still sensed phenol as an attractant. Additional hybrid receptors with signaling elements from both Tar and Tsr indicated that the transmembrane (TM) helices and HAMP domain determined the sign of the phenol-sensing response. Several amino acid replacements in the HAMP domain of Tsr, particularly attractant-mimic signaling lesions at residue E248, converted Tsr to an attractant sensor of phenol. These findings suggest that phenol may elicit chemotactic responses by diffusing into the cytoplasmic membrane and perturbing the structural stability or position of the TM bundle helices, in conjunction with structural input from the HAMP domain. We conclude that behavioral responses to phenol, and perhaps to temperature, cytoplasmic pH, and glycerol, as well, occur through a general sensing mechanism in chemoreceptors that detects changes in the structural stability or dynamic behavior of a receptor signaling element. The structurally sensitive target for phenol is probably the TM bundle, but other behaviors could target other receptor elements. [ABSTRACT FROM AUTHOR]

The C-terminal P5 domain of the histidine kinase CheA is essential for coupling CheA autophosphorylation activity to chemoreceptor control through a binding interaction with the CheW protein. To locate P5 determinants critical for CheW binding and chemoreceptor control, we surveyed cysteine replacements at 39 residues predicted to be at or near the P5 surface in Escherichia coli CheA. Two-thirds of the Cys replacement proteins exhibited in vitro defects in CheW binding, either before or after modification with a bulky fluorescein group. The binding-defective sites were widely distributed on the P5 surface and were often interspersed with sites that caused no functional defects, implying that relatively minor structural perturbations, often far from the actual binding site, can influence its conformation or accessibility. The most likely CheW docking area included loop 2 in P5 folding subdomain 1. All but four of the binding-defective P5-Cys proteins were defective in receptor-mediated activation, suggesting that CheW binding, as measured in vitro, is necessary for assembly of ternary signaling complexes and/or subsequent CheA activation. Other Cys sites specifically affected receptor-mediated activation or deactivation of CheA, demonstrating that CheW binding is not sufficient for assembly and/or operation of receptor signaling complexes. Because P5 is quite similar to CheW, whose structure is known to be dynamic, we suggest that conformational flexibility and dynamic motions govern the signaling activities of the P5 domain. In addition, relative movements of the CheA domains may be involved in CheW binding, in ternary complex assembly, and in subsequent stimulus-induced conformational changes in receptor signaling complexes. [ABSTRACT FROM AUTHOR]

The histidine autokinase CheA functions as the central processing unit in the Escherichia coli chemotaxis signaling machinery. CheA receives autophosphorylation control inputs from chemo receptors and in turn regulates the flux of signaling phosphates to the CheY and CheB response regulator proteins. Phospho-CheY changes the direction of flagellar rotation; phospho-CheB covalently modifies receptor molecules during sensory adaptation. The CheA phosphorylation site, His-48, lies in the N-terminal P1 domain, which must engage the CheA ATP-binding domain, P4, to initiate an autophosphorylation reaction cycle. The docking determinants for the P1-P4 interaction have not been experimentally identified. We devised mutant screens to isolate P1 domains with impaired autophosphorylation or phosphotransfer activities. One set of P1mutants identified amino acid replacements at surface-exposed residues distal to His-48. These lesions reduced the rate of P1 transphosphorylation by P4. However, once phosphorylated, the mutant P1 domains transferred phosphate to CheY at the wild-type rate. Thus, these P1mutants appear to define interaction determinants for P1-P4 docking during the CheA autophosphorylation reaction. [ABSTRACT FROM AUTHOR]

Chemoreceptors such as Tsr, the serine receptor, function in trimer-of-dimer associations to mediate chemotactic behavior in Escherichia coli. The two subunits of each receptor homodimer occupy different positions in the trimer, one at its central axis and the other at the trimer periphery. Residue N381 of Tsr contributes to trimer stability through interactions with its counterparts in a central cavity surrounded by hydrophobic residues at the trimer axis. To assess the functional role of N381, we created and characterized a full set of amino acid replacements at this Tsr residue. We found that every amino acid replacement at N381 destroyed Tsr function, and all but one (N381G) of the mutant receptors also blocked signaling by Tar, the aspartate chemoreceptor. Tar jamming reflects the formation of signaling-defective mixed trimers of dimers, and in vivo assays with a trifunctional cross-linking reagent demonstrated trimer-based interactions between Tar and Tsr-N381 mutants. Mutant Tsr molecules with a charged amino acid or proline replacement exhibited the most severe trimer formation defects. These trimer-defective receptors, as well as most of the trimer-competent mutant receptors, were unable to form ternary signaling complexes with the CheA kinase and with CheW, which couples CheA to receptor control. Some of the trimer-competent mutant receptors, particularly those with a hydrophobic amino acid replacement, may not bind CheW/CheA because they form conformationally frozen or distorted trimers. These findings indicate that trimer dynamics probably are important for ternary complex assembly and that N381 may not be a direct binding determinant for CheW/CheA at the trimer periphery. [ABSTRACT FROM AUTHOR]

In isotropic environments, an Escherichia coli cell exhibits coordinated rotational switching of its flagellar motors, produced by fluctuations in the intracellular concentration of phosphorylated CheY (CheY-P) emanating from chemoreceptor signaling arrays. In this study, we show that these CheY-P fluctuations arise through modifications of chemoreceptors by two sensory adaptation enzymes: the methyltransferase CheR and the methylesterase CheB. A cell containing CheR, CheB, and the serine chemoreceptor Tsr exhibited motor synchrony, whereas a cell lacking CheR and CheB or containing enzymatically inactive forms did not. Tsr variants with different combinations of methylation-mimicking Q residues at the adaptation sites also failed to show coordinated motor switching in cells lacking CheR and CheB. Cells containing CheR, CheB, and Tsr [NDND], a variant in which the adaptation site residues are not substrates for CheR or CheB modifications, also lacked motor synchrony. TsrΔNWETF, which lacks a C-terminal pentapeptide-binding site for CheR and CheB, and the ribose-galactose receptor Trg, which natively lacks this motif, failed to produce coordinated motor switching, despite the presence of CheR and CheB. However, addition of the NWETF sequence to Trg enabled Trg-NWETF to produce motor synchrony, as the sole receptor type in cells containing CheR and CheB. Finally, CheBc, the catalytic domain of CheB, supported motor coordination in combination with CheR and Tsr. These results indicate that the coordination of motor switching requires CheR/CheB-mediated changes in receptor modification state. We conclude that the opposing receptor substrate-site preferences of CheR and CheB produce spontaneous blinking of the chemoreceptor array's output activity. [ABSTRACT FROM AUTHOR]

The Escherichia coli Aer protein contains an N-terminal PAS domain that binds flavin adenine dinucleotide (FAD), senses aerotactic stimuli, and communicates with the output signaling domain. To explore the roles of the intervening F1 and HAMP segments in Aer signaling, we isolated plasmid-borne aerotaxis-defective mutations in a host strain lacking all chemoreceptors of the methyl-accepting chemotaxis protein (MCP) family. Under these conditions, Aer alone established the cell's run/tumble swimming pattern and modulated that behavior in response to oxygen gradients. We found two classes of Aer mutants: null and clockwise (CW) biased. Most mutant proteins exhibited the null phenotype: failure to elicit CW flagellar rotation, no aerosensing behavior in MCP-containing hosts, and no apparent FAD-binding ability. However, null mutants had low Aer expression levels caused by rapid degradation of apparently nonnative subunits. Their functional defects probably reflect the absence of a protein product. In contrast, CW-biased mutant proteins exhibited normal expression levels, wild-type FAD binding, and robust aerosensing behavior in MCP-containing hosts. The CW lesions evidently shift unstimulated Aer output to the CW signaling state but do not block the Aer input-output pathway. The distribution and properties of null and CW-biased mutations suggest that the Aer PAS domain may engage in two different interactions with HAMP and the HAMP-proximal signaling domain: one needed for Aer maturation and another for promoting CW output from the Aer signaling domain. Most aerotaxis-defective null mutations in these regions seemed to affect maturation only, indicating that these two interactions involve structurally distinct determinants. [ABSTRACT FROM AUTHOR]

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 σS 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. [ABSTRACT FROM AUTHOR]

The four transmembrane chemoreceptors of Escherichia coli sense phenol as either an attractant (Tar) or a repellent (Tap, Trg, and Tsr). In this study, we investigated the Tar determinants that mediate its attractant response to phenol and the Tsr determinants that mediate its repellent response to phenol. Tar molecules with lesions in the aspartate-binding pocket of the periplasmic domain, with a foreign periplasmic domain (from Tsr or from several Pseudomonas chemoreceptors), or lacking nearly the entire periplasmic domain still mediated attractant responses to phenol. Similarly, Tar molecules with the cytoplasmic methylation and kinase control domains of Tsr still sensed phenol as an attractant. Additional hybrid receptors with signaling elements from both Tar and Tsr indicated that the transmembrane (TM) helices and HAMP domain determined the sign of the phenol-sensing response. Several amino acid replacements in the HAMP domain of Tsr, particularly attractant-mimic signaling lesions at residue E248, converted Tsr to an attractant sensor of phenol. These findings suggest that phenol may elicit chemotactic responses by diffusing into the cytoplasmic membrane and perturbing the structural stability or position of the TM bundle helices, in conjunction with structural input from the HAMP domain. We conclude that behavioral responses to phenol, and perhaps to temperature, cytoplasmic pH, and glycerol, as well, occur through a general sensing mechanism in chemoreceptors that detects changes in the structural stability or dynamic behavior of a receptor signaling element. The structurally sensitive target for phenol is probably the TM bundle, but other behaviors could target other receptor elements.