Repellent response functions of the Trg and Tap chemoreceptors of Escherichia coli.

The chemoreceptors responsible for the repellent response of Escherichia coli to phenol were investigated. In the absence of all four known methyl-accepting chemoreceptors (Tar, Tsr, Trg, and Tap), cells showed no response to phenol. However, when Trg, which mediates the attractant response to ribose and galactose, was introduced via a plasmid, the cells acquired a repellent response to phenol. About 1 mM phenol induced a clear repellent response; this response was suppressed by 1 mM ribose. Thus, Trg mediates the repellent response to phenol. Mutant Trg proteins with altered sensing for ribose and galactose showed a normal response to phenol, indicating that the interaction site for phenol differs from that for the ribose- and galactose-binding proteins. Tap, which mediates the attractant response to dipeptides, mediated a weaker repellent response to phenol. Tsr, which mediates the attractant response to serine, mediated an even weaker response to phenol. Trg and Tap were also found to function as intracellular pH sensors. Upon a pH decrease, Trg mediated an attractant response, whereas Tap mediated a repellent response. These results indicate that all the receptors in E. coli have dual functions, mediating both attractant and repellent responses.     

Uropathogenic Escherichia coli strains generally lack functional Trg and Tap chemoreceptors found in the majority of E. coli strains strictly residing in the gut

The prevalence and function of four chemoreceptors, Tsr, Tar, Trg, and Tap, were determined for a collection of uropathogenic, fecal-commensal, and diarrheagenic Escherichia coli strains. tar and tsr were present or functional in nearly all isolates. However, trg and tap were significantly less prevalent or functional among the uropathogenic E. coli strains (both in 6% of strains) than among fecal-commensal strains (both in > or =50% of strains) or diarrheal strains (both in > or =75% of strains) (P < 0.02).     

Thermosensing properties of Escherichia coli tsr mutants defective in serine chemoreception.

Tsr, a chemoreceptor for serine and repellents in Escherichia coli, also functions as a thermoreceptor. The relationship between the chemoreceptor and thermoreceptor functions of Tsr was examined in five tsr mutants with altered serine detection thresholds. The thermosensing abilities of the mutant Tsr proteins were not affected by the alterations in their affinities to serine. In contrast, the ability of serine to inactivate thermoreceptor function was altered in these mutants. The minimal serine concentration required for thermoreceptor inactivation was directly related to the decreased affinity of the mutant Tsr for serine. The amino acid replacements in the mutant receptors were deduced from DNA sequence analyses and occurred at two different locations in the presumed periplasmic domain of Tsr. Two mutations caused histidine or cysteine replacements at arginine 64, whereas three others caused isoleucine or proline replacements at threonine 156.     

Thermosensing Function of the Escherichia coli Redox Sensor Aer

Escherichia coli chemoreceptors can sense changes in temperature for thermotaxis. Here we found that the aerotaxis transducer Aer, a homolog of chemoreceptors lacking a periplasmic domain, mediates thermoresponses. We propose that thermosensing by the chemoreceptors is a general attribute of their highly conserved cytoplasmic domain (or their less conserved transmembrane domain).Most organisms have evolved mechanisms to sense and respond to changes in temperature since they can live only within a limited temperature range. Although studies of such thermosensing systems are generally difficult, thermotaxis of Escherichia coli is exceptionally well characterized (5, 6). Wild-type E. coli cells are attracted to warmer and repelled by colder environments. These thermotactic behaviors are mediated by the E. coli chemotaxis signaling system, which regulates the cell''s direction of flagellar rotation. Attractant increases, sensed by transmembrane chemoreceptors (Tsr, Tar, Trg, and Tap), also known as methyl-accepting chemotaxis proteins (MCPs), promote counterclockwise (CCW) rotation and forward swimming, whereas repellent increases, sensed by the same receptors, promote clockwise (CW) rotation and random turns or tumbles (4). Early studies revealed that the chemoreceptors of E. coli also mediate thermotactic responses (5, 6). Tsr, Tar, and Trg function as warm sensors, which produce CCW signals upon temperature upshift and CW signals upon temperature downshift. In contrast, Tap functions as a cold sensor that produces signals of the opposite output to temperature changes (5, 7, 9). Tsr and Tar show altered thermosensing properties after adaptation to their attractants, serine and aspartate, respectively. Tsr loses temperature-sensing ability, whereas Tar shifts from a warm sensor to a cold sensor (7, 8, 10-12). To obtain deeper molecular insights into the thermosensing mechanism of receptors, in this study we focused on Aer, a redox sensor of E. coli. Aer has a cytoplasmic kinase control module like that of the other chemoreceptors, but instead of a periplasmic domain, Aer has a large amino-terminal, cytoplasmic PAS domain that binds flavin adenine dinucleotide (FAD) (Fig. ​(Fig.1;1; for a review, see reference 17).Open in a separate windowFIG. 1.Schematic illustration of the domain organizations of the redox sensor Aer and the aspartate chemoreceptor Tar. Tar, like any other MCP, is a homodimeric transmembrane protein, each subunit of which consists of, from the amino terminus to the carboxy terminus, the short cytoplasmic sequence, the first transmembrane helix (TM1), the periplasmic ligand-binding domain, the second transmembrane helix (TM2), the HAMP domain, and the kinase-control module. In contrast, Aer lacks a periplasmic domain but consists of the cytoplasmic PAS domain that binds FAD near the amino terminus, TM1, TM2, and the cytoplasmic domains of high similarity with MCPs. Aer also lacks methylation sites that are conserved in the kinase control modules of MCPs (depicted with closed circles in Tar) and required for chemotactic adaptation. Note that the monomers of Aer and Tar are shown here for simplicity although the minimum functional unit for Aer and Tar is thought to be a homodimer.In this study, we developed a new temperature-control device (Fig. ​(Fig.2).2). It consisted of a chamber formed from two coverslips (18 mm × 18 mm), with short pieces of glass capillaries (1.0 cm in length) fixed with epoxy adhesive serving as spacers between the two slips. Four silicon tubes were placed at the four corners of the chamber and sealed with epoxy adhesive. The two inlet tubes on the left side were connected to one larger tube (2-mm inside diameter) with a Y adapter. The two outlet tubes on the right side were connected together in the same way. Temperature changes inside the chamber were initiated by switching the water flow between two tanks maintained at different temperatures. The water flow was produced by siphon action, and the flow rate was adjusted by shifting the relative height of the tanks. Although the temperature range used in this study varied from 20°C to 30°C, this device could, in principle, be used to vary temperatures over different ranges. With this new device, we observed normal and inverted thermoresponses mediated by Tar (7) and confirmed that the responses were essentially identical to those observed in previous studies (data not shown).Open in a separate windowFIG. 2.Side (A) and top (B) views of the temperature control device used in this study. A glass slide with cell suspension was placed onto the thermo-control chamber mounted on the microscope stage. Immersion oil was applied at the interface between the thermo-control chamber and the glass slide to facilitate effective thermal conduction and to hold the glass slide with surface tension. Arrows indicate the flow of water into and out of the thermo-control chamber. See text for detail.To test whether Aer can mediate thermotactic responses, we introduced an aer-expressing plasmid, pSB20 (1), into a receptor-less strain, UU2612. (All strains and Aer-carrying plasmids used in this study were kindly provided by J. S. Parkinson.) Expression of Aer was induced with 100 μM isopropyl-β-d-thiogalactopyranoside (IPTG), unless otherwise mentioned, and confirmed by immunoblotting (data not shown). Thermoresponses were measured essentially as described previously (11). Briefly, cells were suspended in motility medium and appropriate concentrations of chemoeffectors were added when necessary. A drop of the cell suspension was placed on a glass slide, sealed with VALAP (equal mixture of petrolatum, lanolin, and paraffin; Wako Chemical, Tokyo, Japan), and mounted on the temperature control device. The aperture between the glass slide and the thermo-control chamber was filled with immersion oil to keep close contact (Fig. ​(Fig.2).2). Temperature changes were monitored by a thin thermocouple inserted into the cell suspension or immersion oil. Swimming patterns of the cells were measured quantitatively as described earlier (11). The transformants (UU2612/pSB20) swam with extreme smooth bias and did not show any change in swimming behavior upon temperature shifts (Fig. ​(Fig.3A,3A, open circles). It has been known that extreme signaling bias of a receptor sometimes disguises its intrinsic ability to sense a certain stimulus (9, 12). Since glycerol at high concentrations serves as a general repellent for all MCPs (9, 13), we tested whether it also works as a repellent for Aer. When 5% (wt/vol) or higher concentrations of glycerol were added, UU2612/pSB20 cells showed continuous tumbling responses (data not shown). In contrast, cells carrying the parent vector (pCJ30) did not show any response to glycerol (data not shown). In the presence of 5% (wt/vol) glycerol, UU2612/pSB20 cells showed smooth and tumbling responses upon temperature upshift and downshift, respectively (Fig. ​(Fig.3A,3A, closed circles). We also examined another repellent for Aer, 2,3-demethoxy-5-methyl-1,4-benzoquinone (hereinafter referred to as “benzoquinone”) (14). In the presence of 0.7 μM benzoquinone, cells showed similar responses upon temperature shifts (Fig. ​(Fig.3B).3B). In contrast, the same strain carrying the parent vector (pCJ30) did not show any response to the repellents or to subsequent temperature shifts (data not shown). These results suggest that Aer functions as a warm sensor.Open in a separate windowFIG. 3.Response of otherwise receptorless cells expressing Aer to changes in temperature. Suspensions of UU2612 (ΔMCP Δaer) cells carrying plasmid pSB20 (aer⁺) in motility medium without (A, open circles) or with 5% glycerol (A, closed circles) or 0.7 μM benzoquinone (B, closed circles) were prepared. The temperature was increased and decreased as shown in lower panels. Arrows indicate the onset of the temperature ramps: A, at 1 and 7 min, 20°C to 30°C, and at 3.5 and 10.5 min, 30°C to 20°C; B, at 1 min, 20°C to 30°C, and at 3.5 min, 30°C to 20°C. Note that temperature changes of the samples were delayed. Swimming behaviors of the cells were recorded, and the time courses of their smooth-swimming fractions were monitored.Moreover, the cells appeared to adapt to thermal stimulation (Fig. 3A and B): after the early responses, their smooth-swimming fraction tended to resume toward the original values without any further change in temperature. It has been reported that Aer does not have conventional methylation sites and can mediate aerotaxis in a methylation-independent manner (2), whereas methylation and demethylation of sensory receptors, catalyzed by the methyltransferase CheR and the methylesterase CheB, respectively, are required for chemotactic adaptation. To determine whether the adaptation observed in thermoresponses mediated by Aer is methylation dependent, Aer was expressed in a receptorless cheR cheB deletion strain, UU2610. The cells showed clear adaptation of their thermoresponses in the presence of 5% glycerol or 0.7 μM benzoquinone (see Fig. S1 in the supplemental material), suggesting that the adaptation seen in thermoresponses mediated by Aer is methylation independent.Because Aer is a redox sensor (17), it is important to consider the effect of oxygen depletion on the swimming behavior of the cells under our experimental conditions. When cells were exposed to two sets of identical thermal shifts (from 0 min to 6 min and from 6 min to 12 min), essentially the same response curves were obtained (Fig. ​(Fig.3A).3A). It is therefore unlikely that the responses observed with the device are affected by oxygen depletion, at least under the conditions tested.To exclude the possibility that repellents are required for thermosensing of Aer, we examined a CW-biased mutant Aer, Aer-S28G. Aer-S28G produces higher CW output than wild-type Aer but can still mediate an aerotactic response (J. S. Parkinson, personal communication). Without glycerol, cells expressing Aer-S28G as a sole receptor showed smooth and tumbling responses upon temperature upshift and downshift, respectively (Fig. ​(Fig.4A).4A). Aer-mediated thermoresponses were also observed without any repellent when wild-type Aer was coexpressed with a CW-biased cytoplasmic Tar fragment that lacks thermosensing ability, though the responses were weak (data not shown). Since changes in temperature can also affect the rate of respiration, it is possible that such upstream elements are involved in Aer thermosensing. To test this possibility, we examined the thermosensing property of an Aer mutant that is defective in FAD binding, Aer-R57H (19). UU2610 cells were transformed with plasmid pSB20 carrying the mutant aer gene. UU2610/pSB20 Aer-R57H cells in which the mutant gene induced with 12 μM IPTG was used for the induction still showed a repellent (tumbling) response to 10% glycerol but did not respond to 100 μM benzoquinone, as expected (data not shown). The cells showed inverted responses upon temperature changes (Fig. ​(Fig.4B),4B), suggesting that Aer-R57H functions as an intrinsic cold sensor and FAD binding is not absolutely required for the thermosensing function of Aer, although the cause of the inversion remains to be elucidated.Open in a separate windowFIG. 4.Thermoresponses of cells expressing the CW-biased Aer mutant (A, S28G) or the FAD-binding Aer mutant (B, R57H) as a sole thermosensor. Suspensions of UU2612 (ΔMCP Δaer) cells carrying plasmid pSB20-Aer-S28G or UU2610 (ΔMCP Δaer ΔcheRB) cells carrying plasmid pSB20-Aer-R57H without any repellent were prepared. The temperature was changed as shown in the lower panel of Fig. ​Fig.3B;3B; arrows indicate the onsets of the temperature ramps.It is likely that temperature sensing involves reversible conformational changes of the receptor induced by changes in temperature, a mechanism similar to sensing of chemoeffectors. Although temperature can affect any part of the receptor, there could be a critical domain whose structure is particularly sensitive to temperature. A temperature-induced structural change in such a domain would propagate to the whole protein to switch signal output. Now that the periplasmic domain can be excluded, the most likely candidates for a temperature-sensing element are the transmembrane helices, the HAMP domain, and the kinase control module (Fig. ​(Fig.1).1). In the previous study, we found that mutations in the second transmembrane domain (TM2) of Tar cause inversion of its thermosensing profile (10), suggesting the importance of the TM segments for thermosensing. Changes in temperature might cause reorientation and/or alteration of packing interactions of the transmembrane helices (TM1 and TM2) that are similar to those caused by aspartate binding to Tar (3). The HAMP domain (named after histidine kinases, adenylyl cyclases, MCPs, and phosphatases), which connects TM2 to the cytoplasmic kinase control module, is commonly used in transmembrane sensors in various species of prokaryotes and thought to function as a flexible sensory transduction module (18). The HAMP domains of chemoreceptors might modulate signal output by undergoing stimulus-induced changes in dynamic behavior (20), a conformational property that should also respond to temperature changes. The conserved kinase control module also seems a likely target for temperature control, because modification of this module by methylation of Tar causes conversion of its thermosensing ability (9, 11, 12). After analyses using Tar mutants with altered methylation sites, we proposed a model that methylation may shift the equilibrium of the signaling states by altering the interaction between α-helices of the kinase control module within a Tar homodimer or between Tar homodimers (11, 12). Similarly, it has been proposed that signaling states of chemoreceptors include modulation of dynamics in adaptation/protein-interaction regions, which constitute the kinase control module (15, 16). Such dynamics can be readily affected by temperature. It should be intriguing to examine Aer/Tar fragments carrying or lacking the TM2 region, the HAMP domain, and/or the kinase control module in various combinations. Finally, it is unlikely that the FAD binding of Aer plays a key role in thermosensing because the FAD-binding mutant Aer-R57H retained the thermosensing function, although it mediated inverted thermoresponses (Fig. ​(Fig.4B4B).In conclusion, we found that Aer has an intrinsic thermosensing ability and therefore the periplasmic domain is not essential for the thermosensing function of a receptor. Since changes in temperature are supposed to induce conformational changes of the receptors equivalent to those induced by binding of chemical attractants and repellents, further characterization of temperature sensing of MCPs and Aer should shed new light on molecular mechanisms underlying receptor signaling.      

Multiple covalent modifications of Trg, a sensory transducer of Escherichia coli

The sensory transducers of Escherichia coli are integral membrane proteins that mediate the tactic response of cells to chemical stimuli. Adaptation to environmental stimuli is correlated with methylation of the transducer proteins. Two transducer genes, tsr and tar, exhibit extensive homologies while no homology has been detected between a third transducer, trg, and those genes. The Tsr and Tar proteins have been shown to contain multiple sites for methylation as well as two sites for another modification that requires an active cheB gene product and is designated the CheB-dependent modification. In this study, covalent modifications of the Trg protein were characterized by analysis of tryptic peptides. We found that methylation occurred at several sites on the Trg protein and that the protein contained at least three sites for CheB-dependent modification, two of which were located on a tryptic peptide that contains both methionine and lysine. This tryptic peptide is analogous to the methionine- and lysine-containing methyl-accepting peptides isolated from the Tsr and Tar proteins and like those peptides may contain several methyl-accepting sites. We estimated the pKa of the group created by the CheB-dependent modification on the methionine- and lysine-containing peptide of Trg to be between pH 2.2 and 5.8. This result supports the idea that the CheB-dependent modification is an enzymatic deamidation of glutamine to glutamic acid.     

Sites of covalent modification in Trg, a sensory transducer of Escherichia coli

The Trg protein mediates chemotactic response of Escherichia coli to the attractants ribose and galactose. Like other transducers, Trg is a transmembrane protein that undergoes post-translational covalent modification. The modifications are hydrolysis (deamidation) of certain glutamine side chains to create glutamate residues and methylation of specific glutamates to form carboxyl methyl esters. Analysis of radiolabeled, tryptic peptides by high performance liquid chromatography and gas-phase sequencing allowed direct identification of the modified residues of Trg. The protein has 5 methyl-accepting residues. Four, at positions 304, 310, 311, and 318, are contained in a 23-residue tryptic peptide ending in lysine. The fifth, at position 500, is within a 25-residue tryptic peptide ending in arginine. At two sites, 311 and 318, glutamines are deamidated to create methyl-accepting glutamates. There is not a required order of modification among the sites. However, there is a substantial preference for methylation on the arginine peptide and, among sites on the lysine peptide, for the middle pair. Comparison of sequences surrounding modified residues identified in this work for Trg and previously for Tsr and Tar suggests a consensus sequence for methyl-accepting sites of Ala/Ser-Xaa-Xaa-Glu-Glu*-Xaa-Ala/OH-Ala-OH/Ala, where OH signifies Ser or Thr and the asterick marks the site of modification.     

Differential activation of Escherichia coli chemoreceptors by blue-light stimuli

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 +/- 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 muM 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.     

Plasmids in Escherichia coli controlling citrate-utilizing ability.

The citrate-utilizing ability of 19 out of 22 citrate-positive Escherichia coli strains isolated from pig sewage was transferred via conjugation to E. coli K-12. The conjugal transfer of citrate-utilizing (Cit) abilities was thermosensitive and concurrent with transfer of drug resistance. Weakly citrate-positive colonies were readily obtained in conjugation experiments. Their Cit characters could be transmitted to the other E. coli strains at a similar frequency in the retransfer experiments, and the transconjugants obtained still showed same characteristic growth on Simmons citrate agar plates. The 19 thermosensitive plasmids conferring citrate utilization and drug resistance were Fi-, and 16 of these plasmids belonged to incompatibility group H1. However, occasionally two conjugative plasmids (pOH3122-1 and pOH3124-1) carrying only the citrate utilization were also obtained in the conjugation experiments, and they were Fi+ and compatible with 19 reference R plasmids. In the two citrate-positive E. coli strains, it was suggested that the conjugative Cit plasmid showing Fi+ character and the more thermosensitive H1 plasmid conferring both the Cit character and drug resistance coexisted in the strain. The characterization of citrate utilization plasmids derived from pig farm sewage is discussed.     

Phenol sensing by Escherichia coli chemoreceptors: a nonclassical mechanism

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.     

Thermosensing properties of mutant aspartate chemoreceptors with methyl-accepting sites replaced singly or multiply by alanine.

The aspartate chemoreceptor Tar has a thermosensing function that is modulated by covalent modification of its four methylation sites (Gln295, Glu302, Gln309, and Glu491). Without posttranslational deamidation, Tar has no thermosensing ability. When Gln295 and Gln309 are deamidated to Glu, the unmethylated and heavily methylated forms function as warm and cold sensors, respectively. In this study, we carried out alanine-scanning mutagenesis of the methylation sites. Although alanine substitutions influenced the signaling bias and the methylation level, all of the mutants retained aspartate-sensing function. Those with single substitutions had almost normal thermosensing properties, indicating that substitutions at any particular methylation site do not seriously impair thermosensing function. In the posttranslational modification-defective background, some of the alanine substitutions restored thermosensing ability. Warm sensors were found among mutants retaining two glutamate residues, and cold sensors were found among those with one or no glutamate residue. This result suggests that the negative charge at the methylation sites is one factor that determines thermosensor phenotypes, although the size and shape of the side chain may also be important. The warm, cold, and null thermosensor phenotypes were clearly differentiated, and no intermediate phenotypes were found. Thus, the different thermosensing phenotypes that result from covalent modification of the methylation sites may reflect distinct structural states. Broader implications for the thermosensing mechanism are also discussed.     

Signaling interactions between the aerotaxis transducer Aer and heterologous chemoreceptors in Escherichia coli

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 trifunctional 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.     

Purification of receptor protein Trg by exploiting a property common to chemotactic transducers of Escherichia coli

The methyl-accepting chemotactic transducers of Escherichia coli were found to bind strongly to Cibacron blue-Sepharose. Among potential elutants tested, only S-adenosylmethionine at moderate concentrations and NaCl at concentrations greater than 1.5 M caused dissociation of these detergent-solubilized transmembrane proteins from the dye. Release by S-adenosylmethionine may be a generalized effect rather than the result of a specific binding site for that compound on transducers. A truncated trg gene was created that coded for the carboxyl-terminal three-fifths of the transducer, which constitutes the cytoplasmic domain common to all four transducers in E. coli. This domain bound to Cibacron blue-Sepharose and was eluted in a pattern similar to that exhibited by intact Trg, indicating that interaction with the dye occurred in this conserved domain. Adherence to Cibacron blue and elution by high salt formed the core of an efficient purification scheme, developed for Trg but applicable to all transducers in E. coli and perhaps to methyl-accepting chemotaxis proteins in other species. Determination of the amino acid sequence at the beginning of purified Trg confirmed that it contained a longer hydrophilic segment at its amino terminus than other transducers of E. coli. The initial methionine of Trg is neither cleaved nor modified, in contrast to the Tar transducer in which the amino terminus was found previously to be blocked. Circular dichroic measurements of purified Trg indicated that the secondary structural organization of the protein is predominantly alpha-helix.     

Recipient ability of bacteriophage-resistant mutants of Escherichia coli K-12.

The ability of a wide range of bacteriophage-resistant mutants to act as recipients in conjugation with F'lac pro and R100-1 donors has been studied. A number of mutant types defective in recipient ability with F'lac pro, as well as mutants which were hyperrecptive with R100-1, have been detected.     

Characterization of vaginal lactobacilli coaggregation ability with Escherichia coli

The coaggregation abilities of probiotic strains might enable it to form a barrier that prevents colonization by pathogenic bacteria. In the present study, the characterization of the coaggregation ability of 19 vaginal lactobacilli was studied. Coaggregation ability of all lactobacilli with Escherichia coli ATCC 11229 was positive. Only the highest coaggregation percentage of Lactobacillus acidophilus S1 was obtained with E. coli ATCC 11229 under both aerobic (71%) and anaerobic conditions (62%). The coaggregation abilities of strains occurred higher at acidic pH than at basic pH values. Moreover, the coaggregation abilities of tested strains against E. coli decreased after heat treatment (70 or 85 °C). Also, the relationship between hydrophobicity and coaggregation of strains was found to be significant. The effect of sonication, some enzymes (lipase and pepsin) and sodium periodate on coaggregation ability of L. acidophilus S1, which is one of the highest potentials on coaggregation ability, was investigated. Sodium periodate did not have a significant effect on coaggregation ability of L. acidophilus S1. The sonicated cell showed lower coaggregation than the control, the supernatant fluid of this sonicated cells showed similar coaggregation ability to the control. Coaggregation abilities of bacteriotherapeutic lactobacilli with pathogenic bacteria can be used for preliminary screening in order to identify potentially probiotic bacteria suitable for human use against urogenital tract infections.     

Substrate-binding ability of Escherichia coli ribonucleic acid polymerase in relation to its protein composition.

The metabolism of [3H]progesterone in the rabbit endometrium and myometrium was studied in vitro. The major metabolities identified were 5alpha-pregnane-3,20-dione, 20alpha-hydroxypregn-4-en-3-one, 3beta-hydroxy-5alpha-preganan-20-one and 5alpha-pregnane-3beta,20alpha-diol. Other minor metabolites tentatively identified were 3alpha-hydroxy-5beta-pregnan-20-one,20alpha-hydroxy-5beta-pregnan-3-one and 5beta-pregnane-3alpha,20alpha-diol. The ability of the endometrium to metabolize progesterone on a unit weight bais was about 2.7 times that of the myometrium. The metabolism of [3H]progesterone in the rabbit uterus under the influnce of oestradiol-17beta and progesterone was studied. The ability of the oestradiol-treated rabbit uterus to metabolize progesterone was increased to 3.47 times that of the overiectomized control uterus, whereas the oestradiol-progesterone-treated rabbit uterus metabolized only 1.86 times that of the control. Study of the metabolism of progesterone with uterine subcellular preparations revealed that the 5alpha-reductase enzyme was present mainly in the nuclear fraction; 20alpha-hydroxysteroid dehydrogenase was found in the cytosol fraction and 3beta-hydroxysteroid dehydrogenase in the particulate fraction of the uterus. The metabolic pathways of progesterone in the rabbit uterine tissue are discussed.     

Site-specific tamoxifen-DNA adduct formation: lack of correlation with mutational ability in Escherichia coli.

We have mapped sites of tamoxifen adduct formation, in the lacI gene using the polymerase STOP assay, following reaction in vitro with alpha-acetoxytamoxifen and horseradish peroxidase (HRP)/H(2)O(2) activated 4-hydroxytamoxifen. For both compounds, most adduct formation occurred on guanines. However, one adenine, within a run of guanines, generated a strong polymerase STOP site with activated 4-hydroxytamoxifen, and a weaker STOP site with alpha-acetoxytamoxifen at the same location. In Escherichia coli the lac I gene reacted with 4-hydroxytamoxifen was more likely to be mutated (2 orders of magnitude) than when reacted with alpha-acetoxytamoxifen, despite the greater DNA adduct formation by alpha-acetoxytamoxifen. This correlates with the greater predicted ability of activated 4-hydroxytamoxifen adducts to disrupt DNA structure than alpha-acetoxytamoxifen adducts. For lac I reacted with activated 4-hydroxytamoxifen, a hot spot of base mutation was located in the region of the only adenosine adduct. No mutational hot spots were observed with alpha-acetoxytamoxifen. Our data clearly shows a lack of correlation between gross adduct number, as assayed by (32)P-postlabeling and mutagenic potential. These data indicate the importance of minor adduct formation in mutagenic potential and further that conclusions regarding the mutagenicity of a chemical may not be reliably derived from the gross determination of adduct formation.