PubMed Health⌕ Search

Biomedical subjects

G L Hazelbauer

Publications and source records attributed to G L Hazelbauer.

At least 37 records · Page 2Linked to original sources
PubMed1993

Strategies for differential sensory responses mediated through the same transmembrane receptor.

Trg mediates chemotaxis of Escherichia coli to galactose and ribose by recognition of respective, sugar-occupied binding proteins. Although both attractants act through one transmembrane receptor, maximal response is approximately 50% greater to ribose. This phenomenon was investigated by mutational analysis of a 20-residue segment of Trg implicated in ligand interaction and signalling. Among 17 defective receptors, responses to the two chemoattractants were reduced equivalently for seven and differentially for 10, in some cases reversing the preference order. Mutational substitutions with equivalent effects occurred throughout the segment, but those with a greater effect on galactose or ribose response were segregated to the amino-terminal two-thirds or the carboxy-terminal one-third, respectively, a segregation corresponding in large part to a functional division based on signalling phenotypes. A model for binding protein-mediated recognition revealed two strategies for differential responses. The wild-type preference for ribose probably reflects a balance of receptor affinities and a limiting supply of binding proteins. Mutants with reversed preference probably have differentially reduced receptor affinities and those with an accentuated ribose preference probably have altered signalling abilities. Two-step recognition of ligand allows functional separation of detection and response.

Bacterial Proteins↗
PubMed1993

Proteins antigenically related to methyl-accepting chemotaxis proteins of Escherichia coli detected in a wide range of bacterial species.

The four methyl-accepting chemotaxis proteins of Escherichia coli, often called transducers, are transmembrane receptor proteins that exhibit substantial identity among the sequences of their cytoplasmic domains. Thus, antiserum raised to one of these proteins recognizes the others and might be expected to recognize related proteins in other bacteria. We used antiserum raised to the transducer Trg in immunoblot experiments to probe a wide range of bacterial species for the presence of antigenically related proteins. Such proteins were detected in over 20 different species, representing 6 of the 11 eubacterial phyla defined by analysis of rRNA sequences as well as one archaebacterial group. Species containing proteins antigenically related to the transducers of E. coli included members of all four subdivisions of the phylum in which E. coli is placed, members of four of the six subdivisions of spirochetes, and two gliding bacteria. These observations provide substantial support for the notion that methyl-accepting taxis proteins are widely distributed among the diversity of bacterial species.

Antibodies, Bacterial↗
PubMed1992

Ligand occupancy mimicked by single residue substitutions in a receptor: transmembrane signaling induced by mutation.

We used mixed, mutagenic oligonucleotides to create single amino acid substitutions in the bacterial chemoreceptor Trg. Mutagenesis was directed at a 20-residue segment of the periplasmic domain implicated in ligand recognition. Transmembrane signaling by the mutant receptors was assayed in vivo by monitoring adaptational covalent modification. Among 20 functionally altered but stable receptors there were two distinct signaling phenotypes. Insensitive receptors did not signal upon stimulation and thus appeared defective in productive ligand interaction. Mimicked-occupancy receptors exhibited transmembrane signaling without ligand. Many mimicked-occupancy receptors produced additional signaling upon ligand binding and in appropriate conditions mediated effective chemotaxis; most insensitive receptors did not. Like normal receptors with one binding site occupied, mimicked-occupancy proteins adapted to persistent transmembrane signaling by increased methylation and thus could respond to other stimuli. Signaling phenotypes were strikingly segregated by residue position. Substitutions mimicking ligand occupancy occurred in half the segment, and those creating insensitive phenotypes occurred in the other half. These observations could be related to the three-dimensional structure of the periplasmic domain of the Tar(s) chemoreceptor. Insensitive substitutions occurred near the distal end of helix 1, where bulky protein ligands could interact; occupancy-mimicking substitutions were on the same helix at positions buried in the subunit interface between helices 1 and 1'. Thus perturbation of the interface induced transmembrane signaling, implicating changes at that interface in signal transduction, a conclusion consistent with differences in crystal structures of unoccupied and ligand-occupied Tar(s).

Amino Acid Sequence↗
PubMed1991

Structural features of methyl-accepting taxis proteins conserved between archaebacteria and eubacteria revealed by antigenic cross-reaction.

A number of eubacterial species contain methyl-accepting taxis proteins that are antigenically and thus structurally related to the well-characterized methyl-accepting chemotaxis proteins of Escherichia coli. Recent studies of the archaebacterium Halobacterium halobium have characterized methyl-accepting taxis proteins that in some ways resemble and in other ways differ from the analogous eubacterial proteins. We used immunoblotting with antisera raised to E. coli transducers to probe shared structural features of methyl-accepting proteins from archaebacteria and eubacteria and found substantial antigenic relationships. This implies that the genes for the contemporary methyl-accepting proteins are related through an ancestral gene that existed before the divergence of arachaebacteria and eubacteria. Analysis by immunoblot of mutants of H. halobium defective in taxis revealed that some strains were deficient in covalent modification of methyl-accepting proteins although the proteins themselves were present, while other strains appeared to be missing specific methyl-accepting proteins.

Archaea↗
PubMed1990

Effects of glutamines and glutamates at sites of covalent modification of a methyl-accepting transducer.

Chemotactic transducer proteins of Escherichia coli contain four or five methyl-accepting glutamates that are crucial for sensory adaptation and gradient sensing. Two residues arise from posttranslational deamidation of glutamines to yield methyl-accepting glutamates. We addressed the significance of this arrangement by creating two mutated trg genes: trg(5E), coding for a transducer in which all five modification sites were synthesized as glutamates, and trg(5Q), in which all five were glutamines. We found that the normal (3E,2Q) configuration was not an absolute requirement for synthesis, assembly, or stable maintenance of transducers. Both mutant proteins were methylated, although Trg(5Q) had a reduced number of methyl-accepting sites because two glutamines at adjacent residues were blocked for deamidation and thus could not become methyl-accepting glutamates. The glutamine-glutamate balance had striking effects on signaling state. Trg(5E) was in a strong counterclockwise signaling configuration, and Trg(5Q) was in a strong clockwise signaling induced by ligand binding, and alanines substituted at modification sites had an intermediate effect. Chemotactic migration by growing cells containing trg(5E) or trg(5Q) exhibited reduced effectiveness, probably reflecting perturbations of the counterclockwise/clockwise ratio caused by newly synthesized transducers not modified rapidly enough to produce a balanced signaling state during growth. These defects were evident for cells in which other transducers were not available to contribute to balanced signaling or were present at lower levels than the mutant proteins.

Bacterial Proteins↗
PubMed1990

Characterization of Halobacterium halobium mutants defective in taxis.

Mutant derivatives of Halobacterium halobium previously isolated by using a procedure that selected for defective phototactic response to white light were examined for an array of phenotypic characteristics related to phototaxis and chemotaxis. The properties tested were unstimulated swimming behavior, behaviorial responses to temporal gradients of light and spatial gradients of chemoattractants, content of photoreceptor pigments, methylation of methyl-accepting taxis proteins, and transient increases in rate of release of volatile methyl groups induced by tactic stimulation. Several distinct phenotypes were identified, corresponding to a mutant missing photoreceptors, a mutant defective in the methyltransferase, a mutant altered in control of the methylesterase, and mutants apparently defective in intracellular signaling. All except the photoreceptor mutant were defective in both chemotaxis and phototaxis.

Bacterial Proteins↗
PubMed1989

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.

Amino Acids↗
PubMed1989

Methyl-accepting taxis proteins in Halobacterium halobium.

Methyl-accepting taxis proteins were identified and characterized in Halobacterium halobium, an archaebacterial species that is both chemotactic and phototactic. The data suggest direct involvement of methylation and demethylation in mechanisms of both chemotaxis and phototaxis and identify adaptation as the sensory process in which those reactions are likely to be involved. Analysis by electrophoresis and fluorography revealed methyl-accepting species, of apparent Mr between 90,000 and 135,000, that exhibited characteristics of sensory components. Those methyl-3H-labeled species were absent in a mutant blocked in taxis. Methylation of specific bands increased after positive chemostimuli and decreased after negative stimuli. Other methyl-3H-labeled bands, from 17 to 29 kd, exhibited features of biosynthetic intermediates, not of sensory components. Assay of rates of demethylation by measuring release of volatile forms of radiolabeled methyl groups revealed transient changes following chemo- or photostimuli that persisted for periods roughly equivalent to adaptation times. Negative chemostimuli induced increased rates of demethylation, as expected from fluorographic analysis, but positive chemostimuli also resulted in an increase. Photostimuli of either sign were followed by increases in rates of demethylation of shorter duration and lesser magnitude than chemostimuli-induced increases, a relationship that corresponded to differences in adaptation time.

Bacterial Proteins↗
PubMed1989

Adaptational "crosstalk" and the crucial role of methylation in chemotactic migration by Escherichia coli.

We investigated roles of methylation in bacterial chemotaxis by characterizing a methyl-accepting transducer protein incapable of methylation because of amino acid substitutions at the modification sites. Mutant Trg protein recognized ligand and generated excitatory signals that affected flagella but was unable to mediate efficient adaptation or net cellular migration in a relevant chemical gradient. Defects caused by lack of methyl-accepting sites on Trg were suppressed by a sufficient cellular content of other transducer molecules with functional methyl-accepting sites. These observations establish directly that methylation is crucial for transducer-mediated chemotaxis and that neither phosphotransfer reactions among the soluble Che proteins nor other interaction among those chemotactic components can effectively fulfill the functions of methylation. Suppression was correlated with adaptational "crosstalk" in which unoccupied methyl-accepting transducers acquired methyl groups, thus apparently substituting effectively for blocked methyl-accepting sites on the transducer. A plausible model for this phenomenon is that increased methylation of unstimulated transducers results from global inhibition of the demethylating enzyme in a cell with a normally active methyltransferase and no available methyl-accepting sites on the stimulated, mutant transducer. Thus methylation can perform its roles in adaptation and gradient sensing even if modification occurs on molecules different from those that recognize the stimulating compound. This observation emphasizes the central role of methylation and the modular nature of the chemosensory system.

Bacterial Proteins↗
PubMed1988

Site-directed mutations altering methyl-accepting residues of a sensory transducer protein.

The Trg protein is one of a family of transducer proteins that mediate chemotactic response in Escherichia coli. Transducers are methyl-accepting proteins that gain or lose methyl esters on specific glutamyl residues during sensory adaptation. In this study, the significance of multiple sites of methylation on transducer proteins was addressed by using oligonucleotide-directed, site-specific mutagenesis to substitute an alanyl residue at each of the five methyl-accepting sites in Trg. The resulting collection of five mutations, each inactivating a single site, was analyzed for effects on covalent modification at the remaining sites on Trg and for the ability of the altered proteins to mediate sensory adaptation. Most of the alanyl substitutions had substantial biochemical effects, enhancing or reducing methyl-accepting activity of other sites, including one case of activation of a site not methylated in wild-type protein. Analysis of the altered proteins provided explanations for many features of the complex pattern of electrophoretic forms exhibited by Trg. The mutant proteins were less efficient than normal Trg in mediating adaptation. Correlation of biochemical and behavioral data indicated that reduction in the number of methyl-accepting sites on the transducer lengthened the time required to reach an adapted state.

Alanine↗
PubMed1987

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.

Amino Acid Sequence↗
PubMed1986

Transfer of chromosomal mutations to plasmids via Hfr-mediated conduction.

Nonconjugative plasmids can be transferred from an Hfr donor to a recipient by Hfr-mediated conduction. We found that this phenomenon can be employed to obtain a plasmid in which a mutation in a chromosomal gene has been transferred to the copy of that gene contained in the hybrid plasmid.

Bacterial Proteins↗
PubMed1986

Mutations specifically affecting ligand interaction of the Trg chemosensory transducer.

The Trg transducer mediates chemotactic response to galactose and ribose by interacting, respectively, with sugar-occupied galactose- and ribose-binding proteins. Adaptation is linked to methylation of specific glutamyl residues of the Trg protein. This study characterized two trg mutations that affect interaction with binding protein ligands but do not affect methylation or adaptation. The mutant phenotypes indicated that the steady-state activity of methyl-accepting sites is independent of ligand-binding activity. The mutation trg-8 changed arginine 85 to histidine, and trg-19 changed glycine 151 to aspartate. The locations of the mutational changes provided direct evidence for functioning of the amino-terminal domain of Trg in ligand recognition. Cross-inhibition of tactic sensitivity by the two Trg-linked attractants implies competition for a common site on Trg. However, the single amino acid substitution caused by trg-19 greatly reduced the response to galactose but left unperturbed the response to ribose. Thus Trg must recognize the two sugar-binding proteins at nonidentical sites, and the complementary sites on the respective binding proteins should differ. trg-8 mutants were substantially defective in the response to both galactose and ribose. An increase in cellular content of Trg-8 protein improved the response to galactose but not to ribose. It appears that Trg-8 protein is defective in the generation of the putative conformational change induced by ligand interaction. The asymmetry of the mutational defect implies that functional separation of interaction sites could persist beyond the initial stage of ligand binding.

Bacterial Proteins↗
PubMed1986

Mutation plus amplification of a transducer gene disrupts general chemotactic behavior in Escherichia coli.

Transducers are transmembrane receptor proteins that generate intracellular signals on stimulation and participate in adaptation by appropriate changes in the level of methylation. The transducer mutation trg-21 conferred a Trg- phenotype and defective taxis to galactose and ribose but a normal response to other attractants when present in a single chromosomal copy. Amplification of trg-21 by a multicopy plasmid made host cells generally nonchemotactic. The dominant phenotype resulted from a strong counterclockwise rotational bias of flagellar motors in Che- cells. Apparently, the Trg21 transducer sends a continuous counterclockwise signal to flagella independent of tactic stimulation. It appears that the cell has a homeostatic capacity that is sufficient to compensate for the effect of mutant transducers produced from a single chromosomal copy of trg-21, but the capacity is exceeded in cells that have multiple copies of the gene. The Trg21 protein did not have a significant effect on methylesterase activity, indicating that the two global effects of a stimulated transducer, that is, on flagellar rotation and on modification enzymes, can occur independently. The mutant protein exhibited essentially normal turnover of methyl groups but had a drastic defect in deamidation which thus reduced the number of methyl-accepting sites. The trg-21 mutation substitutes a threonine for Ala-419. This alanine is a conserved residue in all sequenced transducers and is in a region of the carboxy-terminal domain in which homology among the transducers is very high. The Trg21 phenotype implicates this conserved region in the generation of the excitatory signal which is directed at the flagella.

Bacterial Proteins↗
PubMed1985

A signal sequence mutant defective in export of ribose-binding protein and a corresponding pseudorevertant isolated without imposed selection.

Ribose-binding protein is exported to the periplasmic compartment of Escherichia coli by a process that involves proteolytic cleavage of an amino-terminal extension of amino acids from the precursor form of the protein. In a collection of mutants isolated as defective in the Rbs transport system, a strain was identified that contained only precursor ribose-binding protein, none of which was exported to its normal location in the periplasm. The mutated rbsB contained a base substitution that results in a change of leucine to a proline at position-17 in the signal sequence. A pseudorevertant of the mutant contained proteolytically processed, active ribose-binding protein in the periplasm. The pseudorevertant rbsB carried a second mutation: serine at position-15 in the signal sequence was changed to phenylalanine. Isolation of a signal sequence mutant and a corresponding pseudorevertant without specific selection or site-directed mutagenesis emphasizes the possibility of obtaining export mutants without the use of procedures that could bias or limit the range of mutations found. Explanation of the extreme phenotype of the mutant and the effective correction of that phenotype in the pseudorevertant requires extension of current notions of critical features of signal sequences.

Amino Acid Sequence↗
PubMed1985

Chemotactic transducer proteins of Escherichia coli exhibit homology with methyl-accepting proteins from distantly related bacteria.

Transducers are transmembrane, methyl-accepting proteins central to the chemotactic systems of the enteric bacteria Escherichia coli and Salmonella typhimurium. Methyl-accepting proteins have been reported in a number of species in addition to these enteric bacteria. Those species include Bacillus subtilis and Spirochaeta aurantia, representatives of groups that diverged from ancestral enteric bacteria and from each other very early in bacterial evolution. An antiserum that reacts with all transducers of E. coli precipitated specifically methyl-accepting proteins from B. subtilis and S. aurantia, indicating that these proteins share antigenic determinants with transducers of E. coli. In addition, analysis of tryptic peptides by high-pressure liquid chromatography revealed similarities in the regions of methyl-accepting sites for proteins from all three species. These observations imply that structural features have been preserved in the three species from transducers contained in a common ancestor of eubacteria. It is thus reasonable to predict that other flagellated, chemotactic bacteria will be found to contain methyl-accepting proteins homologous to transducers of enteric bacteria.

Bacillus subtilis↗
PubMed1984

Structure of the Trg protein: Homologies with and differences from other sensory transducers of Escherichia coli.

Transducer proteins are central to chemotaxis in Escherichia coli. Three transducer genes comprise a homologous gene family, while a fourth gene, trg, is more distantly related. We have determined the nucleotide sequence of trg. The deduced sequence of the Trg protein has features in common with other transducers as well as regions of significant divergence. The protein sequence suggests the same transmembrane structure postulated for other transducers: an extra cytoplasmic NH2-terminal domain connected by a membrane-spanning region to an intracellular COOH-terminal domain. The COOH-terminal domain of Trg exhibits substantial sequence identity with the corresponding domains of the other transducers, particularly near the sites of covalent modification. Trg appears to have the same five methyl-accepting sites identified in the Tsr protein. Two of those sites are glutamines that are deamidated to yield methyl-accepting glutamates, while the remainder are synthesized as glutamates. Conservation in number but not in position of modified glutamines in Trg compared to the other transducers is consistent with the notion that uncharged glutamines at a specific number of modification sites serve to balance the signaling state of newly synthesized transducers. The NH2-terminal domain of Trg exhibits no significant homology with other transducers, implying that trg may be a fusion of the common COOH-terminal transducer sequence with an unrelated NH2-terminal sequence. The location of specific mutations within trg provides support for the suggestion that ligand-binding sites are in the NH2-terminal domains.

Amino Acid Sequence↗