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G L Hazelbauer

Publications and source records attributed to G L Hazelbauer.

At least 55 records · Page 3Linked to original sources
PubMed1984

Molecular cloning and characterization of genes required for ribose transport and utilization in Escherichia coli K-12.

We isolated spontaneous and transposon insertion mutants of Escherichia coli K-12 that were specifically defective in utilization or in high-affinity transport of D-ribose (or in both). Cotransduction studies located all of the mutations near ilv, at the same position as previously identified mutations causing defects in ribokinase ( rbsK ) or ribose transport ( rbsP ). Plasmids that complemented the rbs mutations were isolated from the collection of ColE1 hybrid plasmids constructed by Clarke and Carbon. Analysis of those plasmids as well as of fragments cloned into pBR322 and pACYC184 allowed definition of the rbs region. Products of rbs genes were identified by examination of the proteins produced in minicells containing various rbs plasmids. We identified four rbs genes: rbsB , which codes for the 29-kilodalton ribose-binding protein; rbsK , which codes for the 34-kilodalton ribokinase ; rbsA , which codes for a 50-kilodalton protein required for high-affinity transport; and rbsC , which codes for a 27-kilodalton protein likely to be a transport system component. Our studies showed that these genes are transcribed from a common promoter in the order rbsA rbsC rbsB rbsK . It appears that the high-affinity transport system for ribose consists of the three components, ribose-binding protein, the 50-kilodalton RbsA protein, and the 27-kilodalton RbsC protein, although a fourth, unidentified component could exist. Mutants defective in this transport system, but normal for ribokinase , are able to grow normally on high concentrations of the sugar, indicating that there is at least a second, low-affinity transport system for ribose in E. coli K-12.

Bacterial Proteins↗
PubMed1983

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.

Bacterial Proteins↗
PubMed1983

Characterization of the mgl operon of Escherichia coli by transposon mutagenesis and molecular cloning.

We used transposon insertion mutagenesis, molecular cloning, and a novel procedure for in vitro construction of polar and nonpolar insertion mutations to characterize the genetic organization and gene products of the beta-methylgalactoside (Mgl) transport system, which utilizes the galactose-binding protein. The data indicate that the mgl operon contained three genes, which were transcribed in the order mglB, mglA, and mglC. The first gene coded for the 31,000 Mr galactose-binding protein, which was synthesized as a 3,000-dalton-larger precursor form. The mglA product was a 50,000 Mr protein which was tightly associated with the membrane, and the mglC product was a 38,000 Mr protein which was apparently loosely associated with the membrane and was probably located on the internal face of the cytoplasmic membrane. Identification of gene products was facilitated by in vitro insertion of a fragment of Tn5 containing the gene conferring kanamycin resistance into a restriction site in the operon. The fragment proved to have a polar effect on the expression of promoter-distal genes only when inserted in one of the two possible orientations. The three identified gene products were necessary and apparently sufficient for transport activity, but only the binding protein was required for chemotaxis towards galactose. The transport system appeared to contain the minimum number of components for a binding protein-related system: a periplasmic recognition component, a transmembrane protein, and a peripheral membrane protein that may be involved in energy linkage.

Bacterial Proteins↗
PubMed1983

Limited homology between trg and the other transducer proteins of Escherichia coli.

Transducers are transmembrane proteins that are central to the chemotactic system of Escherichia coli. The proteins transduce ligand recognition into an excitatory signal and function in adaptation as methyl-accepting proteins. The transducer genes tsr, tar, and tap have extensive homology with each other. However, previous studies revealed little indication of homology between those three transducer genes and a fourth gene, trg. We investigated the relationship between trg and the other genes by blot-hybridization experiments and the relationship between Trg and the other transducer proteins by immune precipitation and experiments with an antiserum raised to purified Trg protein. In experiments in which 35% mismatch would be tolerated, weak hybridization of trg was detected to a DNA fragment containing tar and tap but not to a fragment containing tsr. In experiments in which only 30% mismatch would be tolerated, no trg hybridization was apparent either to total chromosomal DNA or to DNA from hybrid plasmids carrying the other transducer genes. An anti-Trg serum formed immune precipitates with the Tsr and Tar proteins as well as with the Trg protein to which it was raised. We conclude that there is homology between Trg and the other transducer, but the homology is more limited than that shared among the other transducers. Furthermore, we found no indication of additional transducer genes closely related to trg. Thus, the trg gene is a somewhat distant cousin within a single transducer gene family of E. coli.

Bacterial Proteins↗
PubMed1982

Methyl-accepting chemotaxis proteins are distributed in the membrane independently from basal ends of bacterial flagella.

Chemotactic behavior of Escherichia coli involves communication between methyl-accepting chemotaxis proteins and basal ends, the rotary motors of bacterial flagella. Both the proteins and the basal ends are embedded in the cytoplasmic membrane, but the spatial relationship between the two has not been determined. This communication describes a procedure for obtaining a preparation of membrane vesicles enriched in basal ends and thus in the regions of membrane immediately surrounding them. Methyl-accepting chemotaxis proteins were neither enriched nor depleted in this membrane fraction but instead were distributed throughout the membrane. Thus functional linkages between these proteins and flagellar motors must be mediated by processes other than direct physical interaction.

Bacterial Proteins↗
PubMed1982

A gene coding for a periplasmic protein is located near the locus for termination of chromosome replication in Escherichia coli.

Hybrid plasmids carrying trg, the genetic locus in closest proximity to terC, coded for several polypeptides in addition to the Trg protein. Polypeptides of 59,000 and 61,000 apparent molecular weight were the most prominent products synthesized in minicells containing the hybrid plasmids. Analysis of the effects of deletions generated by a restriction endonuclease identified a region of DNA immediately adjacent to trg as the putative gene coding for the two polypeptides. Studies with whole cells and minicells showed that the 59,000-dalton polypeptide is a periplasmic protein. Analysis by limited proteolysis indicated that the two polypeptides are related, and a number of observations support the notion that the 61,000-dalton protein is a precursor form of the 59,000-dalton mature exported protein. The identification and characterization of a protein, in addition to Trg, which is produced by a gene in close proximity to terC emphasizes the fact that the region does contain intact and active genes.

Bacterial Proteins↗
PubMed1982

Cloning of trg, a gene for a sensory transducer in Escherichia coli.

Clones of trg, a gene which codes for a chemotactic transducer, were isolated linked to ColE1 and pBR322 vectors. Studies with the hybrid plasmids demonstrated unequivocally that trg is the structural gene for methyl-accepting chemotaxis protein III. The Trg protein was found to be structurally complex, electrophoresing as a series of seven bands on high-resolution sodium dodecyl sulfate-polyacrylamide gels. The multiplicity of bands is a function of the activity of cheR, which codes for a methyltransferase, and of cheB, which codes for a demethylase. It appears that Trg, a quantitatively minor transducer, resembles the two major transducer proteins, Tsr and Tar, in that all three are multiply methylated and also multiply modified in a second way which requires an active cheB gene. However, preliminary analysis of the Trg protein indicated that it is significantly less related structurally to the Tsr or Tar protein than those two transducers are to each other. This implies that the features of multiple methylation and cheB-dependent modification are likely to be critical for the common physiological functions in chemotactic excitation and adaptation performed by all three transducers.

Bacterial Proteins↗
PubMed1981

Multiple forms of methyl-accepting chemotaxis proteins distinguished by a factor in addition to multiple methylation.

Methyl-accepting chemotaxis proteins are central to both the excitation and adaptation phases of chemotactic behavior. Using null mutations in the genes coding for the two major methyl-accepting proteins (tsr and tar), we identified the gene products among the membrane proteins of Escherichia coli visualized on one- and two-dimensional gels. On two-dimensional gels, both the tsr and the tar proteins appeared as a group of multiple spots arranged in two to four diagonal arrays. The multiplicity of forms could not be completely explained by the previously documented heterogeneity of the methylated proteins resulting from different numbers of methylated glutamyl residues per polypeptide chain. We suggest that there is at least one other way besides extent of methylation in which the polypeptides of a methylated protein can differ.

Bacterial Proteins↗
PubMed1981

Methyl-accepting chemotaxis protein III and transducer gene trg.

A comparison of the two-dimensional gel patterns of methyl-3H- and 35S-labeled membrane proteins from trg+ and trg null mutant strains of Escherichia coli indicated that the product of trg is probably methyl-accepting chemotaxis protein III. Like the other known methyl-accepting chemotaxis proteins, the trg product is a membrane protein that migrates as more than one species in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, implying that it too is multiple methylated. It appears likely that all chemoreceptors are linked to the tumble regulator through a single class of membrane protein transducers which are methyl-accepting proteins. Three transducers are coded for by genes tsr, tar, and, probably, trg. Another methyl-accepting protein, which is not related to any of these genes, was observed.

Bacterial Proteins↗
PubMed1980

Multiple methylation of methyl-accepting chemotaxis proteins during adaptation of E. coli to chemical stimuli.

In bacterial chemotaxis, adaptation is correlated with methylation or demethylation of methyl-accepting chemotaxis proteins (MCPs). Each protein migrates as a characteristic set of multiple bands in sodium dodecylsulfate polyacrylamide gel electrophoresis. The changes in MCP methylation that accompany adaptation are not the same for all bands of a set. Adaptation to a type II repellent stimulus results in an overall decrease in MCP II methylation, but also in an increase in the amount of radioactive methyl groups in the upper band of the set. We demonstrate that this increase is not due to new methylation, but rather to reduced electrophoretic mobility of previously methylated molecules that have lost some but not all of their methyl groups. We suggest that the pattern of multiple bands is a direct reflection of multiple sites for methylation on MCP molecules, and that the distribution of radiolabel among the bands is determined by the total extent of methylation. The patterns of methylated peptides produced by limited proteolysis of different MCP bands imply that methylation of the multiple sites on a molecule may occur in a specific order.

Adaptation, Physiological↗
PubMed1979

Transposon-insertion mutants of Escherichia coli K12 defective in a component common to galactose and ribose chemotaxis.

From a collection of 8,000 transposon-insertion mutants of Escherichia coli K12 we identified two mutations, trg-1::Tn5 and trg-2::Tn10, that simultaneously eliminate chemotactic response to ribose and galactose, two attractants recognized by independent receptors. We show that these transposon-insertions confer a Trg phenotype, indicating that this specific pattern of tactic defects is a null phenotype. The two mutation sites are cotransductionally linked to an extend consistent with placement in the same gene. The Trg phenotype of a family of deletion mutants produced by curing trg-2::Tn10 implies that trg is a single gene. Experiments with appropriate F-primes and Hfr's locate the trg locus at approximately 31 min on the linkage map, with a marker order: pyrF-rac-(P.O. 43)-trg-man. We also found one trg mutant whose Trg phenotype was not linked to a transposon-insertion but is probably the result of a mutator activity in the parent strain. Selection of transposon-insertions near, but not in trg allowed demonstration of a very close linkage between the spontaneous trg-3 and the transposon-generated trg's, indicating all three mutations are probably in the same gene. In our manipulations of transposon-insertions we found that Tn5 had a tendency to translocate from its initial site of insertion while Tn10 was relatively stable. The trg-product is probably a chemotactic signal transducer, which interacts directly with two independent receptor proteins and transmits information to the central chemotactic machinery.

Chemotaxis↗
PubMed1979

Mutants in transmission of chemotactic signals from two independent receptors of E. coli.

We have characterized chemotactic mutants of E. coli that appear to be defective in a common linkage of two independent receptors to the central chemotactic components. The mutants do not respond to gradients of ribose or galactose and thus are called trg (taxis to ribose and galactose), after Ordal and Adler (1974b). These trg mutants are indistinguishable from their parent in tactic response to other attractants, swimming pattern, growth rates, and transport of ribose and galactose. The mutant cells contain the usual amounts of ribose and galactose receptors, and those proteins function normally in their other role, transport of their respective ligands. The mutations, generated by insertion of translocatable drug-resistance elements (transposons)8 are located near 31 min on the map of the E. coli chromosome, a locus far removed from the genes coding for the ribose and galactose receptors. Trg mutants do not resemble either specific receptor mutants or che mutants. The nature of the requirement for the trg product in the response to ribose and galactose is not defined, but evidence for interference of tactic signals from the ribose and galactose receptors (Strange and Koshland, 1976) supports the idea that the product functions directly in the transmission of tactic signals from the two receptors to the flagella.

Biological Transport↗
PubMed1979

Relation of chemotactic response to the amount of receptor: evidence for different efficiencies of signal transduction.

We determined the content of galactose-glucose-, maltose-, and ribose-binding proteins in cells of Escherichia coli K-12 grown in a variety of media and also measured the respective transport and chemotactic activities that depend on those binding proteins. Correlation of the level of induction of a particular binding protein with the extent of tactic activity mediated by that protein indicates that the magnitude of the tactic response to a particular stimulating compound is a direct function of the number of receptors per cell. In contrast, comparison of the magnitudes of response to substances recognized by independent receptors indicates that some stimulus-receptor complexes are more effective in eliciting tactic responses than are others. Thus, the magnitude of response to any particular stimulating compound is a function both of the number of receptors per cell and of the effectiveness of the stimulus-receptor complex. Considerations of available information about the tactic response to maltose suggest that the effectiveness of a stimulus-receptor complex is related to the transducer with which the receptor interacts. The tar product appears to be a relatively effective transducer of the signals it accepts from receptors for aspartate, alpha-methylaspartate, and maltose, whereas the trg product appears to be a relatively ineffective transducer of signals it accepts from receptors for galactose and ribose.

Bacterial Proteins↗