PubMed Health⌕ Search

Biomedical subjects

K Oosawa

Publications and source records attributed to K Oosawa.

30 records · Page 2Linked to original sources
PubMed1989

Activation of bacterial porin gene expression by a chimeric signal transducer in response to aspartate.

The Tar chemoreceptor of Escherichia coli is a membrane-bound sensory protein that facilitates bacterial chemotaxis in response to aspartate. The EnvZ molecule has a membrane topology similar to Tar and is a putative osmosensor that is required for osmoregulation of the genes for the major outer membrane porin proteins, OmpF and OmpC. The cytoplasmic signaling domain of Tar was replaced with the carboxyl portion of EnvZ, and the resulting chimeric receptor activated transcription of the ompC gene in response to aspartate. The activation of ompC by the chimeric receptor was absolutely dependent on OmpR, a transcriptional activator for ompF and ompC.

Aspartic Acid↗
PubMed1988

Phosphorylation of three proteins in the signaling pathway of bacterial chemotaxis.

Six cytoplasmic che gene products are required for signal transduction in bacterial chemotaxis, but the nature of their biochemical interactions is not known. We show that in vitro the CheA protein becomes autophosphorylated in the presence of ATP. In addition, the phosphate group on CheA can be rapidly transferred to CheB, a protein involved in adaptation to stimuli, or to CheY, a protein involved in the excitation response. The phosphorylation of CheB and CheY is transient; they readily dephosphorylate. We have also found that CheZ, a protein that appears to antagonize CheY function in vivo, accelerates the hydrolysis of the phosphate on CheY. These results suggest that signal transduction in bacterial chemotaxis may involve the flow of phosphate through a cascade of phosphorylated protein intermediates.

Bacterial Proteins↗
PubMed1988

Mutants defective in bacterial chemotaxis show modified protein phosphorylation.

To examine the correlation between CheA phosphorylation and bacterial chemotaxis, cheA mutations leading to defects in chemotaxis were mapped and characterized. Mutant CheA proteins were tested in vitro for phosphorylation and were grouped into four classes: nonphosphorylated, partially phosphorylated, phosphorylated but not dephosphorylated by CheB and CheY, and phosphorylated and dephosphorylated. Nearly all the mutants were found to be defective in an aspect of phosphorylation. Furthermore, the mutant phenotypes were found to cluster in different regions of the cheA gene. We suggest that the CheA protein has three functional domains: one for interaction with CheB and CheY, a second for regulating phosphorylation and controlling the stability of the protein, and a third for receiving input signals regulating CheA activity.

Bacterial Proteins↗
PubMed1988

Cloning of the C-terminal cytoplasmic fragment of the tar protein and effects of the fragment on chemotaxis of Escherichia coli.

A gene encoding only the C-terminal portion of the receptor-transducer protein Tar of Escherichia coli was constructed. The gene product was detected and localized in the cytoplasmic fraction of the cell by immunoblotting with anti-Tar antibodies. The C-terminal fragments from wild-type and mutant tar genes were characterized in vivo. The C-terminal fragment generated from tar-526, a mutation that results in a dominant "tumble" phenotype, was found to be deamidated and methylated by the CheB and CheR proteins, respectively. The C-terminal fragment derived from a wild-type gene was poorly deamidated, and the C-terminal fragment derived from tar-529, a dominant mutant with a "smooth swimming" phenotype, was not apparently modified. Cells carrying the C-terminal fragment with the tar-526 mutation as the sole receptor-transducer protein showed a high frequency of tumbling and chemotaxis responses to changes in intracellular pH. These results suggest that the cytoplasmic C-terminal fragment of Tar retains some of the functions of the whole protein in vivo.

Aspartic Acid↗
PubMed1987

Protein phosphorylation is involved in bacterial chemotaxis.

The nature of the biochemical signal that is involved in the excitation response in bacterial chemotaxis is not known. However, ATP is required for chemotaxis. We have purified all of the proteins involved in signal transduction and show that the product of the cheA gene is rapidly autophosphorylated, while some mutant CheA proteins cannot be phosphorylated. The presence of stoichiometric levels of two other purified components in the chemotaxis system, the CheY and CheZ proteins, induces dephosphorylation. We suggest that the phosphorylation of CheA by ATP plays a central role in signal transduction in chemotaxis.

Bacterial Proteins↗
PubMed1987

Phenol: a complex chemoeffector in bacterial chemotaxis.

Earlier observations that phenol is a repellent for Salmonella typhimurium but an attractant for Escherichia coli were confirmed. This behavioral difference was found to correlate with a difference in the effect phenol had on receptor methylation levels; it caused net demethylation in S. typhimurium but net methylation in E. coli. On the basis of mutant behavior and measurement of phenol-stimulated methylation, the attractant response of E. coli was shown to be mediated principally by the Tar receptor. In S. typhimurium, two receptors were found to be sensitive to phenol, namely, an unidentified receptor, which mediated the repellent response and showed phenol-stimulated demethylation; and the Tar receptor, which (as with E. coli) mediated the attractant response and showed phenol-stimulated methylation. In wild-type S. typhimurium, the former receptor dominated the Tar receptor, with respect to both behavior and methylation changes. However, when the amount of Tar receptor was artificially increased by the use of Tar-encoding plasmids, S. typhimurium cells exhibited an attractant response to phenol. No protein analogous to the phenol-specific repellent receptor was evident in E. coli, explaining the different behavioral responses of the two species toward phenol.

Aspartic Acid↗
PubMed1986

Analysis of mutations in the transmembrane region of the aspartate chemoreceptor in Escherichia coli.

Site-specific mutagenesis was used to replace an alanine with a lysine residue and to create a deletion of seven amino acids into the first transmembrane region (TMI region) of the aspartate chemoreceptor in Escherichia coli. The mutations resulted in the loss of aspartate chemotaxis on tryptone motility plates. However, both mutant proteins were able to associate with the membrane and to bind aspartate. They were both refractory to methylation or to modification of the C-terminal region of the protein by the cheB gene product. These results suggested that the integrity of the TMI domain of the protein was required to maintain the function of the cytoplasmic portion of the receptor. The Lys-19 mutant retained the ability to generate a repellent response. Analysis of suppressor mutations of the Lys-19 mutation suggested that formation of an ion pair or specific changes in a 40 amino acid stretch in the cytoplasmic region of the protein (from amino acid 264 to amino acid 303) could suppress the effects of the Lys-19 mutation. The TMI region of the protein may be involved in transmembrane transmission of signals from the periplasmic portion of the cell to the cytoplasmic portion of the Tar protein.

Amino Acid Sequence↗
PubMed1986

Characterization of Escherichia coli chemotaxis receptor mutants with null phenotypes.

Hydroxylamine mutagenesis was used to alter the tar gene that encodes the transmembrane Tar protein required for chemotaxis. Mutants defective in chemotaxis were selected, and the mutation was characterized by DNA sequencing. Two classes of mutations were found: nonsense and missense. The nonsense mutations were distributed throughout the gene, while the missense mutations were found to cluster in a region that includes 185 amino acids at the C-terminal end of the Tar protein. Partial characterization of mutant phenotypes suggested that some are completely defective in signaling while responding to attractants and repellents by differential methylation. Other mutants are undermethylated and constantly tumble, while yet another class of mutants is overmethylated and biased toward constant swimming with little or no tumbling. These mutants will be useful in experiments designed to understand the mechanism of chemotaxis.

Amino Acids↗
PubMed1984

Demethylation of methyl-accepting chemotaxis proteins in Escherichia coli induced by the repellents glycerol and ethylene glycol.

The addition of glycerol or ethylene glycol caused not only severe tumbling but also a drastic decrease in the methylation level of methyl-accepting chemotaxis proteins (MCPs) in Escherichia coli. Experiments with various mutants having defects in their MCPs showed that the demethylation occurred in all three kinds of MCPs, MCPI, II, and III. The addition of an attractant to the glycerol- or ethylene glycol-treated cells resulted in a distinct increase in the methylation level of the relevant MCP, indicating that glycerol and ethylene glycol do not directly damage the methylation-demethylation system in the cell. The time courses of adaptation and MCP demethylation upon addition of these repellents were consistent with each other. Furthermore, both the response time and the extent of MCP demethylation were increased in parallel with increasing concentrations of glycerol or ethylene glycol. These results indicate that the adaptation to these repellents is performed by the demethylation of MCPs. Thus, glycerol and ethylene glycol are novel repellents, which utilize not just one but all three kinds of MCPs for both information processing and adaptation.

Bacterial Proteins↗
PubMed1983

Glycerol and ethylene glycol: members of a new class of repellents of Escherichia coli chemotaxis.

By using the chemical-in-plug method, we found that glycerol and ethylene glycol caused negative chemotaxis in wild-type cells of Escherichia coli; the threshold concentration was about 10(-3) M for both chemicals. As with other known repellents, the addition of glycerol or ethylene glycol induced a brief tumble response in wild-type cells but not in generally nonchemotactic mutants. Experiments with mutants defective in various methyl-accepting chemotaxis proteins (MCPs) revealed that the presence of any one of three kinds of MCPs (MCP I, MCP II, or MCP III) was necessary to give a tumble response to these repellents. Consistently, it was found that the methylation-demethylation system of MCPs was involved in the adaptation of the cells to these repellents. The effect of glycerol or ethylene glycol was not enhanced by lowering the pH of the medium, and glycerol did not alter the membrane potential of the cells. All of these results suggest that glycerol and ethylene glycol are members of a new class of repellents which produce a tumble response in the cells by perturbing the MCPs in the membrane.

Alcohols↗
PubMed1983

Mapping of the pin locus coding for a site-specific recombinase that causes flagellar-phase variation in Escherichia coli K-12.

Although the vh2 mutation almost entirely prevents phase variation in Salmonella spp., an Escherichia coli strain that carried the Salmonella H1 and H2 region, including the vh2 mutation, showed phase variation. From this strain, EJ1076, a number of mutants defective in phase variation were isolated, and the symbol pin was assigned to their mutant gene. The pin locus was mapped between purB and trp near purB by interrupted matings using Tn10 sites inserted near pin. The locus was cotransduced with purB by P1 vir at a frequency of around 0.33. All the mutations tested were clustered at this locus. Three E. coli K-12 strains probably derived via different lines from the wild type have been tested for the presence of pin+ by introducing the two Salmonella H regions; two were pin+, and one was a pin mutant.

Chromosome Inversion↗