Biomedical subjects

H Aiba

Publications and source records attributed to H Aiba.

At least 91 records · Page 5Linked to original sources

PubMed1993

Osmoregulation of the fatty acid receptor gene fadL in Escherichia coli.

The fadL gene of Escherichia coli codes for an outer membrane protein that is involved in the uptake of long-chain fatty acids. Uptake is regulated by environmental osmolarity, and decreases when the cells are grown under conditions of high osmolarity. A temperature-sensitive mutant that requires fatty acid for growth at 42 degrees C was unable to grow at the high temperature even in the presence of fatty acid if the medium contained 10% sucrose. Promoter activity of the fadL gene in vivo was repressed by high osmolarity in a FadR repressor null mutant. Furthermore, in vitro transcription of the fadL gene was strongly repressed by the addition of OmpR and EnvZ proteins. The results of gel retardation and DNase I protection experiments indicated that OmpR, after incubation with the protein kinase EnvZ, specifically binds to at least four sites around the fadL promoter, two upstream and two downstream from the transcriptional start site. These results suggest that transcription of the fadL gene is osmotically regulated by the OmpR-EnvZ two-component system.

Bacterial Outer Membrane Proteins↗

PubMed1993

Sensor and regulator proteins from the cyanobacterium Synechococcus species PCC7942 that belong to the bacterial signal-transduction protein families: implication in the adaptive response to phosphate limitation.

A 1.2kb DNA fragment was cloned from Synechococcus sp. PCC7942, which is able phenotypically to complement a phoR creC Escherichia coli mutant for the expression of alkaline phosphatase. A 2.5 kb DNA fragment encompassing the putative gene was then cloned and its complete nucleotide sequence determined. Nucleotide sequencing revealed that the intact gene encodes a protein of 46,389 Da, and that the deduced amino acid sequence shows a high degree of homology to those of the bacterial sensory kinase family. In the determined nucleotide sequence, another gene was adjacently located, which encodes a protein of 29,012 Da. This protein shows a high degree of homology to those of the response regulator family. Thus, we succeeded in the cloning of a pair of genes encoding the sensory kinase and response regulator, respectively, in a cyanobacterium. Mutant strains that lack these genes were constructed, and demonstrated to be defective in their ability to produce alkaline phosphatase and some inducible proteins in response to phosphate-limitation in the medium. These results are probably involved, either directly or indirectly, in the signal-transduction mechanism underlying regulation of the phosphate regulon in Synechococcus sp. PCC7942. Hence, the genes encoding the sensory kinase and response regulator were designated as sphS and sphR, respectively (Synechococcus phosphate regulon). The SphS protein was demonstrated in vitro to undergo phosphorylation in the presence of ATP.

Adaptation, Physiological↗

PubMed1993

A lowered concentration of cAMP receptor protein caused by glucose is an important determinant for catabolite repression in Escherichia coli.

A decreased intracellular concentration of cAMP is insufficient to account for catabolite repression in Escherichia coli. We show that glucose lowers the amount of cAMP receptor protein (CRP) in cells. A correlation exists between CRP and beta-galactosidase levels in cells growing under various conditions. Exogenous cAMP completely eliminates catabolite repression in CRP-overproducing cells, while it does not fully reverse the effect of glucose on beta-galactosidase expression in wild-type cells. When the CRP concentration is reduced by manipulating the crp gene, beta-galactosidase expression decreases in proportion to the concentration of CRP. These findings indicate that the lowered concentration of CRP caused by glucose is one of the major factors for catabolite repression. We propose that glucose causes catabolite repression by lowering the intracellular levels of both CRP and cAMP.

Bacterial Proteins↗

PubMed1993

A cyanobacterial gene encoding a protein with extensive homology to mammalian phosphoribosylpyrophosphate synthetase.

Phosphoribosylpyrophosphate (PRibPP) is the primary precursor in the biosynthesis of purine and pyrimidine nucleotides. The synthesis of PRibPP is catalysed by a ubiquitous PRibPP synthetase in many organisms. We provide evidence that a cyanobacterium, Synechococcus sp., has a gene encoding a protein with striking homology to mammalian PRibPP synthetases.

Amino Acid Sequence↗

PubMed1992

A new aspect of transcriptional control of the Escherichia coli crp gene: positive autoregulation.

Transcription of the Escherichia coli crp gene is negatively regulated by CRP-cAMP that binds to a specific site located downstream of the crp promoter. A second binding site for CRP-cAMP (CRP site II) exists upstream of the crp promoter. Using an in vitro transcription assay, we have demonstrated that CRP-cAMP activates transcription of crp in certain conditions. A promoter which carries an altered CRP-binding site II is no longer activated by CRP-cAMP, indicating that CRP site II mediates the activation of crp transcription. The concentrations of cAMP that are required for positive autoregulation are higher than those for negative autoregulation. Evidence for positive and negative autoregulation in vivo is presented by a quantitative S1 nuclease analysis.

Base Sequence↗

PubMed1992

A novel sensor-regulator protein that belongs to the homologous family of signal-transduction proteins involved in adaptive responses in Escherichia coli.

Expression of the Escherichia coli outer membrane porins, OmpC and OmpF, is regulated in response to changes in the medium osmolarity through the functions of the regulatory factors, EnvZ and OmpR. A 3.0 kilobase pair DNA fragment cloned from E. coli is able phenotypically to suppress the defect in ompC and ompF expression caused by an envZ deletion mutation, provided that a certain gene located in this fragment is expressed on a high copy-number plasmid. Nucleotide sequencing revealed that the putative gene encodes a protein of 102,452 Da. The deduced amino acid sequence of the protein shows a high degree of homology to those of both EnvZ and OmpR, i.e. it contains both a 'sensory kinase domain' and a 'response regulator domain' in its primary amino acid sequence. The protein identified in this study is probably a novel member of the homologous family of proteins involved in bacterial adaptive responses. Hence, the gene encoding this novel sensor-regulator protein was designated as barA (bacterial adaptive responses) and mapped at 60 min on the E. coli genetic map. The BarA protein in isolated membranes was demonstrated in vitro to undergo phosphorylation in the presence of ATP.

Adaptation, Biological↗

PubMed1991

Functional map of the alpha subunit of Escherichia coli RNA polymerase: two modes of transcription activation by positive factors.

The role of the alpha subunit of Escherichia coli RNA polymerase in transcription activation by positive factors was investigated using two reconstituted mutant RNA polymerases (containing C-terminally truncated alpha subunits) and three positive factors [the cAMP receptor protein (CRP), OmpR, and PhoB]. The mutant RNA polymerases did not respond to transcription activation by activator proteins that bind upstream of the respective promoters. Transcription by these mutant enzymes was, however, activated in the cases where activators bind to target sites that overlap the promoter -35 region. Two different mechanisms are proposed for the positive control of transcription by activator proteins, one requiring the C-terminal domain of the alpha subunit, and the other not requiring it.

Bacterial Outer Membrane Proteins↗

PubMed1991

Nuclear magnetic resonance study on the structure and interaction of cyclic AMP receptor protein and its mutants: a deuterium-labeling and photo-CIDNP study.

The identification and assignment of the proton magnetic resonances of some aliphatic and aromatic amino acid residues of cyclic AMP receptor protein (CRP) are reported. The signals of the leucine and valine residues at around 0 ppm were identified on the basis of intermolecular nuclear Overhauser effects, deuterium labeling, and partial proteolytic digestion. On the addition of cAMP, methyl proton signals due to Val-49 and three leucine residues were detected as upfield-shifted signals at around -0.2 ppm. These signals can be used as indicators of the proper binding of cAMP because they are not observed on the addition of cGMP or 2'-deoxy-cAMP. They are also not observed on cAMP binding to mutant CRP*5 (Ser-62-Phe), which can only be activated by a high concentration of cAMP, but they are observed on cAMP binding to other mutant CRP*s (four species), which can be activated by lower concentrations of cAMP. The resonance of some aromatic protons, i.e., C-2H of two tryptophans, C-2H and C-4H of six histidines, and C-2,6H and C-3,5H of six tyrosine residues in CRP, were assigned by means of deuterium labeling and NOE measurements. The 1H NMR spectrum of labeled CRP [Trp(ring-d5), Phe(ring-d5), and Tyr(3,5-d2)] showed good resolution in the aromatic region. The addition of cAMP to this CRP in D2O caused pronounced line broadening of resonances arising from the residues in the cAMP-binding domain, but the resonances of the DNA-binding domain were not affected.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1991

Molecular mechanism of negative autoregulation of Escherichia coli crp gene.

Transcription of the Escherichia coli crp gene encoding cAMP receptor protein (CRP) is negatively regulated by CRP-cAMP complex that binds to a specific site located downstream from the transcription start site. The binding of CRP-cAMP to this site activates transcription from a second divergent overlapping promoter. The mechanism of this negative autoregulation of the crp gene has been investigated by in vitro transcription, gel shift, DNase I footprinting, and exonuclease III protection assays. We demonstrated that the crp and divergent promoters are reciprocally and coordinately regulated by CRP-cAMP. The abortive initiation assay revealed that the divergent RNA itself is not required for the inhibition of crp transcription. Detailed binding studies revealed that CRP-cAMP stimulates the binding of RNA polymerase to the divergent promoter and thus blocks the occupation of the crp promoter by RNA polymerase.

Base Sequence↗

PubMed1991

Osmoregulatory expression of the porin genes in Escherichia coli: evidence for signal titration in the signal transduction through EnvZ-OmpR phosphotransfer.

The OmpR protein of Escherichia coli is a positive regulator specific for the ompF and ompC genes. The function of OmpR is modulated through phosphotransfer signaling mediated by the kinase, EnvZ. We previously demonstrated that OmpR contains two functional domains, which are physically separable; one is responsible for the interaction with EnvZ, whereas the other participates in interactions with cognate promoter DNAs. In this study, these domains of OmpR were overproduced in wild-type cells harboring the endogenous intact ompR gene on their chromosome. It was found that when the N-terminal domain of OmpR, which contains the phosphorylation site, was overproduced, expression of the ompF and ompC genes was markedly inhibited, irrespective of the osmolarity of the growth medium. Based on our current model for the molecular mechanism underlying signal transduction through Envz-OmpR phosphotransfer (T. Mizuno and S. Mizushima, Mol. Microbiol. 4, (1990), 1077-1082), we provide evidence that this phenomenon is best interpreted by the concept of 'signal titration' in the phosphotransfer signaling pathway.

Bacterial Outer Membrane Proteins↗

PubMed1991

Signal transduction and osmoregulation in Escherichia coli. A novel type of mutation in the phosphorylation domain of the activator protein, OmpR, results in a defect in its phosphorylation-dependent DNA binding.

The transcriptional factors, OmpR and EnvZ, are crucially involved in the osmotic regulation of ompF and ompC expression in Escherichia coli. The DNA binding ability of the positive regulator, OmpR, is modulated through its phosphorylation and dephosphorylation mediated by EnvZ in response to the medium osmolarity. In this study, two examples of a novel type of mutant ompR allele, ompR96A and ompR115S, whose phenotype is OmpF- OmpC- irrespective of the medium osmolarity, were characterized. These mutations result in amino acid conversions, Glu96 to Ala and Arg115 to Ser, respectively, within the phosphorylation domain of OmpR. Nevertheless, these mutant proteins were capable of undergoing phosphorylation and dephosphorylation normally, just like wild-type OmpR. However, the phosphorylation-dependent enhancement of their in vitro DNA binding ability was found to be severely affected. It was thus revealed that these mutant OmpR represent a novel type in terms of the mechanism of phosphorylation-dependent activation of the function of OmpR, i.e. those are normally phosphorylated but not activated to bind to the cognate promoter DNAs. In this respect, it was further suggested that OmpR oligomerization may be involved in the mechanism underlying the phosphorylation-dependent enhancement of the DNA binding ability of OmpR. The mutant proteins characterized in this study seem to be defective in this particular oligomerization process observed in vitro.

Adenosine Triphosphate↗

PubMed1991

Transmembrane signal transduction and osmoregulation in Escherichia coli. Functional importance of the periplasmic domain of the membrane-located protein kinase, EnvZ.

The EnvZ protein is presumably a membrane-located osmotic sensor which is involved in expression of the ompF and ompC genes in Escherichia coli. Previously, we developed an in vitro method for analyzing the intact form of the EnvZ protein located in isolated cytoplasmic membranes, and demonstrated that this particular form of the EnvZ protein exhibits the ability not only as to OmpR phosphorylation but also OmpR dephosphorylation. In this study, to gain an insight into the structural and functional importance of the putative periplasmic domain of the EnvZ protein, a set of mutant EnvZ proteins, which lack various portions of the periplasmic domain, were characterized in terms of not only their in vivo osmoregulatory phenotypes but also in vitro EnvZ-OmpR phosphotransfer reactions. It was revealed that these deletion mutant EnvZ proteins are normally incorporated into the cytoplasmic membrane. Cells harboring these mutant EnvZ proteins showed a pleiotropic phenotype, namely, OmpF- Mal- LamB- PhoA-, and produced the OmpC protein constitutively irrespective of the medium osmolarity. It was also suggested that all of these mutant EnvZ proteins were defective in their in vitro OmpR dephosphorylation ability, while their OmpR phosphorylation ability remained unaffected. These results imply the functional importance of the periplasmic domain of the EnvZ protein for modulation of the kinase/phosphatase activity exhibited by the cytoplasmic domain in response to an environmental osmotic stimulus.

Bacterial Outer Membrane Proteins↗

PubMed1991

Transcriptional terminator is a positive regulatory element in the expression of the Escherichia coli crp gene.

Plasmids were constructed that contain deletions in the stem region of the presumed rho-independent terminator of the cloned crp gene of Escherichia coli. The level of cyclic AMP binding activity and the amount of CRP in cells harboring the deletion plasmids were found to be significantly lower than those in cells harboring the wild-type crp plasmid. Quantitative S1 assays indicated that the steady-state levels of crp mRNA were markedly reduced in cells harboring the deletion plasmids. Evidence was also presented to show that the crp mRNAs produced from deletion plasmids are less stable than that from the intact crp gene. In vitro transcription assays revealed that the putative crp terminator is indeed a rho-independent terminator. Using the galK expression system and Northern blot analysis we showed that the crp terminator is functional in vivo. Moreover it was shown that the deletion mutations in the stem region of the crp terminator cause a significant readthrough. We conclude that the 3'-flanking sequence of the crp gene acts to stabilize its own mRNA as well as to terminate transcription.

Base Sequence↗

PubMed1990

Helical phase dependent action of CRP: effect of the distance between the CRP site and the -35 region on promoter activity.

A plasmid carrying a CRP-dependent promoter fused to the lac structural genes was manipulated to construct a set of spacing mutants that have varying lengths between the CRP binding site and the -35 region. The lengths of the spacer were changed over 45 bp by inserting or deleting nucleotides. DNase I footprinting analysis revealed that the spacer length did not affect the binding of cAMP-CRP to the CRP site. The effect of the spacer length on transcription activation by cAMP-CRP was tested in vivo by beta-galactosidase and quantitative S1 assays with crp+ and delta crp cells harboring plasmids. Insertions or deletions of non-integral helical turns, which displace the CRP site onto the opposite face of DNA helix compared to the original promoter, eliminated completely the activation of transcription. In contrast, changing the spacer length by integral helical turns allowed the promoter to respond to CRP, although the degree of activation varied with the length of the spacer. We conclude that stereospecific positioning of CRP and RNA polymerase on the DNA helix is strictly required for CRP action. The data support a model that CRP stimulates transcription by directly contacting RNA polymerase.

Base Sequence↗

PubMed1990

Phosphorylation of a bacterial activator protein, OmpR, by a protein kinase, EnvZ, stimulates the transcription of the ompF and ompC genes in Escherichia coli.

The OmpR of Escherichia coli is a positive regulator specific for the ompF and ompC genes, which encode outer membrane proteins OmpF and OmpC, respectively. The EnvZ protein is a protein kinase which specifically phosphorylates the OmpR protein. In this study, the results of in vitro transcription experiments revealed that the phosphorylation of the OmpR protein by the EnvZ protein stimulates the transcription of both the ompF and ompC genes.

Bacterial Outer Membrane Proteins↗

PubMed1990

A nuclear magnetic resonance study of the cyclic AMP receptor protein (CRP): assignments of the NH protons of histidine and tryptophan residues and the effect of binding of cAMP to CRP.

Seven well-resolved signals could be observed in the field lower than 9.5 ppm in the 1H-NMR spectrum of the H2O solution of cyclic AMP receptor protein (CRP). The signals of the tryptophan and histidine residues were identified on the basis of the CPMG spin echo spectra, the intra-residue NOE, 15N labeling, deuterium labeling, and the results of pH titration. The assignments of peaks to specific tryptophan and histidine residues are discussed in relation to the amino acid sequence and X-ray crystallographic data, and were confirmed by experiments involving partial subtilisin digestion. The four signals E (11.25 ppm), F (11.15 ppm), G (10.75 ppm), and H (10.65 ppm) were tentatively assigned to the resonances of the histidine residue at position 159, the arginine residue at position 82, and the tryptophan residues at positions 85 and 13, respectively. On the addition of cAMP and cGMP, signals F and G shifted up- and downfield respectively and conformational changes in the structure of CRP could be detected. The conformational transition mostly occurs when one cAMP molecule binds to one of the dimer subunits, but is completed only when both cAMP binding sites are saturated.

Carrier Proteins↗

PubMed1990

Transmembrane signal transduction and osmoregulation in Escherichia coli: I. Analysis by site-directed mutagenesis of the amino acid residues involved in phosphotransfer between the two regulatory components, EnvZ and OmpR.

Previously, the transfer of a phosphoryl group between the EnvZ and OmpR proteins, which are involved in expression of the ompF and ompC genes in response to the medium osmolarity, was demonstrated in vitro. In this study, the histidine (His) residue at position 243 of the EnvZ protein, and the aspartate (Asp) residues at positions 12 and 55 of the OmpR protein were changed, respectively, by means of site-directed mutagenesis. We characterized the mutant proteins in terms of not only their in vitro phosphotransfer reactions but also their in vivo osmoregulatory phenotypes. The mutant EnvZ protein was defective in its in vitro ability not only as to EnvZ-autophosphorylation but also OmpR-phosphorylation and OmpR-dephosphorylation. This particular mutant EnvZ protein seemed to exhibit null functions as to the in vivo osmoregulatory phenotype. The mutant OmpR protein with the amino acid change at position 12 was clearly phosphorylated in vitro, but at a very low rate as compared with the wild-type OmpR protein. In vitro phosphorylation of the mutant OmpR protein with the amino acid change at position 55 was more severely affected. This mutant OmpR protein appeared to exhibit null functions as to the in vivo osmoregulatory phenotype. These results suggest that the histidine residue at position 243 of the EnvZ protein and the aspartate residues at positions 12 and 55 of the OmpR protein are deeply involved in the phosphotransfer between the EnvZ and OmpR proteins.

Bacterial Outer Membrane Proteins↗

PubMed1990

Transmembrane signal transduction and osmoregulation in Escherichia coli: II. The osmotic sensor, EnvZ, located in the isolated cytoplasmic membrane displays its phosphorylation and dephosphorylation abilities as to the activator protein, OmpR.

The EnvZ protein is presumably a membrane-located osmotic sensor, which specifically phosphorylates the activator protein, OmpR, involved in expression of the ompF and ompC genes in Escherichia coli. In this study, we developed an in vitro system for analyzing the intact form of the EnvZ protein located in the isolated cytoplasmic membrane. This particular form of the EnvZ protein exhibited its in vitro ability not only as to OmpR-phosphorylation but also OmpR-dephosphorylation. It was found that when a high concentration of a mono-cation (K+, Na-, or Li+) was present during the in vitro reactions, OmpR-dephosphorylation was preferentially inhibited and consequently the phosphorylated from of the OmpR protein was accumulated under the in vitro conditions used, although the K+ ion appears to be essential for the OmpR-phosphorylation reaction. Procaine, a local anesthetic, is known to affect the osmotic regulation of the ompF and ompC genes in vivo. In this study, procaine was also found to preferentially inhibit OmpR-dephosphorylation mediated by the EnvZ protein in vitro.

Bacterial Outer Membrane Proteins↗