[Protein phosphorylation and signal transduction in bacteria].
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Ammonia↗
Biomedical subjects
H Aiba
Publications and source records attributed to H Aiba.
Signal transduction and osmoregulation in Escherichia coli. A single amino acid change in the protein kinase, EnvZ, results in loss of its phosphorylation and dephosphorylation abilities with respect to the activator protein, OmpR.
The EnvZ protein is a bacterial protein kinase, which specifically phosphorylates the activator protein, OmpR, involved in expression of the ompF and ompC genes in Escherichia coli. The phosphotransfer between the EnvZ and OmpR proteins was postulated to be involved in the signal transduction in response to an environmental osmotic stimulus. In this study, we isolated a novel type of envZ mutant, in which a base substitution resulted in a Tyr-to-Ser conversion at amino acid residue 351 of the EnvZ protein. This single amino acid conversion was found to dramatically affect the functions of the EnvZ protein. The mutant EnvZ protein was defective in its ability not only as to OmpR-phosphorylation but also as to OmpR-dephosphorylation. The envZ mutant, termed envZ30, was isolated as a pseudorevertant, which phenotypically suppresses an ompR3-type mutant exhibiting an OmpF- OmpC-constitutive phenotype. These results will be discussed in relation to the structure and function of the protein kinase, EnvZ.
Bacterial Outer Membrane Proteins↗
Semisynthetic promoters activated by cyclic AMP receptor protein of Escherichia coli.
Semisynthetic promoters activated by Escherichia coli cyclic AMP receptor protein (CRP) were created by combining a synthetic CRP-binding site (crb) and nucleotide sequences derived from cryptic promoter regions. A 22-bp oligodeoxyribonucleotide corresponding to an idealized crb was randomly placed into DNA regions that precede a promoterless lacZ gene on a plasmid. Several plasmid clones were obtained which allowed the expression of lacZ in crp+ cya+ cells carrying a chromosomal deletion of lac genes. The beta-galactosidase and the quantitative S1-nuclease assays of crp+ and delta crp cells harboring these plasmids indicated that the transcription from newly created promoters is dependent on CRP. Sequence analysis revealed that these promoters are divided into two types based on the location of the crb relative to the transcription start point (tsp). The distance from the center of the crb to the tsp is 70 bp in the first type and 38 bp in the second type. The sequences of all these promoters exhibit poor homology with the consensus promoter sequence.
Base Sequence↗
Evidence for the physiological importance of the phosphotransfer between the two regulatory components, EnvZ and OmpR, in osmoregulation in Escherichia coli.
Previously, the transfer of the phosphoryl group between the EnvZ and OmpR proteins, which are involved in activation of the ompF and ompC genes in response to the medium osmolarity, has been demonstrated in vitro. In this study, we characterized mutant EnvZ and OmpR proteins in terms of their in vitro phosphorylation and dephosphorylation. The proteins isolated from the mutants, envZ11 and ompR3, were found to be defective in seemingly the same aspect, i.e. OmpR dephosphorylation. The protein isolated from the ompR77 mutant, which is a suppressor mutant specific for envZ11, was found to be defective in another aspect, i.e. OmpR phosphorylation. These results imply that the phosphotransfer reactions observed in vitro play roles in the mechanism underlying the osmoregulatory expression of the ompF and ompC genes in vivo. We provide evidence that the EnvZ protein is involved not only in OmpR phosphorylation but also in OmpR dephosphorylation.
Bacterial Proteins↗
Location of phosphorylation site and DNA-binding site of a positive regulator, OmpR, involved in activation of the osmoregulatory genes of Escherichia coli.
The OmpR protein of Escherichia coli is a positive regulator involved in activation of the ompF and ompC genes which encode the major outer membrane proteins OmpF and OmpC, respectively. By employing recombinant DNA techniques, we isolated the N- and C-terminal halves of the OmpR molecule. From the results of biochemical analyses of these fragments, it was concluded that the N-terminal portion contains a site involved in phosphorylation by an OmpR-specific protein kinase EnvZ, whereas the C-terminal part possesses a DNA-binding site for the ompC and ompF promoters.
Bacterial Outer Membrane Proteins↗
Transfer of phosphoryl group between two regulatory proteins involved in osmoregulatory expression of the ompF and ompC genes in Escherichia coli.
EnvZ is a cytoplasmic membrane protein which is involved in osmoregulatory expression of the ompF and ompC genes in Escherichia coli possibly by sensing the environmental osmotic signal. A truncated form of the EnvZ protein (EnvZ*), comprising 82% of EnvZ starting from the C terminus, was purified to homogeneity. The purified EnvZ* was autophosphorylated with ATP. The phosphoryl group on EnvZ* could then be rapidly transferred to OmpR, which is a positive regulator of the ompF and ompC genes and which was proposed to interact with EnvZ in the process of osmoregulation. In the presence of ATP, the phosphorylated OmpR was rapidly dephosphorylated. These results suggest that the transfer of the phosphoryl group between EnvZ and OmpR plays an important role in the signaling pathway in osmoregulation.
Adenosine Triphosphate↗
Molecular analysis by deletion and site-directed mutagenesis of the cis-acting upstream sequence involved in activation of the ompF promoter in Escherichia coli.
Expression of the ompF gene coding for an outer membrane protein of Escherichia coli is regulated by a transcriptional activation mechanism that requires the ompR gene product that acts on nucleotides located upstream of the -35 and -10 regions of the ompF promoter. We previously demonstrated that this cis-acting upstream sequence displays a sequence-directed curvature of the DNA helix. To characterize the structure and function of this upstream sequence, a series of deletion mutants and base-substitution mutants of the upstream sequence of the ompF promoter were constructed, and their abilities as to OmpR-binding and activities of the ompF promoter were examined after they had been connected to the lacZ gene. The nucleotides extending from position -91 to -79 are essential not only for sequence-specific recognition of the ompF promoter by the OmpR protein, but also for OmpR-dependent activation of the ompF promoter. It was also demonstrated that the nucleotides extending from position -111 to -92 play a role in stimulation of the ompF expression. A local structural alteration in the ompF promoter was observed in some of the base-substitution mutants. Based on the results, the structure and function of the upstream sequence of the ompF promoter are discussed in relation to activation of the ompF promoter by the OmpR protein.
Bacterial Outer Membrane Proteins↗
Phosphorylation of a bacterial activator protein, OmpR, by a protein kinase, EnvZ, results in stimulation of its DNA-binding ability.
The Escherichia coli OmpR protein is an activator protein specific for the ompF and ompC genes, which respectively encode the outer membrane proteins, OmpF and OmpC. The EnvZ protein is a protein kinase specific for the OmpR protein. In this study, we compared the in vitro DNA-binding ability of the phosphorylated form of the OmpR protein with that of the non-phosphorylated form by means of non-denaturing gel retardation analysis and DNase I footprinting analysis. The results indicate that the phosphorylation of the OmpR protein results in stimulation of its in vitro DNA-binding ability as to both the ompF and ompC promoter DNAs.
Bacterial Outer Membrane Proteins↗
[Regulation of gene expression in prokaryote: signal transduction and phosphorylation].
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Escherichia coli↗
Regulatory effect of a synthetic CRP recognition sequence placed downstream of a promoter.
A series of plasmids were constructed in which a promoter was introduced into a lac-based operon fusion vector. A perfectly symmetrical oligonucleotide of 22-bp corresponding to an idealized binding site for cAMP receptor protein (CRP) of E. coli was chemically synthesized. The synthetic CRP site was placed between the promoter and the lacZ structural gene with varying distances from the promoter. Specific binding of cAMP-CRP complex to the synthetic CRP site was shown by a gel retardation and a DNase I footprinting assays. Plasmid constructs were transformed into crp+ and crp- cells carrying a chromosomal deletion of the lac genes. The regulatory effect of the inserted CRP site was examined by comparing the beta-galactosidase activity and the levels of RNA transcript in two cells harboring the plasmids. We found a strong inhibitory effect of the CRP site in the presence of cAMP and CRP when it was placed close to the promoter. When the CRP site was placed far downstream of the promoter, a moderate repression of transcription was observed.
Base Sequence↗
Function of micF as an antisense RNA in osmoregulatory expression of the ompF gene in Escherichia coli.
To analyze the function of micF as an antisera RNA in the osmoregulatory expression of the ompF gene in Escherichia coli, we performed two experiments. In the first experiment, two strains were constructed in which the transcription initiation site of the ompF gene and the transcription termination site of the micF gene were separated by 186 and 4,100 base pairs, respectively, on the chromosome. These two strains showed almost the same profile of ompF expression as the wild-type strain in which the two genes are separated by 10(6) base pairs. When a high-copy-number plasmid carrying the micF gene was introduced into these strains, ompF expression was completely repressed, whereas no repression was observed with a low-copy-number plasmid carrying the micF gene. These results indicate that the distance between the two genes on the chromosome is not critical for the function of micF. In the second experiment, expression of the ompF gene was examined by pulse-labeling in both the micF+ and the micF deletion strains. Upon a shift from a low- to a high-osmolarity medium, suppression of OmpF protein synthesis occurred more quickly and more extensively in the micF+ strain than in the micF deletion strain. The steady-state synthesis of the OmpF protein was also completely suppressed in the micF+ strain in the high-osmolarity medium, whereas the suppression was incomplete in the micF deletion strain. From these results we conclude that (i) the micF gene contributes to the fast and complete response of the OmpF synthesis to the medium osmolarity, and that (ii) the distance between the micF and ompF genes on the chromosomes is not critical for the function of the micF gene. The results suggest, rather, that the ratio of the copy numbers of the two genes is critical for the function of the micF gene.
Bacterial Outer Membrane Proteins↗
Photodynamic guanine modification by hematoporphyrin is specific for single-stranded DNA with singlet oxygen as a mediator.
Photodynamic modification of DNA by hematoporphyrin (Hp) was characterized by the DNA sequencing technique using 32P-labeled DNA fragments, and the reaction mechanism was investigated by ESR spectroscopy. Mild photodynamic treatment of single-stranded DNA with Hp induced an alteration of guanine residues, and subsequent treatment with piperidine led to chain cleavages at each guanine residue. On the other hand, methylene blue plus light modified the guanine residues in both single-stranded and double-stranded DNA. ESR studies using 2,2,6,6-tetramethylpiperidine and 2,2,6,6-tetramethyl-4-piperidone as singlet oxygen traps demonstrated that Hp plus light produced almost the same amount of singlet oxygen as methylene blue plus light and that the photochemically generated singlet oxygen reacts significantly with guanylate but only slightly with other mononucleotides. An ESR spin destruction method revealed that photoexcited Hp generated porphyrin radical, but guanylate did not react with this radical. These results indicate that photoexcited Hp reacts with oxygen to generate singlet oxygen which oxidizes the guanine residues of single-stranded DNA and that the difference in photoreactivities of DNA with Hp and methylene blue may be explained in terms of the structural difference in their intercalating abilities.
DNA Restriction Enzymes↗
Negative control of transcription by cAMP and cAMP receptor protein in Escherichia coli.
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Cyclic AMP↗
Expression of the adenylate cyclase gene during cell elongation in Escherichia coli K-12.
Expression of the adenylate cyclase gene (cya) in synchronized Escherichia coli cells was investigated by using the cya-lacZ protein and operon fusion plasmids. The regulation of cya expression during the cell cycle is characterized as follows: cya is expressed during cell elongation; expression is repressed during cell division; regulation is exerted at the transcriptional level. To test cya expression during cell elongation, we constructed a plasmid (pLCR1) in which the lacUV5 promoter operator was fused to the structural gene of cya and investigated the effect of cya expression by isopropyl-beta-D-thiogalactopyranoside (IPTG) on the cell division of cells containing pLCR1. By the addition of IPTG, cell division was inhibited and filaments were formed. Such an inhibitory effect was antagonized by adding cyclic GMP to the culture medium and was not observed in the crp mutant.
Adenylyl Cyclases↗
Mutations that alter the allosteric nature of cAMP receptor protein of Escherichia coli.
Mutations which permit cAMP binding protein (CRP) to act in the absence of cAMP have been isolated by in vitro mutagenesis of a plasmid containing the cloned crp gene. Adenylate cyclase deficient cells harbouring the mutant (crp*) plasmids exhibited a variety of fermentation profiles on MacConkey indicator plates containing various sugars. beta-galactosidase synthesis in cells carrying the crp* plasmids was activated most by the addition of cGMP as well as cAMP. The sites of mutations which are responsible for the cAMP independent phenotype were determined by in vitro recombination and DNA sequencing. The amino acid substitutions in the mutant proteins were found in two specific regions of the crp gene encoding residues 53-62 and 141-148 of CRP polypeptide. The first region may participate in cAMP binding, while the second appears to be the inter-domain region of the N-terminal cAMP-binding and C-terminal DNA-binding domains.
Alleles↗
Evidence for negative control of cya transcription by cAMP and cAMP receptor protein in intact Escherichia coli cells.
The transcriptional regulation of cya by cAMP and its receptor protein (CRP) has been studied by S1 nuclease and RNA dot blot assays. The crp- Escherichia coli cells were shown to produce about 5-fold more cya mRNA than do the wild type cells. The effect of cAMP and CRP on the cya transcription was directly examined by introducing a crp plasmid into the cells and/or by adding cAMP exogenously. The levels of cya mRNA in crp+ cells decreased with increasing concentrations of cAMP in the growth medium. The repressive effect of cAMP on cya transcription was strongly enhanced in cells carrying a multicopy crp plasmid. These results indicate that the cya transcription is negatively regulated by cAMP-CRP complex in intact cells.
Base Composition↗
Transcriptional organization of the convergent overlapping dnaQ-rnh genes of Escherichia coli.
The transcriptional organization was determined for the DNA polymerase III epsilon subunit (dnaQ) and the ribonuclease H (rnh) genes, which are closely spaced and organized in a face-to-face system (Maki, H., Horiuchi, T., and Sekiguchi, M. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 7137-7141). Nuclease S1 mapping revealed that transcription starts from a single promoter for rnh and two promoters for dnaQ and proceeds in opposite directions. The 5' terminus of the rnh RNA overlaps about 100 and 20 nucleotide sequences with the 5' terminus of the dnaQ-1 and dnaQ-2 RNA, respectively. The levels of in vivo transcription for the three RNA species increased altogether by more than 10-fold in cells carrying a multicopy plasmid with intact dnaQ-rnh genes, keeping the relative level for the convergent transcription units nearly constant.
DNA Polymerase III↗
Transcription of the Escherichia coli adenylate cyclase gene is negatively regulated by cAMP-cAMP receptor protein.
The regulatory region of the Escherichia coli cya gene was analyzed by using S1 nuclease mapping and in vitro transcription experiments. The cya gene was transcribed, both in vivo and in vitro, from one major promoter (P2) and two weak promoters (P1 and P1') that are located about 200 base pairs upstream of P2. The transcription from P2 was specifically inhibited by cAMP-CRP (cAMP receptor protein) in vitro. This regulatory mechanism was shown to be physiologically relevant through quantitative analyses of the cya mRNA in intact cells by S1 and dot blot assays. DNase I protection experiments revealed that cAMP-CRP binds to the cya DNA region between +11 and -20, in which a consensus CRP binding sequence is present. Moreover, it was found that cAMP-CRP alters the binding of RNA polymerase to the promoter region, thus inhibiting the transcription of the cya gene.
Adenylyl Cyclases↗