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Biomedical subjects

A Nakazawa

Publications and source records attributed to A Nakazawa.

At least 163 records · Page 9Linked to original sources
PubMed1983

Molecular cloning of regulatory gene xylR and operator-promoter regions of the xylABC and xylDEGF operons of the TOL plasmid.

The regulatory gene xylR of the TOL plasmid, which functions positively on both xylABC and xylDEGF operons in the presence of m-xylene or m-methylbenzyl alcohol, was cloned onto an Escherichia coli vector, pACYC177. A fused operon consisting of the operator-promoter region of the xylABC operon and the xylE gene was cloned onto pBR322. The xylE product, catechol 2,3-dioxygenase, was induced by m-xylene or m-methylbenzyl alcohol in the cells containing the fused operon when a 2.8-kilobase segment of the TOL plasmid was provided in trans. Therefore, the segment appeared to contain the regulatory gene xylR. The xylR gene was mapped very close to the other regulatory gene, xylS, determined previously. The xylR gene was not effective on activation of the xylDEGF operon unless an additional region containing xylS was provided together with the inducer. These results indicate that both xylR and xylS are essential to the m-methylbenzyl alcohol-dependent induction of the xylDEGF operon. The map positions of xylR and xylS were precisely determined by subcloning or insertion inactivation. In addition, the operator-promoter regions of the xylABC and xylDEGF operons were mapped to the 0.6- and 0.4-kilobase regions of the TOL plasmid, respectively.

Catechol 2,3-Dioxygenase↗
PubMed1983

Plasmid-encoded regulation of colicin E1 gene expression.

A plasmid-encoded factor that regulates the expression of the colicin E1 gene was found in molecular cloning experiments. The 2,294-base-pair AvaII fragment of the colicin E1 plasmid (ColE1) carrying the colicin E1 structural gene and the promoter-operator region had the same information with respect to the repressibility and inducibility of colicin E1 synthesis as the original ColE1 plasmid. An operon fusion was constructed between the 204-bp fragment containing the colicin E1 promoter-operator and xylE, the structural gene for catechol 2,3-dioxygenase encoded on the TOL plasmid of Pseudomonas putida. The synthesis of the dioxygenase from the resulting plasmid occurred in recA+, but not in recA- cells and was derepressed in the recA lexA(Def) double mutant. These results indicate that the ColE1 plasmid has no repressor gene for colicin E1 synthesis and that the lexA protein functions as a repressor. Colicin E1 gene expression was adenosine 3',5'-phosphate (cAMP) dependent. Upon the removal of two PvuII fragments (2,000 bp in length) from the ColE1 plasmid, the induced synthesis of colicin E1 occurred in the adenylate-cyclase mutant even without cAMP. The 3,100-bp Tth111I fragment of the ColE1 plasmid cloned on pACYC177 restored the cAMP dependency of the deleted ColE1 plasmid. Since the deleted fragments correspond to the mobility region of ColE1, the cAMP dependency of the gene expression should be somehow related to the plasmid mobilization function.

Cloning, Molecular↗
PubMed1982

Translocation of colicin E1 through cytoplasmic membrane of Escherichia coli.

The product of the malE-lacZ gene fusion was reported to compete with some proteins including outer membrane lipoprotein in the protein translocation across the Escherichia coli membrane. The fusion product also inhibited colicin E1 export. Furthermore, globomycin, which accumulated prolipoprotein in the membrane, inhibited the translocation of colicin E1 in the wild-type cells, but not in lipoprotein-negative mutant cells. Since colicin E1 contains the internal signal-like sequence [Proc. Natl. Acad. Sci. USA (1982) 79, 2827-2831], these results suggest that colicin E1 is exported by the aid of this sequence at a common site for maltose-binding protein and lipoprotein translocation.

Biological Transport↗
PubMed1982

Sugar transport by the bacterial phosphotransferase system. Phosphoryl transfer reactions catalyzed by enzyme I of Salmonella typhimurium.

The phosphorylation of Enzyme I is the first step in the phosphotransfer reaction sequence catalyzed by the phosphoenolpyruvate:glycose phosphotransferase system (PTS) from Salmonella typhimurium. The characterization of phospho approximately Enzyme I and the reactions in which it participates are described in this report. About 1 mol of phosphoryl group was incorporated per mol of Enzyme I monomer when the homogeneous enzyme was incubated with [32]phosphoenolpyruvate and Mg2+. The phosphoryl group in phospho approximately Enzyme I is linked at the N-3 position in the imidazole ring of a histidine residue. Phospho approximately Enzyme I donates its phosphoryl group to pyruvate (to form phosphoenolpyruvate (P-enolpyruvate)) and to the histidine-containing phosphocarrier protein of the phosphotransferase system (HPr) (to form phospho approximately HPr). In the presence of HPr and appropriate sugar-specific proteins, the phosphoryl group can be transferred from Enzyme I to methyl alpha-glucoside (to form sugar-phosphate). The phosphorylation of Enzyme I by phosphoenolpyruvate requires divalent cation, but the phosphoryl group is transferred from phospho approximately Enzyme I to HPr in the presence of 20 mM EDTA. Kinetic studies show a biphasic rate for Enzyme I phosphorylation, suggesting that the enzyme is phosphorylated in the associated state. Equilibrium experiments were conducted on the following Reactions A and C. (formula: see text). The apparent K' for Reaction B was calculated from K'A and K'C. K'C was found to be about 11. K'A was studied both at very low and high substrate (P-enolpyruvate and pyruvate) concentrations relative to their respective Km values. At low substrate concentrations, the reaction appeared independent of pH in the range of 6.5 to 8.0, and when analyzed according to the simplest expression that could be written for total species of each component (Reaction A), the apparent average K' was 1.5. At high substrate concentrations, about 50% of the Enzyme I was phosphorylated, and this value changed only slightly with large changes in the P-enolpyruvate to pyruvate ratio. Expressions for K'A are derived which partially explain these results by including enzyme-substrate complexes in the equilibrium expression. The K' values were used to derive apparent standard free energy changes for the hydrolysis of the phosphoproteins of the PTS. Since these are similar to those for the hydrolysis of P-enolpyruvate, the phosphate transfer potentials of the PTS phosphoproteins are among the highest of known biological phosphate derivatives. In addition, unlike the reactions which occur during anaerobic glycolysis and electron transport, the high phosphate transfer potential is conserved in the PTS reaction sequence until the last step, the translocation of the sugar substrate across the membrane concomitant with its phosphorylation. Potential regulation of the PTS, in particular the effect of the intracellular ratio of P-enolpyruvate to pyruvate, is considered.

Biological Transport↗
PubMed1982

Sugar transport by the bacterial phosphotransferase system. Isolation and characterization of a phosphocarrier protein HPr from wild type and mutants of Salmonella typhimurium.

HPr, the histidine-containing phosphocarrier protein of the phosphotransferase system, has been isolated and purified from wild type Salmonella typhimurium and from two mutants of this organism. Comparison of the sequences of these three forms of HPr indicates that the two mutants contain substitutions at residue 4 in the polypeptide chain; in place of glutamine in the wild type, one mutant (SB3899) contained serine and the other (SB3093) contained lysine. The substitution of lysine for glutamine resulted in increased positive charge of the molecule which is reflected in an expected decreased mobility on polyacrylamide gel electrophoresis and its behavior on isoelectric focusing. However, these changes had no effect on phosphocarrier activity in the phoshoenolpyruvate:glycose phosphotransferase system as measured by the kinetics of the interaction on the mutant HPr proteins with both Enzyme I and Enzyme II. These results have important implications for potential chemical modification of HPr. The HPr from wild type cells was crystallized.

Amino Acid Sequence↗
PubMed1982

Nucleotide sequence of the structural gene for colicin E1 and predicted structure of the protein.

We have determined the nucleotide sequence of the structural gene for colicin E1, which consists of 1,566 base pairs. The amino acid sequence (522 residues) of the protein was derived from the DNA sequence, and the molecular weight was calculated to be 57,279. From the analysis of the predicted secondary structure, there appear to be three consecutive long alpha-helices in the NH2-terminal half of the polypeptide, spanning 40, 100, and 35 amino acid residues. In addition, there is a polypeptide region near the COOH terminus that shows homology to the NH2-terminal signal portions of outer membrane lipoprotein in Escherichia coli and beta-lactamase in Bacillus licheniformis. Most of the homologous amino acids are located in the region where either alpha-helix or beta-sheet would be expected to occur, as determined from the amino acid sequence. These characteristics of the predicted protein structure might correspond to properties of colicin E1 as an ionophore in its antimicrobial action and also as an exported protein during its induced synthesis.

Amino Acid Sequence↗
PubMed1982

Purification and characterization of a protein from Escherichia coli which forms complexes with superhelical and single-stranded DNAs.

From the cells of an Escherichia coli K-12 strain, a 22,000-dalton protein which has an affinity for the superhelical DNA molecule was purified to apparent homogeneity by monitoring the DNA-binding activity using the filter binding assay. In the sedimentation analysis of the DNA-protein complex, the protein has an affinity for the superhelical or single-stranded DNA molecule but neither for the open-circular nor for the linear DNA molecule. The amino acid composition of the protein resembled those of the other prokaryotic histone-like proteins and also to eukaryotic histones H2A and H2B. The protein precipitated upon heating, which is in contrast to the heat-stable feature of the other histone-like proteins. Furthermore, DNA and RNA syntheses in vitro were not affected by the presence of the protein. In view of these characteristics, this protein may play a role in maintaining the bacterial nucleoid structure.

Bacterial Proteins↗
PubMed1982

Direct participation of lexA protein in repression of colicin E1 synthesis.

Spontaneous colicin E1 production by plasmid RSF2124 in a recA lexA(spr) strain of Escherichia coli was about 10-fold greater than that observed in a wild-type strain. The synthesis was repressed nearly to the level of a recA strain in the presence of the plasmid pMCR551, which carries the lexA gene.

Bacterial Proteins↗
PubMed1981

Organization of the lexA gene of Escherichia coli and nucleotide sequence of the regulatory region.

The product of the lexA gene of Escherichia coli has been shown to regulate expression of the several cellular functions (SOS functions) induced by treatments which abruptly inhibit DNA synthesis. We have cloned and mapped the lexA gene on a small segment of approximately 600 base pairs. The lexA promotor was located by transcription R-loop analysis, and the lexA product of 22,000 daltons was identified by protein synthesis in vitro. An unknown gene was found which directed the synthesis of a protein of 35,000 daltons in a region downstream from the lexA gene. Nucleotide sequence of the regulatory region of the lexA gene was determined. The sequence contained inverted repeats homologous to that of the recA regulatory region. These inverted repeats may be recognized by the lexA protein, because the protein is considered to repress both the genes as a common repressor.

Bacterial Proteins↗
PubMed1981

Constriction of a fused operon consisting of the recA and kan (kanamycin resistance) genes and regulation of its expression by the lexA gene.

The kanamycin resistance gene (kan) of transposon Tn5 was cloned into a derivative of plasmid pBR322. A DNA fragment containing the promoter-operator region of the recA gene was inserted into the promoter region of the cloned kan gene to produce a fused operon, recA-kan. Plasmid pMCR685 carrying recA-kan expressed a low level of activity of the kan gene product (kanamycin phosphotransferase; KPT) in the wild-type cells of Escherichia coli, while the plasmid showed an increased level of the activity in the SPr- mutant cells which produce the inactive lexA protein. The KPT activity in the wild-type cells harboring the plasmid increased 6- to 11-fold upon treatment of the cells with mitomycin C or nalidixic acid, both of which are known to induce synthesis of recA protein. Expression of the recA-kan operon fusion was remarkably repressed by the lexA gene cloned into a plasmid carrying the operon fusion. Higher concentrations of mitomycin C were required for maximal induction of KPT activity in the cells harboring the resulting plasmid pMCR687. These results strongly suggest that the lexA gene product can be itself repress the recA gene, and that pMCR687 is a useful vector to clone genes whose expression is harmful to the host cell growth.

Cloning, Molecular↗
PubMed1981

The nucleotide sequence surrounding the promoter region of colicin E1 gene.

The nucleotide sequence of 570 bp, covering the N-terminal portion of the colicin E1 gene, was determined. The sequence of the N-terminal four amino acids of the colicin E1 protein, determined by manual Edman degradation, agreed with that predicted from the nucleotide sequence. From analysis of the 5'-terminal sequences of RNAs synthesized in vitro, the promoter and operator regions of the colicin E1 gene were assigned. These data indicate the existence of two promoters, one of which is located in the coding region for colicin E1. DNA sequence homology of 16 bp was found between the putative operator regions of the colicin E1 and recA genes.

Amino Acid Sequence↗
PubMed1981

Molecular cloning of TOL genes xylB and xylE in Escherichia coli.

The xylB and xylE genes in the TOL plasmid of Pseudomonas putida mt-2, which code for benzyl alcohol dehydrogenase and catechol 2,3-oxygenase, respectively, were cloned onto plasmid pBR322 in Escherichia coli for detailed mapping. The xylB gene was mapped in a 2.9-kilobase region within the BamHI BC fragment of pTN2, an in vivo RP4-TOL recombinant, whereas the xylE gene was mapped in a 1.8-kilobase region within the BamHI BD fragment. The directions of transcription of these genes were deduced from the expression of the cloned genes which had been ligated in orientations opposite pBR322 at its BamHI site within the tetracycline resistance gene. The xylB and xylE genes are inducible by a specific inducer of the TOL pathway genes in the RP4-TOL recombinant, whereas they are not inducible in the pBR322-TOL hybrids. The regulatory regions involved in expression of the xylB and xylE genes do not appear to be located in the vicinity of the structural genes. Catechol 2,3-oxygenase formed in E. coli carrying an xylE-containing plasmid is similar, or identical, to that formed in P. putida carrying the TOL plasmid.

Alcohol Oxidoreductases↗
PubMed1981

Molecular cloning of gene xylS of the TOL plasmid: evidence for positive regulation of the xylDEGF operon by xylS.

The xylDEGF operon and the regulatory gene xylS of the TOL plasmid found in Pseudomonas putida mt-2 were cloned onto Escherichia coli vector plasmids. A 9.5-kilobase fragment, derived from the TOL segment of pTN2 deoxyribonucleic acid, carried the xyl genes D, E, G, and F, which encode toluate oxygenase, catechol 2,3-oxygenase, 2-hydroxymuconic semialdehyde dehydrogenase, and 2-hydroxymuconic semialdehyde hydrolase, respectively. The enzymes were noninducible unless a 3-kilobase PstI fragment, derived also from the TOL segment, was provided in either cis or trans. The PstI fragment appeared to contain the regulatory gene xylS, which produced a positive regulator. The regulator was activated by m-toluate or benzoate, but not by m-xylene or m-methylbenzyl alcohol. the map positions of xylG and xylF were also determined.

Carboxylic Acids↗
PubMed1980

Physical and functional mapping of RP4-TOL plasmid recombinants: analysis of insertion and deletion mutants.

Cleavage sites for the restriction endonucleases XhoI, BamHI, HindIII, and EcoRI were mapped on the pTN2 plasmid, a recombinant of TOL and RP4, which specifies the toluene-degrading enzymes in the same way as the wild-type TOL plasmid. The pTN2 plasmid, purified from a strain of Escherichia coli, contained the entire length of the RP4 plasmid (about 54 kilobase pairs [kb]) and the TOL segment (about 56 kb). The TOL segment is inserted at about 12 and 5 kb away from the EcoRI and BamHI cleavage sites of RP4, respectively. Cleavage sites for XhoI, BamHI, HindIII, and EcoRI were also mapped on an insertion mutant, pTN1, and two deletion mutants, pTN81 and pTN9. Analysis of pTN81 and pTN9 allowed estimation of the region of the gene cluster for benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase, as well as the region required for toluate oxygenase activity. Induction of TOL enzymes directed by pTN1 suggested the location and orientation of transcription of the gene cluster for catechol 2,3-oxygenase, 2-hydroxymuconic semialdehyde dehydrogenase, and 2-hydroxymuconic semialdehyde hydrolase. Analysis of strains carrying both pTN9 and a xylR mutant of the TOL plasmid demonstrated that xylR+ is trans dominant over xylR.

Alcohol Oxidoreductases↗
PubMed1979

Gene expression in vitro of colicin El plasmid.

Among eighteen polypeptides synthesized in vitro from colicin El plasmid, one of the major products with a molecular weight of 59,000 was identified as colicin El by its immunological property, molecular size, and biological activity. In addition to this polypeptide, seven other polypeptides reacted with colicin El antiserum. Using EcoRI-cleaved colicin El DNA, a 56,000 dalton polypeptide of truncated colicin El was synthesized, but no polypeptide that reacted with colicin El antiserum was produced from SmaI-cleaved colicin El DNA. This fact indicates that the direction of transcription of colicin El structural gene is from SmaI site to EcoRI site in vitro. The immunity protein of a molecular weight of 14,300 and a component of relaxation proteins of a molecular weight of 64,000 were deduced by comparing the results of the gene expression in vitro of one-half (pAO100) and a quarter (pAO2) of colicin El plasmid. The directions of transcription-translation in the genes on the plasmid were discussed. The colicin El plasmid appears to have at least three transcriptional units.

Colicins↗