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T Beppu

Publications and source records attributed to T Beppu.

At least 235 records · Page 13Linked to original sources
PubMed1990

The A-factor-binding protein of Streptomyces griseus negatively controls streptomycin production and sporulation.

A-factor, 2-(6'-methylheptanoyl)-3R-hydroxymethyl-4-butanolide, is an autoregulator essential for streptomycin production and sporulation in Streptomyces griseus. S. griseus 2247 that requires no A-factor for streptomycin production or sporulation was found to have a defect in the A-factor-binding protein. This observation implied that the A-factor-binding protein in the absence of A-factor repressed the expression of both phenotypes in the wild-type strain. Screening among mutagenized S. griseus colonies for strains producing streptomycin and sporulating in the absence of A-factor yielded three mutants that were also deficient in the A-factor-binding protein. Reversal of the defect in the A-factor-binding protein of these mutants led to the simultaneous loss of streptomycin production and sporulation. These data suggested that the A-factor-binding protein played a role in repressing both streptomycin production and sporulation and that the binding of A-factor to the protein released its repression. Mutants deficient in the A-factor-binding protein began to produce streptomycin and sporulate at an earlier stage of growth than did the wild-type strain. These mutants produced approximately 10 times more streptomycin than did the parental strain. These findings are consistent with the idea that the intracellular concentration of A-factor determines the timing of derepression of the gene(s) whose expression is repressed by the A-factor-binding protein.

4-Butyrolactone↗
PubMed1990

Autoregulatory factors of secondary metabolism and morphogenesis in actinomycetes.

The Gram-positive bacterial genus Streptomyces possesses interesting biological aspects, such as the ability to produce a wide variety of secondary metabolites and a mycelial form of growth that culminates in sporulation. A close relationship of secondary metabolism and cell differentiation has been well recognized; secondary metabolism might be a physiological expression of cell differentiation. A variety of diffusible low-molecular-weight chemical substances have been found to function in general as regulatory factors, like "hormones" in eukaryotes, for secondary metabolism and cell differentiation. Among these factors, A-factor has been most extensively studied. This review summarizes recent research on the chemical structures, functions, biosyntheses, and mode of action of these regulatory factors.

4-Butyrolactone↗
PubMed1990

Structural specificity for biological activity of trichostatin A, a specific inhibitor of mammalian cell cycle with potent differentiation-inducing activity in Friend leukemia cells.

Biological activities of four chemically synthesized trichostatin-related compounds, (R)-trichostatin A, (S)-trichostatin A, (R)-trichostatic acid, and (S)-trichostatic acid, were investigated. Assays of differentiation-inducing activity in Friend leukemia cells and G2-arresting activity in the cell cycle of normal rat fibroblast cells were used as monitoring systems for comparing the bioactivities of these compounds. The results clearly showed that both of the enantiomers of trichostatic acid had no activity in both the assay systems. In the case of (S)-trichostatin A, the antipode of naturally occurring trichostatin A, 50% effective concentrations were determined to be 50-70-fold higher than those of (R)-trichostatin A. The relationship between this ratio and the value of enantiomeric excess strongly suggests that (S)-trichostatin A is also biologically inactive. These results indicate that the absolute configuration and the presence of the hydroxamate group of trichostatin A are essential for its biological activity.

Animals↗
PubMed1989

Secretion by yeast of the zymogen form of Mucor rennin, an aspartic proteinase of Mucor pusillus, and its conversion to the mature form.

The Mucor rennin gene encoding a prepro-form of the fungal aspartic proteinase from Mucor pusillus was expressed under the control of the yeast GAL7 promoter in Saccharomyces cerevisiae. An inactive zymogen of the enzyme with the 44-amino-acid pro-sequence was identified in the medium during the initial stage of cultivation. Processing of the purified zymogen to the mature enzyme proceeded autocatalytically under the acidic conditions. The rate of processing was accelerated by an increase in the concentration of the zymogen or addition of the mature enzyme. The in vitro processing was inhibited by inhibitors for the aspartic proteinases. The zymogen with no proteinase activity due to a mutation at the active site residue, Asp, was still processed at a relatively slower rate in a wild-type strain of yeast, but no processing occurred in the pep4-3 mutant strain of S. cerevisiae deficient in yeast proteinase A. Thus, Mucor rennin is excreted in a form of zymogen, which is then processed in the yeast secretion pathway mainly by the autocatalytic proteolysis but, alternatively, by a proteinase of yeast.

Amino Acid Sequence↗
PubMed1989

Primary structure of nitrile hydratase deduced from the nucleotide sequence of a Rhodococcus species and its expression in Escherichia coli.

The nitrile hydratase (NHase) of Rhodococcus species N-774, which is composed of two subunits, alpha and beta, catalyzes the hydration of various nitrile compounds to the corresponding amides. The amino acid sequences of the NH2 termini and the fragments obtained by digesting each of the two subunits with lysyl endopeptidase were determined for preparation of synthetic oligonucleotides as hybridization probes. A 4.4-kb SphI fragment which contained DNA sequences hybridizing to several of the probes was cloned in pBR322 in Escherichia coli. The nucleotide sequences together with the determined amino acid sequences indicated that the alpha and beta subunits of NHase consisted of 207 amino acids (Mr, 22918) and 212 amino acids (Mr, 23428), respectively. The open reading frame for the alpha subunit includes that for the beta subunit with a short interval of only 26 base pairs; the two genes are probably translated in a polycistronic manner. Although large amounts of the alpha- and beta-subunit proteins were produced as insoluble forms in E. coli when the cloned genes were placed under the control of the lac promoter, no enzymatic activity was detected. The activity of the enzyme was restored, to some extent, by solubilization of the proteins with 8 M urea and subsequent dialysis for refolding at pH 10 in the presence of Fe2+ and pyrroloquinoline quinone.

Amino Acid Sequence↗
PubMed1989

A 2.0-A structure of the blue copper protein (cupredoxin) from Alcaligenes faecalis S-6.

The structure of a blue copper protein, cupredoxin, from the potent denitrifying bacterium Alcaligenes faecalis S-6, has been determined and refined against 2 A x-ray diffraction data. The agreement between observed and calculated structure factors is 0.159, and estimated errors in coordinates are 0.09-0.15 A. The protein folds in a beta sandwich similar to plastocyanin and azurin and includes features such as a "kink" and a "tyrosine loop" which have been noted previously for these proteins as well as immunoglobulins. The copper is bound by four ligands, in a distorted tetrahedral arrangement, with Cu-S gamma = 2.07 A (Cys-78), Cu-N delta 1 = 2.10 and 2.21 for His-40 and His-81, and Cu-S delta = 2.69 A (Met-86). Two of the ligands are further oriented by hydrogen bonds either to other side chains (Asn-9 to His-40), backbone atoms (NH...S) or a water molecule (to His-40). The methionine ligand has no extra constraints. The C-terminal loop containing three of the ligands is hydrogen-bonded to the strand containing His-40 by hydrogen bonds between the conserved residues Thr-79 and Asn-41. The pronounced dichroism of the crystal is a result of the orientation of the normal to the C beta-S gamma-Cu plane parallel to the crystallographic 6-fold axis.

Alcaligenes↗
PubMed1989

Cloning and characterization of the carbapenem biosynthetic genes from Streptomyces fulvoviridis.

Carbapenem non-producing mutants were isolated from Streptomyces fulvoviridis and divided into six cosynthesis groups. By using one of the mutants as the host and plasmid pIJ385 as the vector, we cloned carbapenem biosynthetic genes from the parental S. fulvoviridis strain. A cloned 6-kb DNA fragment complemented the defects of three mutants each of which had a mutation in different genes. Southern blot hybridization using the cloned 6-kb fragment as probe showed the presence of the nucleotide sequences homologous to the probe in other carbapenem-producing Streptomyces spp. In addition, Streptomyces griseus, a carbapenem non-producer, possessed the sequence homologous to the probe and showed co-synthesis phenomena with some of the carbapenem non-producing mutants of S. fulvoviridis.

Anti-Bacterial Agents↗
PubMed1989

afsB stimulates transcription of the actinorhodin biosynthetic pathway in Streptomyces coelicolor A3(2) and Streptomyces lividans.

The pleiotropic regulatory gene, afsB, from Streptomyces coelicolor A3(2), possibly encoding a DNA-binding protein, is required for actinorhodin production in this organism. Northern blot hybridization using a DNA fragment covering part of the set of cloned actinorhodin biosynthetic gene cluster (act) as the probe showed lack of the act transcripts in an afsB-negative mutant of S. coelicolor A3(2); the transcripts were restored on introduction of a cloned afsB gene. Introduction of the cloned afsB gene into Streptomyces lividans stimulated transcription of the act genes under conditions in which they are normally silent in this strain, leading to production of actinorhodin in large quantity. These data show that afsB exerts its positive regulatory effect by means of transcriptional stimulation of its target genes.

Anthraquinones↗
PubMed1989

Dyspnea resulting from accumulation of pleural effusion after radical neck dissection. A case report.

The patient became dyspneic 3 days after radical neck dissection on the left side. A chest radiography showed bilateral pleural effusion.During the operation, a lymphatic leak was noted. In this case, the factor of an associated perforation of the pleural had not been demonstrated. Fresh frozen plasma was administered and positive end-expiratory pressure was applied. The patient had no residual pulmonary sequelae.

Blood Transfusion↗
PubMed1989

Nucleotide sequence of the membrane-bound aldehyde dehydrogenase gene from Acetobacter polyoxogenes.

The nucleotide sequence of the membrane-bound aldehyde dehydrogenase (ALDH) gene from an industrial vinegar producer, Acetobacter polyoxogenes, was determined. Comparison of the sequence with the NH2-terminal amino acid sequence of the mature ALDH and determination of the actual translational initiation codon by means of in vitro manipulation of the upstream and proximal regions of the cloned gene showed that ALDH was primarily translated as a 773-amino-acid protein and that the 44-amino-acid sequence at the NH2-terminus, which probably serves as a signal peptide, was processed during maturation and localization in the membrane. When ALDH was expressed in a large quantity in Escherichia coli cells after the coding region had been placed downstream of the lac promoter, the ALDH protein, which still contained the signal peptide and had no ALDH activity, was localized in the membrane fraction.

Acetobacter↗
PubMed1989

Alteration of catalytic properties of chymosin by site-directed mutagenesis.

Artificial mutations of chymosin by recombinant DNA techniques were generated to analyze the structure--function relationship in this characteristic aspartic proteinase. In order to prepare the mutant enzymes in their active form, we established procedures for purification of correctly refolded prochymosin from inclusion bodies produced in Escherichia coli transformants and for its subsequent activation. Mutagenesis by linker insertion into cDNA produced several mutants with an altered ratio of milk clotting activity to proteolytic activity and a different extent of stability. In addition to these mutants, several mutants with a single amino acid exchange were also constructed by site-directed mutagenesis and kinetic parameters of these mutant enzymes were determined by using synthetic hexa- and octa-peptides as substrates. Exchange of Tyr75 on the flap of the enzyme to Phe caused a marked change of substrate specificity due to the change of kcat or Km, depending on the substrate used. Exchange of Val110 and Phe111 also caused a change of kinetic parameters, which indicates functional involvement of these hydrophobic residues in both the catalytic function and substrate binding. The mutant Lys220----Leu showed a marked shift of the optimum pH to the acidic side for hydrolysis of acid-denatured haemoglobin along with a distinct increase in kcat for the octa-peptide in a wide pH range.

Amino Acids↗
PubMed1989

Cloning of the Membrane-Bound Aldehyde Dehydrogenase Gene of Acetobacter polyoxogenes and Improvement of Acetic Acid Production by Use of the Cloned Gene.

A genomic clone bank of Acetobacter polyoxogenes NBI1028 constructed in Escherichia coli by use of the expression vector pUC18 was screened with antibody raised against membrane-bound aldehyde dehydrogenase (ALDH; 75 kilodaltons [kDa]) from A. polyoxogenes NBI1028. A clone that synthesized a 41-kDa protein cross-reactive with anti-ALDH antibody was isolated. For cloning of the full-length ALDH structural gene, a cosmid gene bank was screened by Southern blot hybridization with the cloned DNA as a probe, and subcloning from the positive cosmid clone was performed with shuttle vector pMV24. Plasmid pAL25, containing the full-length ALDH structural gene, was isolated and expressed in both E. coli and Acetobacter aceti to produce a fused protein (78 kDa) with a short NH(2)-terminal beta-galactosidase peptide. pAL25 conferred ALDH production on a mutant of A. aceti lacking the enzyme activity. Transformation of A. aceti subsp. xylinum NBI2099 with pAL25 caused 2- and 1.4-fold increases in the production rate and in the maximum concentration of acetic acid in submerged fermentation, respectively.

Journal Article↗
PubMed1989

Characterization of the precursor of Serratia marcescens serine protease and COOH-terminal processing of the precursor during its excretion through the outer membrane of Escherichia coli.

The Serratia marcescens serine protease, which is directed by the gene encoding a precursor composed of a typical NH2-terminal signal sequence, a mature enzyme domain, and a large COOH-terminal domain, was excreted through the outer membrane of Escherichia coli. The precursor, with the expected molecular size (110 kilodaltons), was detected in an insoluble form in the periplasmic space of E. coli cells after induction with isopropyl-beta-D-thiogalactopyranoside of the expression of the gene under the control of the tac promoter. Upon membrane fractionation of the disrupted cells by sucrose density gradient centrifugation, the precursor was recovered from a fraction slightly heavier than the outer membrane fraction but not from the inner membrane fraction. Conversion of the precursor into the mature form, which was accompanied by its excretion into the medium, was observed even in the absence of de novo protein synthesis caused by the addition of chloramphenicol. The mutated gene product lacking all of the COOH-terminal domain was localized in the periplasmic space only and was not excreted into the medium. Additional mutant genes were generated by site-directed mutagenesis to test the role of some amino acids in the excretion of this protease in E. coli. The mutant protein with no protease activity because of the change of the catalytic residue Ser-341 to Thr was still excreted into the medium but with abnormal processing. Both self-processing and host-dependent processing of the precursor seem to be involved in the excretion of the mature enzyme. Replacement of the four Cys residues, two in the mature enzyme and two in the COOH-terminal domain, with Ser in different combinations caused a distinct or complete loss of excretion, suggesting that a certain conformation possibly formed via disulfide bonding was important for the excretion of the S. marcescens protease.

Base Sequence↗
PubMed1989

Nucleotide sequence and transcriptional analysis of the Streptomyces griseus gene (afsA) responsible for A-factor biosynthesis.

The nucleotide sequence of the Streptomyces griseus afsA gene, possibly encoding a key enzyme for A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) biosynthesis, was determined. The translational initiation codon was identified by introducing out-of-frame mutations at appropriate positions by oligonucleotide-directed mutagenesis. The afsA gene was thus found to code for a protein of 301 amino acid residues and 32.6 kilodaltons whose codon usage pattern was in agreement with the general tendency of Streptomyces genes with an extremely high guanine-plus-cytosine content. High-resolution S1 nuclease mapping indicated that the transcriptional start point was the A residue, the first position of the ATG translational initiation codon.

4-Butyrolactone↗