Isolation and characterization of a linear plasmid from Streptomyces rimosus

Abstract A linear plasmid was isolated from a strain of Streptomyces rimosus . This plasmid was separated by agarose gel electrophoresis and its size, about 43 kb, determined both by this method and by electron microscopy. The cleavage pattern of the linear plasmid with 5 restriction endonucleases is given. A protein, which is removed by proteinase K, is probably associated to this plasmid. By ethidium bromides or acridine orange treatment we obtained mutants which had lost their aerial mycelium and their linear plasmid.     

Streptomyces rimosus extracellular proteases

Summary Streptomyces rimosus culture filtrates after oxytetracycline production were shown to contain a number of hydrolytic enzymes in concentrations high enough to indicate their exploatation. Crude enzyme preparations were obtained using precipitation, salting out and spray-drying methods. The presence of acid, neutral, and alkaline protease and their elastolytic, esterolytic and collagenolytic activities were discerned. Substrate specificity, pH and temperature optimum, pH stability and molecular weight were studied. These proteases belonged to the group of serine and metalloenzymes. Carboxyl and thiol proteases were not present.     

Streptomyces rimosus extracellular proteases

Summary A procedure for purification of a serine alkaline proteinase from Streptomyces rimosus waste broth was developed. The procedure includes ultrafiltration, CM-Sephadex C-50 and CM-cellulose chromatography and gel filtration on Sephadex G-75. The recovery of electrophoretically pure enzyme was 25%. The enzyme is active on different protein and synthetic substrates at neutral and alkaline pHs but its activity on hemoglobin and bovine serum albumin, is optimal at pH 4.0–4.5. It has a molecular weight of 22,000 and a pI of 4.90. The enzyme is inhibited by DFP and PMSF but not by TPCK. Its substrate specificity, amino acid composition and some other properties were also determined.     

Streptomyces rimosus extracellular proteases

Summary A trypsin-like proteinase was isolated from Streptomyces rimosus culture filtrates obtained from an oxytetracycline production process. The isolation procedure includes ultrafiltration, chromatography on CM-Sephadex, AH-Sepharose and CM-cellulose and gives a homogeneous protein with 19% yield. The enzyme is an anionic trypsin (M_r 28 000, pI 4.5), is stable from pH 4.5 to 9 and up to 40°C, and contains three disulphide bridges, three histidines and three methionines per molecule. At its pH optimum (pH 8.4–8.8) it splits peptide, ester and arylamide bonds of arginine in the endo-position and, to a smaller extent, in the exo-position. Like other streptomycete trypsins, it is a more efficient catalyst than bovine trypsin and has a relative preference for peptide-arylamides, N-benzyloxycarbonyl-l-norleucyl-l-prolyl-l-arginine-p-nitroanilide being by far its best substrate.     

Endo-deoxyribonuclease from Streptomyces rimosus

From filtrates of an oxytetracycline-producing culture of Streptomyces rimosus a deoxyribonuclease was purified to homogeneity and determined to be a potent endo-DNase. It is a monomeric, basic protein (M_r 21 000; pI 9.5) stable in a broad pH range but unstable to higher temperature. The enzyme has an absolute requirement for Mg^{2 +} or Mn^{2 +}, and for its full activity requires free SH groups and a low-ionic-strength environment. Its N-terminal primary structure differs from that of other nucleases.     

Extracellular metalloendopeptidase of Streptomyces rimosus

Metalloendopeptidase was isolated from Streptomyces rimosus culture filtrates in a homogeneous form. It was determined to be a 15 kDa basic protein, most active around pH 7.5, and susceptible to inhibition by chelating agents, N-bromosuccinimide, thiorphan, and 10⁻⁴ M zinc. The enzyme was highly specific for phenylalanine at the N-side of endopeptide bonds. Determination of amino acid sequence of the enzyme’s NH₂-part allowed the recognition of its structure homology with isolated and predicted metallopeptidases from several Streptomyces species. The data contribute to the definition of M7 family of metalloendopeptidases in streptomycetes.     

Streptomyces rimosus extracellular proteases 3. Isolation and characterization of leucine aminopeptidase

Summary A leucine aminopeptidase was purified to homogeneity fromStreptomyces rimosus culture filtrates, which are waste broth of oxytetracycline bioproduction process. Purification procedure includes ultrafiltration and chromatography on CM-Sephadex, AH-Sepharose and FPLC Mono S column. The enzyme is a monomer with molecular weight of 27,500 Daltons and pI of 7.3, stable in broad pH range and up to 70°C. It is a metallo enzyme dependent on Ca²⁺ ions for its full activity. By its specificity it is a true aminopeptidase active on amino acid amide, arylamide, peptide and ester bonds. The hydrolysing activity shows preference for leucine at the N-terminal position of substrates, also acts on aromatic acids and methionine, but does not release glycine, proline, acidic amino acids orD-amino acid residues.     

Isolation of Arabidopsis Mutants with Enhanced Disease Susceptibility by Direct Screening

To discover which components of plant defense responses make significant contributions to limiting pathogen attack, we screened a mutagenized population of Arabidopsis thaliana for individuals that exhibit increased susceptibility to the moderately virulent bacterial pathogen Pseudomonas syringae pv. maculicola ES4326 (Psm ES4326). The 12 enhanced disease susceptibility (eds) mutants isolated included alleles of two genes involved in phytoalexin biosynthesis (pad2, which had been identified previously, and pad4, which had not been identified previously), two alleles of the previously identified npr1 gene, which affects expression of other defense genes, and alleles of seven previously unidentified genes of unknown function. The npr1 mutations caused greatly reduced expression of the PR1 gene in response to PsmES4326 infection, but had little effect on expression of two other defense genes, BGL2 and PR5, suggesting that PR1 expression may be important for limiting growth of PsmES4326. While direct screens for mutants with quantitative pathogen-susceptibility phenotypes have not been reported previously, our finding that mutants isolated in this way include those affected in known defense responses supports the notion that this type of screening strategy allows genetic dissection of the roles of various plant defense responses in disease resistance.     

High-frequency Protoplast-transfection of Streptomyces parvulus 2297 with Actinophage R4 DNA

High frequency transfection of Streptomyces parvulus with actinophage R4 DNA was performed by modifying the procedure of protoplast transformation of S. coelicolor A3(2) with SCP2 plasmid DNA [Bibb et al., Nature, 274, 398 (1978)]. Optimum conditions for protoplast transfection included the presence of 16~24% (w/v) polyethyleneglycol 4000, and the maximum efficiency of transfection was 3 × 10^?5 per phage DNA molecule. This value was at least 100 times higher than the efficiency of previously reported transfection systems in Streptomyces.     

Inhibition of extracellular lipase from Streptomyces rimosus with 3,4-dichloroisocoumarin

Kinetic characterization of lipase inhibition was performed by activity measurement and mass spectrometry (MS), for the first time with serine-protease inhibitor 3,4-dichloroisocoumarin (DCI). Inhibition of Streptomyces rimosus extracellular lipase (SrLip), a member of the SGNH superfamily, by means of DCI follows the mechanism of two-step irreversible inhibition. The dissociation constant of the noncovalent E?I complex and first-order rate constant for inactivation were determined by incubation (K_i* = 26.6?±?2.8 µM, k₂ = 12.2?±?0.6 min–1) or progress curve (K_i* = 6.5?±?1.5 µM, k₂ = 0.11?±?0.01 min–1) method. Half-times of reactivation for lipase inhibited with 10-fold molar excess of DCI were determined by activity measurement (t_1/2 = 11.3?±?0.2?h), matrix-assisted laser desorption/ionization (MALDI, t_1/2 = 13.5?±?0.4?h), and electro-spray ionization (ESI, t_1/2 = 12.2?±?0.5?h) MS. The active SrLip concentration was determined by incubating the enzyme with near equimolar concentrations of DCI, followed by activity and MS measurement.     

Genetic Recombination in Crosses Between Streptomyces aureofaciens and Streptomyces rimosus

Polsinelli, M. (University of Pavia, Pavia, Italy), and Maria Beretta. Genetic recombination in crosses between Streptomyces aureofaciens and Streptomyces rimosus. J. Bacteriol. 91:63-68. 1966.-Biochemical mutants were obtained from Streptomyces rimosus and S. aureofaciens by ultraviolet irradiation. Crosses were performed between auxotrophic strains of S. rimosus and S. aureofaciens with positive results. Data are reported which indicate that the interaction observed in some crosses is due to gene recombination.     

Genetics of Streptomyces rimosus, the Oxytetracycline Producer

From a genetic standpoint, Streptomyces rimosus is arguably the best-characterized industrial streptomycete as the producer of oxytetracycline and other tetracycline antibiotics. Although resistance to these antibiotics has reduced their clinical use in recent years, tetracyclines have an increasing role in the treatment of emerging infections and noninfective diseases. Procedures for in vivo and in vitro genetic manipulations in S. rimosus have been developed since the 1950s and applied to study the genetic instability of S. rimosus strains and for the molecular cloning and characterization of genes involved in oxytetracycline biosynthesis. Recent advances in the methodology of genome sequencing bring the realistic prospect of obtaining the genome sequence of S. rimosus in the near term.