Jasenka Pigac

Purification and properties of extracellular lipase from Streptomyces rimosus

An extracellular lipase of Streptomyces rimosus R6-554W was isolated from the culture filtrate by column chromatography using diethylaminoethyl-cellulose, carboxymethyl-cellulose, hydroxylapatite, Mono S (fast protein liquid chromatography), and Sephadex G-75. It was shown to be a monomeric, basic protein (Mr = 27 500, pI = 8.45), active toward triolein and p-nitrophenyl esters, with preference for those with medium size (C8–C12) acyl chain length. Interfacial activation was observed with p-nitrophenyl butyrate as substrate. The lipase was most active at 50–60°C and in alkaline conditions around pH 9–10, with p-nitrophenyl palmitate as substrate. It showed high stability at a broad pH range of 4–10 and was fairly thermostable. Dithiothreitol moderately inactivated the enzyme. Phenylmethylsulfonyl fluoride partly inhibited lipase only when added during the hydrolytic reaction.

The 387 kb linear plasmid pPZG101 of Streptomyces rimosus and its interactions with the chromosome

The linear plasmid pPZG101 of Streptomyces rimosus R6 was restriction mapped with the enzymes AseI, BfrI, DraI and XbaI. It is 387 kb in size and the ends are inverted repeats of at least 95 kb in length. Twenty spontaneous morphological variants and seventeen auxotrophic mutants were screened for changes in the plasmid. Two strains were found that had lost all plasmid sequences. Four strains had integrated parts of the plasmid into the chromosome. Restriction analysis suggested that at least three of the integrated strains had retained free plasmid ends. If it is assumed that the chromosome of S. rimosus R6 is linear, this might be explained by replacement of one or both chromosome ends by a plasmid end. One strain, which overproduced oxytetracycline, carried an enlarged linear plasmid of 1 Mb in size that had acquired chromosomal sequences from the oxytetracycline biosynthesis cluster.

Genetics of Streptomyces rimosus, the Oxytetracycline Producer

SUMMARY 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.

Detection and study of cosynthesis of tetracycline antibiotics by an agar method.

SUMMARY: Pairs of non-tetracycline-producing mutants of Streptomyces rimosus or S. aureofaciens were grown side by side on agar. Their ability to produce antibiotic by cosynthesis was tested by placing a strip of agar cut from the combined culture on plates containing Bacillus subtilis. The activity was revealed as an inhibition halo formed on B. subtilis, opposite one or other mutant strain. The strain surrounded by the halo was considered as a converter of an intermediate product secreted by the other strain. Two types of mutants were observed: a rare type probably affecting the main pathway of antibiotic biosynthesis, and a more frequent type probably affecting some regulatory process.

Characterization and persistence of actinophage RP2 isolated from Streptomyces rimosus ATCC 10970.

While searching for true lysogens among oxytetracycline-producing Streptomyces rimosus strains, free phage particles were detected and isolated from a liquid culture of S. rimosus ATCC 10970 (R7). The actinophage, designated RP2, appears to be a typical temperate DNA phage producing turbid plaques on the sensitive strain S. rimosus R6. Electron microscopic examination of RP2 lysates showed that it belongs to group B of Bradleys morphological classification. The rate of RP2 adsoprption at 28 degrees C appeared to be low. The length of the latent period was about 6 h and the average burst size about 120 phage particles. The lysogenic nature of the host-virus system described was established on the basis of the following characteristics: spontaneous lysis frequency of 2 X 10(-6) per cell, resistance to curing with phage-specific antiserum, spontaneous curing frequency of less than 0.05% and immunity to superinfection with the homologous phage. Clear-plaque mutants of RP2, which failed to lysogenize sensitive cultures, arose at a frequency of 10(-5).

Structural characterization of extracellular lipase from Streptomyces rimosus: assignment of disulfide bridge pattern by mass spectrometry

Abstract The cloning, sequencing and high-level expression of the gene encoding extracellular lipase from Streptomyces rimosus R6-554W have been recently described, and the primary structure of this gene product was deduced using a bioinformatic approach. In this study, capillary electrophoresis-on-the-chip and mass spectrometry were used to characterize native and overexpressed extracellular lipase protein from S. rimosus. The exact molecular mass of the wild-type and the overexpressed lipase, determined by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, were in excellent agreement (Δm=0.11 Da and Δm=0.26 Da, respectively) with a value of 24165.76 Da calculated from the structure deduced from the nucleotide sequence, considering the mature enzyme with all six cysteines forming disulfide bridges. The primary structure derived from the nucleotide sequence was completely verified using a combination of tryptic digestion and formic acid cleavage of the protein, followed by peptide mass fingerprinting. Selected peptides were further investigated by MALDI low-energy collision-induced dissociation hybrid tandem mass spectrometry, allowing the unambiguous determination of their predicted amino acid sequence. No post-translational modifications of mature S. rimosus lipase were detected. Comparison of the peptide mass fingerprints from the reduced and non-reduced overexpressed enzyme unequivocally revealed three intramolecular disulfide bonds with the following linkages: C27-C52, C93-C101 and C151-C198.

Genetic Interactions in Streptomyces rimosus Mediated by Conjugation and by Protoplast Fusion

The development of a protoplast fusion technique for oxytetracycline-producing Streptomyces rimosus strains, and its evaluation for the application for a breeding programme, has been described. Treatment of S. rimosus protoplasts with 40% (w/v) PEG 1550 for 30 min gave optimal numbers of recombinants ranging from 1 to 10% of the total progeny. Therefore, by comparison with conjugation, protoplast fusion increased the frequency of recombination by two to three orders of magnitude. The proportion of multiple crossover classes amongst recombinants was higher, by a factor of ten, after protoplast fusion (13.3%) than after conjugation (1.5%). Participation of less frequent complementary genotype doubled from 9.0% in conjugation to 17.9% in protoplast fusion. Overall, this suggested that the opportunities for crossing over in a fusion of S. rimosus protoplasts were spatially and/or temporally extended leading to a loosening of linkage with a near-random assortment of genotypes in a cross. However, by minimizing the multiple crossover classes and calculating allele frequency gradients, it was shown that the protoplast fusion technique allows arrangement of genetic markers on the S. rimosus chromosome. These are ideal characteristics for the recombination of divergent lines in a strain improvement programme.

Analysis of Amplifications and Deletions in Streptomyces Species

Genetic instability is very common in Streptomyces species, and was one of the first reported properties (Beijerinck, 1913). Usually genetic instability has been detected as influencing easily scored phenotypes such as pigment production (Gregory and Huang, 1964), sporulation, auxotrophy (Redshaw et al., 1979) and antibiotic resistance (Freeman et al., 1977). In some cases genetic instability affects antibiotic production and can be a serious problem in industrial fermentations.

Crystallization and preliminary X-ray diffraction studies of a complex of extracellular lipase from Streptomyces rimosus with the inhibitor 3,4-dichloroisocoumarin

A recombinant lipase (triacylglycerol acylhydrolase; EC 3.1.1.3) from the bacterium Streptomyces rimosus was inhibited by the serine protease inhibitor 3,4-dichloroisocoumarin and crystallized by the hanging-drop vapour-diffusion method at 291 K. The crystals belonged to the monoclinic space group P2(1), with unit-cell parameters a = 38.1, b = 78.7, c = 56.6 Å, β = 104.5° and probably two molecules in the asymmetric unit. Diffraction data were collected to 1.7 Å resolution using synchrotron radiation on the XRD beamline of the Elettra synchrotron, Trieste, Italy.