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.

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The linear plasmid pPZGlOl of Streptomyces rimosus R6 was restriction mapped with the enzymes Asel, Bfrl, Dral and Xbal. It is 387 kb in size and the ends are inverted repeats of a t 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.

An extracellular lipase from Streptomyces rimosus R6-554W has been recently purified and biochemically characterized. In this report the cloning, sequencing, and high-level expression of its gene is described. The cloned DNA contained an ORF of 804 bp encoding a 268-amino-acid polypeptide with 34 amino acid residues at the amino terminus of the sequence that were not found in the mature protein. The theoretical molecular mass (24.172 kDa) deduced from the amino acid sequence of the mature enzyme was experimentally confirmed. This lipase showed no overall amino acid sequence similarity to other lipases in the databases. However, two hypothetical proteins, i. e. putative hydrolases, derived from the genome sequencing data of Streptomyces coelicolor A3(2), showed 66% and 33% identity. In addition, a significant similarity to esterases from Streptomyces diastatochromogenes and Aspergillus terreus was found. Sequence analysis revealed that our novel S. rimosus lipase containing a GDS(L)-like consensus motif belongs to family II of lipolytic enzymes, previously unrecognized in Streptomyces. When the lipase gene was expressed in a S. rimosus lipase-deficient strain harboring the lipase gene on a high-copy-number vector, lipase activity was 22-fold higher than in the original strain.

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 Bradley's morphological classification. The rate of RP2 adsorption at 28 "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 yo and immunity to superinfection with the homologous phage. Clear-plaque mutants of RP2, which failed to lysogenize sensitive cultures, arose at a frequency of I N T R O D U C T I O NThe first reports of the occurrence of true lysogeny in actinomycetes appeared simultaneously from three independent laboratories in 1956. It was soon established that most industrial cultures of antibiotic-producing streptomycetes were lysogenic or pseudolysogenic. The distribution of lysogenic Streptomyces species in culture collections has since been extensively studied especially in relation to antibiotic production (Welsch, 1969). However, the genetically well-characterized strain Streptomyces coelicolor A3(2) (Sermonti, 1969;Hopwood et al., 1973) appears to be the only actinomycete in which the genetic control of lysogeny has been investigated. Lornovskaya et al. (1970) were the first to describe a temperate phage isolated from S. coelicolor A3(2), while Dowding & Hopwood (1973) reported the isolation of the free virion VP5, capable of lysogenizing the same strain.While searching for true lysogens among the oxytetracycline-producing S. rimosus strains, we detected free phage particles in the liquid culture of S. rimosus ATCC 10970 (R7). In an attempt to establish this strain and its phage as a host-virus model system for investigating the impact of lysogeny on the industrial production of antibiotics, it has been necessary to grow and characterize the newly detected phage. In this report we describe the conditions necessary for its assay, propagation and preservation, as well as some of the phage characteristics, and show that S. rimosus, which, after S. coelicolor, is genetically the best known Streptomyces species (AlaCeviC, 1969;Friend & Hopwood, 1971 ; AlaEeviC et al., 1973) harbours a prophage (designated RP2) in a truly lysogenic state.

Usually plasmid DNA is introduced into Streptomyces strains by polyethylene glycol-mediated transformation of protoplasts. However, many Streptomyces strains are only poorly or not at all transformable via protoplasts. Therefore, we have optimized the parameters critical for the application of electrotransformation of plasmid DNA into Streptomyces species. The most critical parameters evaluated for electrotransformation of the model strain Streptomyces rimosus R6 were the pretreatment of mycelia, buffer composition, and electric field strength. The electrocompetent mycelia were prepared from 24-hold cultures, treated mildly with lysozyme, resuspended in sucrose-glycerol-polyethylene glycol buffer, and stored in aliquots at ؊70؇C. The electric field strength of 10 kV/cm at 400 ⍀ and a capacitance of 25 F was applied. The method is simple and rapid, yielding transformant colonies in 48 to 72 h. Efficiencies of 10 5 to 10 6 transformants per g of plasmid DNA were reproducibly achieved for S. rimosus R6 and its mutants, and these numbers were 10 2 to 10 3 higher than those attained by polyethylene glycol-assisted transformation of protoplasts. In addition, we show that electroporation can be applied to other Streptomyces species, such as S. lividans 66, S. coelicolor A3(2), and an S. venezuelae strain. This last one could not be transformed by the standard protoplast procedure. Our data suggest that, because of the diversity of streptomycetes, the conditions have to be optimized for each strain.

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The oxytetracycline-producing Streptomyces rimosus strains R6-65 and R7 (ATCC 10970) are lysogenic for the two narrow-host-range phages RP2 and RP3. Both phages are released at low frequency from the lysogenic strains and form plaques on 'cured' S. rimosus strains. RP2 and RP3 are of similar shape with flexible tails and contain double-stranded DNA of about 70 % G + C with cohesive ends (group B1 of bacteriophage classification). The two phages also have identical, very slow, growth kinetics in S. rimosus, with a latent phase of about 6 h and a rise period of about 4 h. RP2 and RP3 are heteroimmune and they differ slightly in their size of phage particles and length of DNA (647 and 62.4 kb for RP2 and RP3, respectively). The restriction maps of the two phages are completely different, and hybridization experiments showed only one short region of sequence similarity (less than 430 bp); the two phages are thus essentially unrelated. Both phages lysogenize their hosts by recombination via defined attachment (aft) sites. The positions of the attP sites have been localized on the restriction maps of RP2 and RP3 to restriction fragments of 800 and 300 bp, respectively. The prophages did not affect the level of oxytetracycline production or the genetic instability of this trait. IntroductionStreptomyces rimosus produces the commercially important antibiotic oxytetracycline. It is the genetically best-characterized species among industrially important Streptomyces. Chromosomal genetic maps have been constructed for three strains: M4018, R7 (ATCC 10970) and R6 (Friend & Hopwood, 1971; AlaEevi6 et al., 1973). Classical genetical methods have also been applied to analyse the genetics of oxytetracycline biosynthesis in strains R6 and M4018 (Pigac & Alakvi6, 1979;Rhodes et al., 1981). In addition, genes for oxytetracycline production and resistance have been cloned and analysed from derivatives of strain M4018 Binnie et al., 1989). S. rimosus strain R6 proved to be genetically unstable, and in this strain the oxytetracycline production and resistance genes are localized at an unstable region of the chromosome (Cullum et al., 1991 ;Gravius et al., 1993). S. rimosus strains R6, R7 and M40 18 contain three related giant linear plasmids, between 280 and 370 kb in size (Cullum et al., 1991 For more than 50 years it has been known that actinophages can attack streptomycetes, but they did not attract much attention until their undesirable interference with industrial fermentations was recognized (Welsch, 1969). More recently, however, considerable advances have been made in the genetic and physical analysis of phages and their interaction with their hosts (Lomovskaya et al., 1980). Streptomyces phages have also been developed as DNA cloning vectors (Chater, 1986).Interest in S. rimosus phages also arose because of problems during industrial fermentations. Ya. I. Rautenstein and collaborators (Rautenstein et al., 1972) reported several cases of lysis of S. rimosus cultures of two lysogenic strains (560 and IK) under industrial co...