No abstract

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.

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.