Genetics of Streptomyces rimosus, the oxytetracycline producer

Abstract

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.

FIG. 1.

Chemical structures of primary tetracyclines (A) and of rimocidin (B).

FIG. 1.

Chemical structures of primary tetracyclines (A) and of rimocidin (B).

FIG. 2.

Colony morphology of two Streptomyces rimosus strains. (Right) S. rimosus ATCC 10970 (NRRL 2234), abbreviated to strain R7. (Left) S. rimosus strain R6, also known as the Zagreb strain, isolated from soil by the Faculty of Food Science and Biotechnology, University of Zagreb. Note the differences in sporulation, an important determinant in developing fermentation parameters and systems for gene exchange.

FIG. 3.

Architecture of the oxytetracycline gene cluster. The four regions otcZ, otcX, otcD, and otcY correspond to loci originally characterized by genetic analysis and cross-feeding studies of blocked mutants. The open boxes show the positions of identified genes in the cluster, with their orientations marked by arrows. (Modified from reference with permission of the publisher.)

FIG. 4.

Proposed gene functions in the biosynthesis of oxytetracycline. Nonaketamide (nascent linear polyketide chain), pretetramid, and the intermediates 6-MPT, 4-hydroxy-6-MPT, 4-keto-ATC, 4-amino-ATC, ATC, 5-DHTC, and DHOTC, as well as OTC, are shown. Nonaketamide carbon atoms are numbered starting from the enzyme (-S-E), while carbon atoms from the first tetracyclic structure (pretetramid) are numbered according to IUPAC nomenclature.

FIG. 5.

Proposed architecture of rimocidin-producing modular (type I) polyketide synthase. Modules are numbered according to the order of chain elongation. KS, ketoacyl synthase; AT, acyltransferase; KR, ketoreductase; DH, dehydratase; ER, enoyl reductase; ACP, acyl carrier protein.

FIG. 6.

Restriction map of the chromosome of Streptomyces rimosus R6-501 for the enzymes AseI (outer arc) and DraI (inner arc). The terminal inverted repeats are drawn as a stem structure. The numbers of the linking clones are indicated adjacent to the corresponding restriction sites; the missing DraI linking clone is indicated by an asterisk. The cosmid clones carrying the ends of the terminal inverted repeats (C-136 and C-123) are also indicated. The OTC cluster and the attB-pSAM2 gene have been localized precisely. The other markers have been localized only to particular AseI and DraI fragments. (Reprinted from reference with permission of the publisher.)

FIG. 7.

Electron micrographs of RP2 (A) and RP3 (B) particles negatively stained with potassium phosphotungstate. Magnification, ×75,000. Bars, 100 nm. (Reprinted from reference 104 [panel A] and reference 193 [panel B] with permission of the publisher.)

FIG. 8.

Model to explain the structure of inverted chromosomes in deletion mutants of Streptomyces rimosus. (A) Two copies of the chromosome undergo recombination in inverse orientation (I), which generates a double chromosome with two origins of replication (II) and a linear molecule that carries two copies of the OTC gene cluster but no known origin of replication (III). (B) PFGE of AseI and XbaI digests of S. rimosus strains. Lanes 1, parental strain S. rimosus R6-500; lanes 2, MV15. (Modified from reference with permission of the publisher.)

FIG. 9.

Comparison between genetic linkage maps of Streptomyces rimosus and other Streptomyces species. Combined genetic maps of S. rimosus R6 and R7 (outer circle) and S. coelicolor A3(2) (middle circle) and part of the map of S. bikiniensis subsp. zorbonensis (inner circle) are shown. (Modified from reference with permission of the publisher.)

FIG. 10.

Electron photomicrographs of ultrathin sections of Streptomyces rimosus R7 hyphae (A) and protoplasts (B). CM, cytoplasmic membrane; CW, cell wall; NE, nuclear equivalent. Bars, 500 nm. (Reprinted from reference .)

FIG. 11.

(A) Morphology of RP1 plaques on (a) Streptomyces rimosus R6 after overnight incubation and (b) Streptomyces rimosus R6Φ^r10 after incubation for 4 days. Magnification, ×0.5. (B) Electron micrograph of actinophage RP1 negatively stained with potassium phosphotungstate. Bar, 100 nm. (Reprinted from reference .)