The Family Streptomycetaceae, Part I: Taxonomy

The Family Streptomycetaceae

Summary The 387 kb linear plasmid pPZG101 of Streptomyces rimosus R6 can integrate into the chromosome or form a prime plasmid carrying the oxytetracycline biosynthesis cluster The integration of plasmid pPZG101 into the linear chromosome of S rimosus R6-501 in mutant MV25 was shown to be due to a single crossover at a 4 bp common sequence pPZG101 had integrated into a 250 kb DNA sequence that was reiterated at a low level This sequence includes the oxytetracycline biosynthesis cluster, so that homologous recombination generated a mixed population carrying different copy numbers of the region The 1 Mb linear plasmid pPZG103 in mutant MV17 had also arisen from a cross-over between pPZG101 and the chromosome, so that one end of pPZG103 consists of c 850 kb of chromosomal sequence including the oxytetracycline biosynthesis cluster The plasmid pPZG101 was shown to consist of a unique central region of about 30 kb flanked by terminal inverted repeats of about 180 kb Analysis of a presumed ancestor plasmid pPZG102 suggested that the long terminal repeats had arisen by a recombination event during the strain development programme

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Recombination between the linear plasmid pPZG101 and the linear chromosome of Streptomyces rimosus can lead to exchange of ends

The daptomycin biosynthetic gene cluster of Streptomyces roseosporus was analyzed by Tn5099 mutagenesis, molecular cloning, partial DNA sequencing, and insertional mutagenesis with cloned segments of DNA. The daptomycin biosynthetic gene cluster spans at least 50 kb and is located about 400 to 500 kb from one end of the ∼7,100-kb linear chromosome. We identified two peptide synthetase coding regions interrupted by a 10- to 20-kb region that may encode other functions in lipopeptide biosynthesis.

Molecular Cloning and Physical Mapping of the Daptomycin Gene Cluster from Streptomyces roseosporus

The chromosomal structures of mutant strains of Streptomyces ambofaciens which have arisen from genetic instability were investigated by using pulsed-field gel electrophoresis and probing with sequences cloned from the unstable region which maps near the ends of the linear chromosomal DNA. The chromosomal structures of seven mutant strains harboring large deletions were classified into three types. (i) Deletions internal to one chromosomal arm were characterized in two of the mutant strains. In these strains, a linear chromosomal structure was retained, as were parts of the terminal inverted repeats sequences (TIRs) and the proteins bound to them. (ii) Four of the mutants presented a deletion including all sequences from the TIRs. A junction fragment homologous to sequences originating from internal region of both arms was characterized. Consequently, the chromosomal DNA of these strains was deduced to be circularized. Furthermore, chromosomal stability was assessed in the progeny of these circular DNA mutants. Additional deletion events were detected in 11 mutants among the 13 strains isolated, demonstrating that circular chromosomes do not correspond to a stabilization of the chromosome structure and that the occurrence of deletion could be independent of the presence of chromosomal ends. (iii) A mutant with DNA amplification was shown to have a linear chromosome with a deletion of all sequences between the amplified region and the end of the chromosome. The other chromosomal arm remained unaffected by deletion and associated with protein.

Occurrence of deletions, associated with genetic instability in Streptomyces ambofaciens, is independent of the linearity of the chromosomal DNA.

Genetic instability in Streptomyces species often involves large deletions sometimes accompanied by DNA amplification. Two such systems in Streptomyces lividans 66 involve the production of mutants sensitive to chloramphenicol and the production of mutants resistant to the galactose analogue 2-deoxygalactose, respectively. Overlapping cosmids were isolated that span the ca. 1 Mb region between the two amplifiable regions. The structure of the region was confirmed by restriction mapping using the rarely cutting enzymes AseI, BfrI and DraI and pulsed-field gel electrophoresis. The region contains a non-clonable gap flanked by inverted repeats; the structure is consistent with the presence of a physical gap, i.e. a linear chromosome.

The Streptomyces lividans 66 chromosome contains a 1 MB deletogenic region flanked by two amplifiable regions.

Thirty-two 2-deoxygalactose-resistant mutants with DNA amplifications were isolated from Streptomyces lividans 66 strains carrying plasmid pMT664, which carries an agarase gene (dagA) and IS466. Thirty-one of the mutants carried amplified DNA sequences from a 70 kb region about 300 kb from one end of the linear chromosome in this species. In 28 of the mutants, all the wild-type sequences between the amplified region and the start of the 30 kb inverted repeat that forms the chromosome end were deleted. Thus, there appeared to be loss of one chromosome end and its replacement by the DNA amplification. In some mutants there amplification of a previously characterised 5.7 kb sequence that lies about 600 kb from the other chromosome end was also noted.

DNA amplifications and deletions in Streptomyces lividans 66 and the loss of one end of the linear chromosome

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The Streptomyces lividans 66 chromosome contains a 1 MB deletogenic region flanked by two amplifiable regions.

DNA amplifications and deletions in Streptomyces lividans 66 and the loss of one end of the linear chromosome