engleski

Chromosome and plasmid terminal sequences and genome heterogeneity in Streptomyces rimosus

The oxytetracycline producer Streptomyces rimosus R6 is the best characterised industrial Streptomyces species (Gravius et al., 1993). Like other Streptomyces it possesses a linear chromosome of about 8 Mb in length. Previous work had shown that the chromosome ends were inverted repeats (Pandza et al., 1997). The strain also contains a linear plasmid. The oldest available strain R6-65 carries the 310 kb plasmid pPZG102 that on the basis of restriction mapping does has little or no terminal inverted repeat. A strain derived from R6-65 in a strain selection programme R6-501 has an identical chromosomal macrorestriction pattern to that of R6-65, but carries the 387 kb linear plasmid pPZG101, which has extensive inverted repeats and seems to have been derived from pPZG102 by replacement of one end by a copy of the other end. Previous work also showed that single cross overs between the chromosome of R6-501 and pPZG101 could form linear molecules with one chromosome and one plasmid end (Pandza et al., 1998).Using a glass bead binding method, the chromosome and plasmid terminal sequences from R6-65 and R6-501 were cloned and sequenced. The chromosome ends and the left and right ends of pPZG102 showed typical conserved Streptomyces terminal sequences with seven palindromes in about 180 bp. The three end sequences all differed from each other, but only at a few nucleotide positions. The changes often occurred as compensating pairs that conserved the palindromes. This suggests that the formation of a single stranded stem-loop structure is important for terminal replication. Although the terminal sequences of the chromosomes of R6-65 and R6-501 were identical as were the sequences of the end of pPZG101 and the conserved end of pPZG102, in both cases the terminal DNA fragments were slightly bigger in the R6-501-derived ends. The reason for this microheterogeneity is as yet unknown. As the pPZG101 and chromosome terminal sequences are nearly identical, it is likely that the same terminal protein can attach to both ends of the chromosome-plasmid hybrids.The strain R6-501 showed an unexpected additional protein-bound linear end that had a typical seven palindrome sequence. It belonged to a previously undetected 4.6 kb linear element. The other end of the element could not be purified by glass bead binding that suggested that it does not carry a stable covalently-bound protein. The sequences of some AseI linking clones of S. rimosus were compared to the genomic sequence of S. coelicolor A3(2). The results suggested that the chromosome divergence within the genus Streptomyces is much greater than previously suspected.

Streptomyces rimosus; linear chromosome; linear plasmid; terminal sequences

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