Isolation and characterization of stable mutants of Streptomyces peucetius defective in daunorubicin biosynthesis.

Daunorubicin and its derivative doxorubicin are antitumour anthracycline antibiotics produced by Streptomyces peucetius. In this study we report isolation of stable mutants of S. peucetius blocked in different steps of the daunorubicin biosynthesis pathway. Mutants were screened on the basis of colony colour since producer strains are distinctively coloured on agar plates. Different mutants showed accumulation of aklaviketone, epsilon-rhodomycinone, maggiemycin or 13-dihydrocarminomycin in their culture filtrates. These results indicate that the mutations in these isolates affect steps catalysed by dnrE (mutants SPAK and SPMAG), dnrS (SPFS and SPRHO) and doxA (SPDHC) gene products.

Genetic instability in Streptomyces.

Genetic instability is very common among Streptomyces strains and the affected strains display several distinctive phenotypes. In several cases DNA rearrangements, specifically deletions and amplifications of specific segments of DNA, were demonstrated. Depending upon the reproducible amplification of a segment in independent isolates one could predict the basic structural elements involved in the amplification process. In the case of S. lividans 66, 5·7 kb amplifiable DNA sequence is located near the end of the linear chromosome. Amplification of this sequence and deletion of the chromosome linked to it leads to the creation of a new end or in some cases even circularisation of the chromosome occurs. A model incorporating these aspects is discussed. The possible involvement of proteins encoded by the amplifiable region is also discussed.

Structural organization, amplification, deletion and rearrangements of DNA sequences associated with an unstable region of the chromosome of Streptomyces coelicolor A3(2).

The chromosome of Streptomyces coelicolor A3(2) carries unstable DNA sequences hybridising with DNA sequences from an unstable chromosomal region of the related species S. lividans. These S. coelicolor sequences are nearly identical to those of S. lividans TK23 in organisation but differ from those of S. lividans 66 TK64 which harbours a tandem duplication of these sequences. Southern hybridisations using heterologous probes and S. coelicolor DNA cleaved with a variety of restriction enzymes permitted us to construct a partial restriction map of the unstable region of the chromosome of S. coelicolor. Genetic analysis shows that the unstable region yields distinguishable variants with several distinct DNA rearrangements.

Molecular cloning and sequencing of mcrA locus and identification of McrA protein in Escherichia coli.

The Mcr systems (previously known as Rgl systems) of Escherichia coli recognize and cleave specific sequences carrying methylated or hydroxymethylated cytosines. We have cloned the mcrA gene and determined its nucleotide sequence. An 831 base pair sequence encodes the McrA protein. Analysis of the sequence data reveals that there arc additional ORFs internal to the above. A phage T7 expression system was used to determine the protein products encoded by the cloned mcrA gene. The results clearly show that a 31 kDa polypeptide is responsible for McrA activity. This is in agreement with the molecular weight deduced from sequence data. McrA protein was found to be localized in the outer membrane of Escherichia coli. To our knowledge this is the first restriction enzyme localized in the outer membrane of Escherichia coli.