Characterization of a unique methyl-specific restriction system in Streptomyces avermitilis

Abstract

Streptomyces avermitilis contains a unique restriction system that restricts plasmid DNA containing N6-methyladenine or 5-methylcytosine. Shuttle vectors isolated from Escherichia coli RR1 or plasmids isolated from modification-proficient Streptomyces spp. cannot be directly introduced into S. avermitilis. This restriction barrier can be overcome by first transferring plasmids into Streptomyces lividans or a modification-deficient E. coli strain and then into S. avermitilis. The transformation frequency was reduced >1,000-fold when plasmid DNA was modified by dam or TaqI methylases to contain N6-methyladenine or by AluI, HhaI, or HphI methylases to contain 5-methylcytosine. Methyl-specific restriction appears to be common in Streptomyces spp., since either N6-methyladenine-specific or 5-methylcytosine-specific restriction was observed in seven of nine strains tested.Streptomyces avermitilis produces avermectins, commercially important macrolide secondary metabolites with potent anthelmintic activities (4). Avermectins are active against almost all arthropod ectoparasites (10) and are effective in controlling numerous agricultural pests (30). Procedures to study the biosynthesis of secondary metabolites by using cloning vectors and recombinant DNA techniques have been developed for several Streptomyces species (for examples, see references 16, 18, and 33). However, certain procedures can be performed only with Escherichia coli (i.e., the use of lambda vectors, cosmids, M13 sequencing vectors, transposon mutagenesis, and regulated expression vectors). To take advantage of the procedures available with E. coli, Streptomyces-E. coli shuttle vectors have been made (20,21,29,37,38). Cloning systems developed for S. avermitilis include vectors derived from phage TG1 (12) and plasmid pVE1 (24) and an efficient transformation procedure (23). Unfortunately, Streptomyces-E. coli shuttle vectors cannot be introduced directly into S. avermitilis because S. avermitilis restricts the entry of DNA isolated from E. coli. This restriction barrier could also pose a problem in any attempt to produce hybrid antibiotics in S. avermitilis (15).Restriction-modification systems are widespread in Streptomyces spp. (1,6,8,17,26,35). Most restriction-modification systems are composed of a methylase and an endonuclease. The modification enzyme (methylase) modifies the host DNA at a specific sequence composed of four or more bases, and the restriction endonuclease cleaves unmodified foreign DNA at or near the specific sequence (for a review, see reference 17). More than 600 restriction endonucleases and 98 methylases are known (17). Three methyl-specific restriction systems have been described elsewhere (19,31,36). In strains with methyl-specific restriction systems, foreign methyl-modified DNA is restricted and the host does not modify DNA. Diplococcus pneumoniae restricts DNA containing N6-methyladenine at the sequence GATC (19).Two other strains restrict DNA containing 5-methylcytosine, but these strains show little or no sequence specifi...

Characterization of a Streptomyces-lividans-type site-specific DNA modification system in the avermectin-producer Streptomyces avermitilis permits investigation of two novel giant linear plasmids, pSA1 and pSA2

“…We are currently determining the nature and cause of this novel type of modification. In terms of DNA metabolism, these two species have previously been compared with regard to their ability to restrict methylated DNA (MacNeil, 1988). This study indicated that while S. lividans has a weak methyl-specific restriction system that can slightly reduce transformation frequencies with A$-methyladenine-containing DNA, S.…”

Efficient transformation of Bacillus thuringiensis requires nonmethylated plasmid DNA

“…The dam-13: :Tn9 allele present in GM2163 was transferred to the isogenic pEG409-containing strains EG1884 and EG1885 by transduction with Plvir (27) Table 3). The only other strain that has been reported to restrict DNA modified by either the Dam or the Dcm methylase is Streptomyces avermitilis (19). Finally, the transformation frequencies for four other strains (HD3, HD4, HD7, and HD567-56) were low regardless of the source of plasmid DNA (Table 3).…”

Genetic construction and functional analysis of hybrid polyketide synthases containing heterologous acyl carrier proteins

“…The (16). B40 and B17 are actVII and actIV actinorhodin-biosynthetic pathway mutants, respectively, which also carry the markers hisAl uraAl strAl (7,23,25 (18). The gra, "fren," otc, and tcm ACP genes were obtained from pIJ5200 (26), pIJ5214 (2), pIJ5210 (24), and pELE37 (10) (19).…”