Streptomyces coelicolor A3(2) Lacks a Genomic Island Present in the Chromosome of Streptomyces lividans 66

Abstract

Streptomyces lividans ZX1 has become a preferred host for DNA cloning in Streptomyces species over its progenitor, the wild-type strain 66 (stock number 1326 from the John Innes Center collection), especially when stable DNA is crucial for in vitro electrophoresis, because DNA from strain 66 contains a novel modification that makes it sensitive to oxidative double-strand cleavage during electrophoresis. Detailed analysis of this modification-deficient mutant (ZX1) revealed that it has several additional phenotypic traits associated with a chromosomal deletion of ca. 90 kb, which was cloned and mapped by using a cosmid library. Comparative sequence analysis of two clones containing the left and right deletion ends originating from strain 66 and one clone with the deletion and fused sequence cloned from strain ZX1 revealed a perfect 15-bp direct repeat, which may have mediated deletion and fusion to yield strain ZX1 by site-specific recombination. Analysis of AseI linking clones in the deleted region in relation to the published AseI map of strain ZX1 yielded a complete AseI map for the S. lividans 66 genome, on which the relative positions of a cloned phage HAU3 resistance (HAU3 r ) gene and the dnd gene cluster were precisely localized. Comparison of S. lividans ZX1 and its progenitor 66, as well as the sequenced genome of its close relative, Streptomyces coelicolor M145, reveals that the ca. 90-kb deletion in strain ZX1 may have originated from an insertion from an unknown source.Streptomyces lividans strain 66 (stock number 1326 from the John Innes Center collection) is one of the most commonly used host strains for DNA cloning in Streptomyces species. There are two main reasons for this. First, S. lividans, unlike its close relative, Streptomyces coelicolor A3(2), lacks a methylation-dependent restriction system that recognizes DNA isolated from normal Escherichia coli strains. Second, well-characterized plasmid-free derivatives of S. lividans are available (17). On the other hand, S. lividans DNA contains a novel modification that makes its DNA susceptible to double-strand cleavage under oxidative stress in vitro, e.g., during normal (7, 13, 36) and pulsed-field gel electrophoresis (PFGE) (22, 37); this S. lividans phenotype is named Dnd for DNA degradation (see Fig. 1A). A mutant strain, ZX1, that does not modify its DNA was isolated from the recombination-deficient S. lividans 66 derivative, JT46 (35), by NTG (N-methyl-NЈnitro-N-nitrosoguanidine) mutagenesis (36). Apart from having lost the ability to modify its DNA, S. lividans ZX1 is sensitive to phage HAU3 (37), while the progenitors of strain ZX1 are resistant (37). The property of recombination deficiency in strain JT46, which greatly stabilizes plasmids (especially those of bifunctional vectors) in vivo and thus minimizes DNA deletions, is retained by strain ZX1. A physical map of S. lividans ZX1 has been published (26), and the linearity of chromosomes in the streptomycetes was first demonstrated using S. lividans ZX1 (27).Not surprisingly, most of ...

Insertion site and distribution of a genomic island conferring DNA phosphorothioation in the Mycobacterium abscessus complex

“…However, studies on Streptomyces (He et al, 2007;Zhou et al, 2004) suggested that absence of the genes was the more likely explanation. Therefore, we designed primers P1 and P2 to amplify a 484 bp region of dndC (MAB_1095) (Fig.…”

“…However, Wallace et al (1993) reported that over 40 % of clinical isolates of M. abscessus were untypable by PFGE due to degradation of the DNA, also known as DNA smearing. The phenomenon of DNA degradation (Dnd) during PFGE occurs with other species of bacteria (Corkill et al, 2000;Ou et al, 2009;Yao et al, 2009), and much of our understanding of the underlying mechanism stems from analyses of natural variants and recombinant strains of Streptomyces that differ in Dnd phenotype (Chen et al, 2010;Dyson & Evans, 1998;Ou et al, 2009;Zhou et al, 1988Zhou et al, , 2004Zhou et al, , 2005. DNA from Streptomyces lividans, but not from Streptomyces coelicolor A3(2), exhibits site-specific degradation in some preparations of Tris buffer during electrophoresis, and it was discovered that this technical problem can be resolved by adding thiourea to the gel running buffer (Chen et al, 2010;Ray et al, 1992Ray et al, , 1995Zhou et al, 1988).…”

“…In species analysed so far, the dnd gene clusters are located on mobile genetic elements, predominantly genomic island (GIs) (Ou et al, 2009;Zhou et al, 2004), which are segments of 'alien' DNA acquired through horizontal gene transfer (Langille et al, 2010). ATCC 19977 T , the type strain of M. abscessus sensu stricto, contains a putative dnd gene cluster (genes MAB_1093c to MAB_1097) on a 19.6 kbp GI (Ripoll et al, 2009).…”

The use of Streptomyces for immunization against mycobacterial infections

“…7 Specifically Streptomyces lividans is an important member of this family, because it is nonpathogenic filamentous bacteria and there is substantial knowledge about its genetic manipulation. 8 In fact, this organism is able to produce large amounts of extracellular proteins, and is often used for cloning, expression, and production of heterologous proteins. [9][10][11] The Mycobacterium tuberculosis genes point to explore effective prevention and treatment means.…”

Phosphorothioation: An Unusual Post-Replicative Modification on the DNA Backbone

“…When transformed into ZX1, cosmid 16C3 (ZX1::16C3) could restore the Dnd phenotype of mutant ZX1, indicating that 16C3 harbored genes associated with the DNA modification. By subsequent sub-cloning and Dnd phenotypic tests, a 6,665 bp dnd locus containing five dnd genes was precisely localized on cosmid 16C3 [11]. , dndD (lane 6) and dndE (lane 7) can abolish the Dnd phenotype, whereas the mutation of dndB (lane 4) aggravates the degradation.…”