Discovery and characterization of a new transposable element, Tn4811, in Streptomyces lividans 66

Abstract

Transposition of a new 5.4-kb transposon, Tn4811, of Streptomyces lividans to the melC operon of Streptomyces antibioticus on plasmid pU702 was discovered. The nucleotide sequence of this copy of Tn4811, which contained an imperfect (9 of 11 bp) terminal inverted repeat, five putative Streptomyces coding sequences for an oxidoreductase and its transcription regulator, and three transposition-related proteins, was determined. SLP-strains of S. lividans contained one copy (A) of Tn4811, while SLP2+ strains contained an additional copy (B) on the SLP2 plasmid. The nucleotide sequences at three insertion junctions of Tn4811 were determined. Copy B lacked 41 bp from the left end. At the other five junctions the duplication of a putative 3-bp target sequence (TGA) was observed. A sequence of less than 3 kb homologous to Tn4811 was present in S. antibioticus. DNA homologous to Tn4811 was not detected in 14 other Streptomyces species.Structural instability of genomic DNA is a widespread feature of gram-positive, filamentous, soil bacteria of the genus Streptomyces (32). Many genetic traits undergo spontaneous loss in laboratory cultures at frequencies of 10' to 10-2. Exposure to certain stresses such as UV irradiation, DNA intercalating agents, cold temperature, or protoplasting and regeneration increases the frequency of these instabilities. The mutations involved in the instabilities are attributed to large deletions of chromosomal DNA, which are frequently accompanied by tandem amplifications of particular sequences nearby (32). The genetic principle(s) underlying the structural instability is not clear. Possible mechanisms involved are homologous recombination, site-specific recombination, and transposition. Homologous recombination systems in Streptomyces species and their contribution to genetic instability have received little study. Tsai and Chen (41) isolated a rec mutant defective in intraplasmid recombination but not in chromosomal recombination (25). Chou and Chen (11), in their investigation of an unstable arg gene, found no effect of this rec mutation on its instability. Site-specific recombination and transposition, although representing forms of fluidity of genomic DNA, have not been implicated in the instability of other DNA sequences (8).About one-third of Streptomyces species can produce melanin pigment (44). The melanin (melC) operon of Streptomyces antibioticus has been cloned (24), sequenced (3), and widely used in many recombinant vectors. The melC operon in the Streptomyces species examined is genetically unstable, undergoing spontaneous deletions at relatively high frequencies-about 10-' (32). Similar to those observed in other unstable genes in Streptomyces species, the deletions of melC were frequently accompanied by extensive tandem amplifications of specific sequences (18,32).

Streptomyces coelicolor A3(2) plasmid SCP2*: deductions from the complete sequence

“…It is bounded by 28 bp imperfect inverted repeats and flanked by a 3 bp duplicated target sequence. The putative gene products encoded by the three genes SCP2.10c, SCP2.11c and SCP2.12c of Tn5714 are most similar to those of S. lividans Tn4811 (Chen et al, 1992). SCP2.10c shows Pfam matches to the Transposase 13 and Transposase 6 families and presumably is the transposase of Tn5714.…”

The unstable melC operon of Streptomyces antibioticus is codeleted with a Tn4811-homologous locus

“…The probe was a 4.9-kb fragment of S. antibioticus (from pLUS788; Table 1) containing the entire 1.6-kb meiC operon (21). All these mutants lacked the entire 4.9-kb sequence ( (9). Interestingly, all 16 mutants had lost the 3.0-kb BclI fragment homologous to Tn4811 (Fig.…”

“…We previously isolated a 5.4-kb transposon, Tn4811, of Streptomyces lividans from within the meiC operon (9). Tn4811 contained five typical Streptomyces coding sequences (9). Open reading frame 1 (ORF1), ORF2, and ORF3 were proposed to be involved in transposition and regulatory functions.…”

“…The mutations are primarily deletions of the meiC sequence (4,16,28,29). Other Mel-variants result from mutations in other loci (9,13). Although the cloned S. antibioticus meiC operon has been a widely used marker (21), it is structurally unstable on recombinant vectors, undergoing deletions and insertions (for examples, see references 9, 24, and 30).…”

“…Although the cloned S. antibioticus meiC operon has been a widely used marker (21), it is structurally unstable on recombinant vectors, undergoing deletions and insertions (for examples, see references 9, 24, and 30). We previously isolated a 5.4-kb transposon, Tn4811, of Streptomyces lividans from within the meiC operon (9). Tn4811 contained five typical Streptomyces coding sequences (9).…”