Linear plasmid SLP2 of Streptomyces lividans is a composite replicon

SLP2 is a 50 kb linear plasmid in Streptomyces lividans that contains short (44 bp) terminal inverted repeats and covalently bound terminal proteins. The nucleotide sequence of SLP2 was determined. The rightmost 15.4 kb sequence is identical to that of the host chromosome, including the Tn4811 sequence at the border, which is interrupted by an insertion sequence (IS) element in SLP2. Examination of the flanking target sequences of Tn4811 suggests a previous recombinational event there. The 43 putative protein coding sequences contained many involved in replication (including two terminal protein homologues), partitioning, conjugal transfer and intramycelial spread. The terminally located helicase-like gene ttrA was necessary for conjugal transfer. The two telomeres diverge significantly in primary sequence, while preserving similar secondary structures. Mini-linear plasmids containing these telomeres replicated in S. lividans using the chromosomally encoded terminal protein. In addition, two pseudotelomere sequences are present near the left telomere. The G+C content and GC or AT skew profiles exhibit complex distributions. These, plus the inferred recombination at the right arm, indicate that SLP2 has evolved through rounds of exchanges involving at least three replicons.     

Characterization of the genetic components of Streptomyces lividans linear plasmid SLP2 for replication in circular and linear modes

The nucleotide sequence of Streptomyces lividans linear plasmid SLP2 consists of 50,410 bp (C. H. Huang, C. Y. Chen, H. H. Tsai, C. Chen, Y. S. Lin, and C. W. Chen, Mol. Microbiol. 47:1563-1576, 2003). Here we report that the basic SLP2 locus for plasmid replication in circular mode resembles that of Streptomyces linear plasmids pSLA2 and SCP1 and comprises iterons(SLP2) and the adjacent rep(SLP2) gene. More efficient replication additionally required the 47-bp sequence between bp 581 and 628 upstream of the iterons. Replacement of either the iterons or the rep gene of SLP2 by the corresponding genes of pSLA2 or SCP1 still allows propagation in Streptomyces, although the transformation frequencies were 3 orders of magnitude lower than the original plasmids, suggesting that these plasmids share similar replication mechanisms. To replicate SLP2 in linear mode, additional SLP2 loci--either mtap(SLP2)/tpg(SLP2) or mtap(SLP2)/ilrA(SLP2)--were required. IlrA(SLP2) protein binds specifically to the iterons(SLP2) in vitro. Interactions were detected between these SLP2-borne replication proteins (Mtap(SLP2), Tpg(SLP2), and IlrA(SLP2)) and the telomeric replication proteins (TpgL, TapL, and TpgL) of the S. lividans chromosome, respectively, but the SLP2 proteins failed to interact. These results suggest that SLP2 recruits chromosomally encoded replication proteins for its telomere replication.     

The chromosomal DNA of Streptomyces lividans 66 is linear

Two copies of a DNA sequence similar or identical to one end of the linear plasmid SLP2 were found on the Streptomyces lividans chromosome. Restriction mapping showed that these sequences represented free ends. Electrophoretic retardation and glass-binding studies indicated that the telomeres carry covalently bound proteins. Moreover, the chromosome migrated as an 8Mb linear DNA in pulsed-field gel electrophoresis. A similar finding with the chromosomes of six other Streptomyces species suggested that a linear chromosome may be characteristic of the genus. The S. lividans chromosome can be circularized by joining the two ends by artificial targeted recombination or by spontaneous deletions spanning both telomeres. Thus the chromosome appears to be able to exist, in viable bacteria, as a linear or a circular molecule.     

The integrated conjugative plasmid pSAM2 of Streptomyces ambofaciens is related to temperate bacteriophages.

Streptomyces ambofaciens ATCC23877 and derivatives contain the 11-kb element pSAM2 present in an integrated state or as a free and integrated plasmid. This element, able to integrate site-specifically in the genome of different Streptomyces species, is conjugative and mobilizes chromosomal markers. Besides these plasmid functions, we have shown that the site-specific recombination system of pSAM2 presents strong similarities with that of several temperate phages. The integration event is promoted by a site-specific recombinase of the integrase family. The int gene encoding this integrase is closely linked to the plasmid attachment site (attP). A small open reading frame (ORF) overlaps the int gene and the predicted protein exhibits similarities with Xis proteins involved in phages excision. The integrated copy of pSAM2 in strain ATCC23877 is flanked by att sequences (attL and attR). Another att sequence (attX) is present in this strain and attX and attL are the boundaries of a 42-kb fragment (xSAM1) absent, as well as pSAM2, from S.ambofaciens DSM40697. Sequences partially similar to pSAM2 int gene are found near the chromosomal integration zone in both S.ambofaciens strains. The possible origin of pSAM2, an element carrying plasmid as well as phage features, is discussed.     

pS10147-2, a3.7 kb multicopy plasmid isolated from Streptomyces coelicolor

The following putative precursors of the pseudomurein were isolated from trichloroacetic acid extracts of Methanobacterium thermoautotrophicum: a uridine diphosphate activated derivative of glutamic acid and the uridine diphosphate activated peptides (see text). The activated glutamic acid residue and the three activated pepetides lack the glycan components N-acetylglucosamine and N-acetyltalosaminuronic acid present in the intact pseudomurein. In this case uridine diphosphate should be directly linked to the amino group of a glutamic acid residue, which represents a new mode of amino acid and peptide activation.     

Gene expression after transformation of Streptomyces lividans protoplasts with plasmid pIJ2 DNA

A new method has been developed for the selection of antibiotic-resistant clones after transformation of Streptomyces protoplasts with plasmid DNA. This method is based on establishing a spatial concentration gradient for the antibiotic, the resistance to which is encoded by the transforming plasmid. By this method, the resistance development of regenerating protoplasts can be followed. The results suggest that antibiotic resistance is inducible. In addition, we were able to show that resident plasmids incompatible with the incoming ones are eliminated when this direct selection principle is used. Moreover, this method, which may facilitate the application of gene technology in Streptomyces, works even though the transformation procedure gives variable results.     

Cloning and expression of a Streptomyces cholesterol oxidase gene in Streptomyces lividans with plasmid pIJ702

The cholesterol oxidase gene (cho) of Streptomyces sp. was cloned into Streptomyces lividans with the vector pIJ702. Deletion analysis of the recombinant plasmid showed that entire coding sequence of the cho gene was located within a 2.5-kilobase segment of the chromosomal DNA obtained from the cholesterol oxidase-producing strain. When cloned cells of S. lividans were grown in an appropriate medium, the cells produced severalfold more cholesterol oxidase extracellularly than did the producing strain.     

The terminal inverted repeats of the linear plasmid SCP1 of Streptomyces coelicolor A3(2) possess a truncated copy of the transposon Tn 4811 of Streptomyces lividans 66

Streptomyces coelicolor A3(2), the best genetically studied streptomycete and Streptomyces lividans 66 are very closely related strains. This is further emphasized by our finding that a truncated copy of Tn4811 of S. lividans is present in the terminal inverted repeats of the S. coelicolor giant linear plasmid SCP1. The copy of Tn4811 in SCP1 lacks the first 1276 bp and shows only minor changes in the nucleotide sequence of the remaining 4.12 kb. Tn4811 exists in both ends of SCP1.     

Cloning and expression of a Streptomyces cholesterol oxidase gene in Streptomyces lividans with plasmid pIJ702.

The cholesterol oxidase gene (cho) of Streptomyces sp. was cloned into Streptomyces lividans with the vector pIJ702. Deletion analysis of the recombinant plasmid showed that entire coding sequence of the cho gene was located within a 2.5-kilobase segment of the chromosomal DNA obtained from the cholesterol oxidase-producing strain. When cloned cells of S. lividans were grown in an appropriate medium, the cells produced severalfold more cholesterol oxidase extracellularly than did the producing strain.     

Cloning and characterization of a linear 2.3 kb mitochondrial plasmid of maize

Summary A linear 2.3 kb DNA molecule found in maize mitochondria was cloned into pUC8. A natural deletion of this plasmid, found in cmsT and some N (fertile) types of maize plants, was mapped to one end of the plasmid. A minor sequence homology to S-2, another linear mitochondrial plasmid, was detected, as well as more significant sequence homology with chloroplast and maize nuclear DNA. Hybridization to teosinte mitochondrial DNA (mtDNA) revealed the presence of part of the maize plasmid in the high molecular weight mtDNA of the maize relatives. RNA dot hybridization indicates that the plasmid is transcribed in mitochondria. The termini of the 2.3 kb linear plasmid contain inverted repeated sequences; of the first 17 nucleotides of the termini, 16 are identical to the terminal inverted repeats of the linear S plasmids found in the mitochondria of cmsS maize plants.     

Replication at the telomeres of the Streptomyces linear plasmid pSLA2

The Streptomyces linear plasmid pSLA2 initiates DNA replication bidirectionally towards its telomeres from a site located near the centre of the molecule; at the telomeres, the recessed ends of lagging strands are filled in by non-displacing DNA synthesis. Here, we report experiments that test three proposed mechanisms for lagging-strand fill-in. We present data inconsistent with recombinational or terminal hairpin models for the formation of full-length duplex pSLA2 DNA. Instead, we find that deletions in short, distantly separated homologous palindromes in the leading-strand 3' overhang prevent propagation of linear pSLA2 DNA, implicating a mechanism of palindrome-mediated leading-strand fold-back in telomere replication. We further show that circularized pSLA2 DNA molecules are opened in vivo precisely at the terminal nucleotides of telomeres, generating functional linear replicons containing native telomeres covalently bound to a protein at their 5' DNA termini. Together, our results support a model in which pairing of multiple widely separated pSLA2 palindromes anchors the 3' end of the leading-strand overhang to a site near the overhang's base — providing a recognition site for terminal-protein-primed DNA synthesis and subsequent endonucleolytic processing. Thus, the replication of Streptomyces plasmid telomeres may have features in common with the mechanism proposed for telomere replication in autonomous parvoviruses.     

The chromosomal integration site for the Streptomyces plasmid SLP1 is a functional tRNATyr gene essential for cell viability

The genetic element SLP1 exists in nature as a single DNA segment integrated into the genome of Streptomyces coelicolor. Upon mating with Streptomyces lividans, a closely related species, SLP1 undergoes precise excision from its chromosomal site and is transferred into the recipient where it integrates chromosomally. Previous work has shown that integration and excision involve site-specific recombination between a chromosomal site, attB, and a virtually identical sequence, attP, on SLP1. We demonstrate here by means of gene replacement that a tRNA(Tyr) sequence that overlaps part of the attB site of S. lividans is both biologically functional and essential for cell viability. The requirement for this tRNA gene has been used to stabilize the inheritance of a segrationally unstable plasmid in cells lacking a chromosomal attB site. The evolution of an essential DNA locus as an attachment site for a chromosomally integrating genetic element represents a novel mechanism of biological adaptation.