Characterization of the Genetic Components of Streptomyces lividans Linear Plasmid SLP2 for Replication in Circular and Linear Modes

Abstract

Unlike most eubacteria, Streptomyces species usually contain linear chromosomes and plasmids (9,16,17). The linear plasmids are 12 to 1,700 kb long (16,27). Their telomeres contain inverted repeat sequences from 44 bp (7) to 180 kb (19), and their 5Ј telomeric ends are linked covalently to terminal proteins (1, 28). Unlike linear replicons of adenoviruses and bacteriophage 29, which also contain terminal proteins linking covalently to 5Ј telomeric DNA ends and undergo replication by a mechanism of strand displacement (23), replication of Streptomyces linear plasmids starts at centrally located loci (5, 25) and continues bidirectionally towards the telomeres-leaving an ϳ280-nucleotide 3Ј single-strand overhang as an intermediate (5). This is converted to a double strand by a postulated "folding back" of multiply short palindromes on the telomere extension (20). The chromosomal telomere-associated protein (TapL, encoded by tapL) binds to the palindromes II/III then to recruit telomere terminal protein (TpgL, encoded by tpgL) (1, 2). Neither tapL nor tpgL homologous genes are carried by the linear plasmids pSLA2 and pSCL1 (1, 2), and the mechanism of recruitment or activation of these chromosomal telomere proteins for plasmid telomere replication is unknown.The centrally located loci of Streptomyces linear plasmids can also maintain propagation in circular mode when the telomeres are deleted (5,8,22,25). The centrally located locus for replication of linear plasmid pSLA2 is composed of the iterons located within the essential genes rep1 (encoding DNA-binding protein) and rep2 (DNA helicase) (6). Experimental evidence shows that the replication origin of linear SCP1 plasmids contains a rep2 pSLA2 -like gene and its adjacent regions contain different iteron sequences (22). Similar loci are also indicated in linear plasmids pSCL1 and SLP2 (11,27). The extent of functional similarity of these individual replicating components, and consequently the extent to which mechanisms of replication are similar among linear plasmids, is not known.The minimal locus required for maintaining the replication of pSLA2 in circular mode cannot allow its propagation in linear mode unless it also contains a new plasmid locus, rlrA pSLA2 (required for linear replication) (21). Plasmids containing rlrA pSLA2 are detrimental for propagation in circular mode, the effect of which can be reversed by an adjacent and divergently transcribed locus, rorA pSLA2 (rlrA override), which resembles korA (kilA override) of Streptomyces circular plasmid pIJ101 (14,26). rlrA pSLA2 and rorA pSLA2 increase inheritance and copy number of pSLA2 circular plasmids, suggesting that they may affect the origin locus (e.g., iterons) (21). Although rorA pSLA2 -homologous genes are found in linear plasmids SCP1, pSLA2-L, and SLP2, no rlrA pSLA2 -homologous loci of the plasmids are found (3,11,18), suggesting that they carry a distinct locus that enables replication in linear mode.SLP2 is a large (50,410-bp) linear plasmid of Streptomyces lividans (7, 10, 11). Here we r...

Linear Plasmid SLP2 Is Maintained by Partitioning, Intrahyphal Spread, and Conjugal Transfer inStreptomyces

“…In the absence of the partitioning, transfer, and spread systems, the plasmid (SLP2tsr⌬par⌬tra⌬spd) was almost completely lost after a sporulation cycle in S. coelicolor. Although we could not create such a triple mutation in SLP2tsr in S. lividans, the mini-SLP2 plasmids created by Xu et al (36), which contained only the basic replication locus of SLP2, were also highly unstable in S. lividans (about 99% loss), supporting the importance of the three systems in SLP2 maintenance.…”

Two Internal Origins of Replication in Streptomyces Linear Plasmid pFRL1

“…Earlier work showed that Streptomyces linear plasmids commonly contain one internal origin of replication (6,12,16,32,36,39,42,46). Here, we found that the 54,288-bp pFRL1 contained two internal replication loci, one next to a telomere and another ϳ10 kb from it.…”

“…The centrally located replication locus of pSLA2 consists of a rep gene (encoding a DNA helicase) and its adjacent iterons (6). Such rep (helicase and adjacent iterons) loci are also found in pSCL1, SCP1, SLP2, and pSHK1 (16,32,36,40,42,46) while other loci consisting of a rep gene encoding a hypothetical protein and its adjacent noncoding sequence (ncs) have been identified in pSLA2-L, pSCL2, pRL2, pRL4, and pFRL2 (12,39,46). The minimal origin required for maintaining the replication of pSLA2 in the circular mode cannot promote its propagation in the linear mode unless it also contains an rlrA gene.…”

Linear plasmids and their replication

“…In the case of the linear plasmid SLP2, efficient replication as a circular derivative requires a 47 bp sequence upstream of 23-mer iterons. Inclusion of these sequences increased the transformation efficiency by about 1000 fold (41). This region is not transcribed as shown by RT PCR and hence may contain regulatory factor(s) that act in cis (41).…”

“…Inclusion of these sequences increased the transformation efficiency by about 1000 fold (41). This region is not transcribed as shown by RT PCR and hence may contain regulatory factor(s) that act in cis (41). SLP2 contains two tpg homologues -tpg SLP2 and a putative pseudogene on the right arm.…”