Identification and Characterization of a pSLA2 Plasmid Locus Required for Linear DNA Replication and Circular Plasmid Stable Inheritance in Streptomyces lividans

Abstract

Streptomyces linear plasmids and linear chromosomes can replicate also in a circular form when their telomeres are deleted. The 17-kb linear plasmid pSLA2 has been a useful model in studies of such replicons. Here we report that the minimal origin initiating replication of pSLA2-derived plasmids as circular molecules cannot propagate these plasmids in a linear mode unless they also contain a novel plasmid-encoded locus, here named rlrA (required for linear replication). In contrast with the need for rlrA to accomplish replication of telomere-containing linear plasmids, expression of rlrA, which encodes two LuxR family regulatory domains, interferes with the establishment of pSLA2 in circular form in Streptomyces lividans transformants. The additional presence of an adjacent divergently transcribed locus, rorA (rlrA override), which strongly resembles the kor (kil override) transcription control genes identified previously on Streptomyces plasmids, reversed the detrimental effects of rlrA on plasmid establishment and additionally stabilized circular plasmid inheritance by spores during the S. lividans life cycle. While the effects of the rlrA/rorA locus of pSLA2 were seen also on linear plasmids derived from the unrelated SLP2 replicon, they did not extend to plasmids whose replication was initiated at a cloned chromosomal origin. Our results establish the existence of, and provide the initial description of, a novel plasmid-borne regulatory system that differentially affects the propagation of linear and circular plasmids in Streptomyces.Streptomyces species are among the few eubacteria known to include both linear chromosomes and linear plasmids (10,17,20). The telomeres of Streptomyces linear replicons contain a series of short inverted repeat DNA sequences (7,9,12,18,22) and are capped by terminal proteins linked covalently to 5Ј ends of double-stranded DNA (1,9,20,33). Unlike adenovirus and bacteriophage 29, which also have terminal protein linked covalently to 5Ј DNA ends but which replicate by a strand displacement mechanism (25), Streptomyces linear plasmids contain a centrally located origin and replicate bidirectionally (4). This process has been shown to leave 280 nucleotides of single-stranded DNA at the 3Ј ends of pSLA2 replication intermediates; these are then filled in (4, 5), possibly by a fold-back mechanism involving the inverted repeats of telomeres (22). As Streptomyces chromosomes also appear to duplicate their genes bidirectionally (21) and Streptomyces coelicolor and Streptomyces lividans chromosomal telomeres are highly similar to those of pSLA2 (12,22), the filling in of recessed 5Ј ends of linear chromosomes is presumed to occur by a similar mechanism.Streptomyces linear replicons can replicate in both linear and circular form when their telomeres are deleted (4,19,20,27,30). The site of initiation of DNA replication for Streptomyces linear plasmid pSLA2 has been identified experimentally (4) and found to include a region containing short direct repeats (iterons) as well as a DNA helicas...

Linear plasmids and their replication

“…The presence of rlrA pSLA2 in a circular derivative is inhibitory to its replication. The presence of rlrA pSLA2 is not however inhibitory for replication in the linear form (32). The inhibitory effect of rlrA pSLA2 in the circular derivative can be overcome by another locus, rorA pSLA2 (33,34).…”

“…Another gene downstream of rep1, rep2, encodes a protein with a helicase-like activity. Additional genes necessary for pSLA2 replication are the tpgs, terminal protein genes (30), tap, the gene for a terminus associated protein (31) loci required for linear replication (rlrA/rorA) (32). The presence of rlrA pSLA2 in a circular derivative is inhibitory to its replication.…”

Three functional replication origins of the linear and artificially circularized plasmid SCP1 of Streptomyces coelicolor

“…Identification of three replication loci on SCP1. PCR fragments comprising rep genes of SCP1 were cloned in E. coli plasmid pQC156, which contained Streptomyces selection markers tsr/melC (Qin et al, 2003), and the resulting plasmids (see Table 1) were introduced by transformation into protoplasts of S. lividans ZX7. Transformation frequencies of plasmids were calculated as transformants (mg DNA) -1 .…”

Characterization of the Replication, Transfer, and Plasmid/Lytic Phage Cycle of the Streptomyces Plasmid-Phage pZL12

“…Plasmid isolation, transformation of Escherichia coli, and Southern hybridization were done in accordance with the method of Sambrook et al (33). Plasmids pSP72, pQC578, and pQC156 were used as cloning vectors (30). E. coli DH5␣ was used as a cloning host.…”