Streptomyces sp. linear plasmids and linear chromosomes usually contain conserved terminal palindromic sequences bound by the conserved telomeric proteins Tap and Tp, encoded by the tap and tpg genes, respectively, as well as plasmid loci required for DNA replication in circular mode when the telomeres are deleted. These consist of iterons and an adjacent rep gene. By using PCR, we found that 8 of 17 newly detected linear plasmids in Streptomyces strains lack typical telomeric tap and tpg sequences. Instead, two novel telomeres in plasmids pRL1 and pRL2 from the eight strains and one conserved telomere in pFRL1 from the other strains were identified, while multiple short palindromes were also found in the plasmids. The complete nucleotide sequence of pRL2 revealed a gene encoding a protein containing two domains, resembling Tap of Streptomyces and a helicase of Thiobacillus, and an adjacent gene encoding a protein similar to Tpg of Streptomyces and a portion of the telomere terminal protein pTP of adenoviruses. No typical iterons-rep loci were found in the three plasmids. These results indicate an unexpected diversity of telomere palindromic sequences and replication genes among Streptomyces linear plasmids.Streptomyces species are gram-positive, high-GϩC, myceliumproducing eubacteria. Unlike the case for most eubacteria, linear plasmids and linear chromosomes are common in Streptomyces species (3,8,11,19,20,28). The linear plasmids vary in size between 12 kb (16) and 1,700 kb (19). Their telomeres contain long inverted repeat sequences of 44 bp (7) to 180 kb (21), and the 5Ј telomeric ends are linked covalently to terminal proteins (Tp) (1, 31). The telomeres of the ϳ8-Mb linear Streptomyces chromosomes are 46 bp to 1 Mb long (14,29). With the exceptions of the telomeres of the large linear plasmid SCP1 and the Streptomyces griseus linear chromosome (9, 18), Streptomyces linear plasmids and linear chromosomes usually contain conserved palindromic DNA sequences at their telomeres (13).Unlike the terminal protein-capped linear replicons of adenoviruses and bacteriophage ⌽29 (25), replication of Streptomyces linear plasmids starts at centrally located loci (27) and proceeds bidirectionally toward the telomeres (5). This leaves an ϳ280-nucleotide (nt) single-strand overhang at the 3Ј telomeric end of pSLA2 as a replication intermediate (5). To convert the 3Ј overhang to a double strand, the terminal 144 nt of the telomere contain short palindromes 1 to 5 (22), with palindromes 2/3 being bound by the conserved telomere-associated protein (Tap) to recruit the conserved telomere terminal protein (Tp) (1, 2).Streptomyces linear plasmids can also propagate in circular mode when the telomeres are deleted (5, 10, 24, 27). The centrally located locus for replication of pSLA2 consists of a rep-2 gene (encoding a DNA helicase) and its adjacent iterons within a transcribed rep-1 gene (6). The replication loci of plasmids SCP1 and pSLA2-L also consist of rep genes and different iteron sequences (10, 24). Such iterons-rep loci ...

Myxobacteria are very important due to their unique characteristics, such as multicellular social behavior and the production of diverse and novel bioactive secondary metabolites. However, the lack of autonomously replicating plasmids has hindered genetic manipulation of myxobacteria for decades. To determine whether indigenous plasmids are present, we screened about 150 myxobacterial strains, and a circular plasmid designated pMF1 was isolated from Myxococcus fulvus 124B02. Sequence analysis showed that this plasmid was 18,634 bp long and had a G؉C content of 68.7%. Twenty-three open reading frames were found in the plasmid, and 14 of them were not homologous to any known sequence. Plasmids containing the gene designated pMF1.14, which encodes a large unknown protein, were shown to transform Myxococcus xanthus DZ1 and DK1622 at high frequencies (ϳ10 5 CFU/g DNA), suggesting that the locus is responsible for the autonomous replication of pMF1. Shuttle vectors were constructed for both M. xanthus and Escherichia coli. The pilA gene, which is essential for pilus formation and social motility in M. xanthus, was cloned into the shuttle vectors and introduced into the pilA-deficient mutant DK10410. The transformants subsequently exhibited the ability to form pili and social motility. Autonomously replicating plasmid pMF1 provides a new tool for genetic manipulation in Myxococcus.Myxobacteria are gram-negative gliding bacteria that are phylogenetically located in the delta division of the Proteobacteria (29,34,41). The two most intriguing characteristics of myxobacteria are their complicated multicellular social behavior, which provides an excellent model for studies of cell-to-cell communication and evolution (6,18,39,47), and their excellent capacity for production of diverse and novel bioactive secondary metabolites. Their production of bioactive secondary metabolites makes myxobacteria an important source of potential new drugs, although this possibility has not been well explored (36). The study and utilization of myxobacteria have been limited by the formidable isolation and culture techniques required (35) and the difficulty of performing genetic manipulations. In the past few decades, genetic studies of myxobacteria were performed mainly with the model species Myxococcus xanthus using transduction (7, 23) and the more efficient electroporation protocols (19). Besides these studies, Sorangium strains were also studied using conjugation protocols (13,14,22,31,33). Sorangium is a special cellulose degrader among the 17 myxobacterial genera (34, 50) and produces almost one-half of the known secondary metabolites produced by myxobacteria (8). Because no naturally occurring self-replicating plasmid has been discovered previously and no broad-hostrange vectors can replicate in myxobacterial cells, all the genetic transfer systems used have been based on integration of introduced plasmids or phages into the recipient chromosomes. Consequently, some genetic manipulations are hard to perform in myxobacteria or are not v...

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...