Characterization of replication and conjugation of plasmid pWTY27 from a widely distributed Streptomyces species

Abstract

Background: Streptomyces species are widely distributed in natural habitats, such as soils, lakes, plants and some extreme environments. Replication loci of several Streptomyces theta-type plasmids have been reported, but are not characterized in details. Conjugation loci of some Streptomyces rolling-circle-type plasmids are identified and mechanism of conjugal transferring are described.

Results: We report the detection of a widely distributed Streptomyces strain Y27 and its indigenous plasmid pWTY27 from fourteen plants and four soil samples cross China by both culturing and nonculturing methods. The complete nucleotide sequence of pWTY27 consisted of 14,288 bp. A basic locus for plasmid replication comprised repAB genes and an adjacent iteron sequence, to a long inverted-repeat (ca. 105 bp) of which the RepA protein bound specifically in vitro, suggesting that RepA may recognize a second structure (e.g. a long stem-loop) of the iteron DNA. A plasmid containing the locus propagated in linear mode when the telomeres of a linear plasmid were attached, indicating a bi-directional replication mode for pWTY27. As for rolling-circle plasmids, a single traA gene and a clt sequence (covering 16 bp within traA and its adjacent 159 bp) on pWTY27 were required for plasmid transfer. TraA recognized and bound specifically to the two regions of the clt sequence, one containing all the four DC1 of 7 bp (TGACACC) and one DC2 (CCCGCCC) and most of IC1, and another covering two DC2 and part of IC1, suggesting formation of a high-ordered DNA-protein complex.

Conclusions: This work (i) isolates a widespread Streptomyces strain Y27 and sequences its indigenous theta-type plasmid pWTY27; (ii) identifies the replication and conjugation loci of pWTY27 and; (iii) characterizes the binding sequences of the RepA and TraA proteins.

Figure 1

Identification of a pWTY27 locus required for replication inStreptomyces lividans. (a). Identification of a replication locus. Plasmids were constructed in E. coli (see Methods and Table 1), and introduced by transformation into S. lividans ZX7. Positions of these cloned fragments on pWTY27 and transformation frequencies are shown. The ncs is indicated by striped boxes, relevant genes by open arrowheads and the two replication genes by filled arrowheads. (b). RT-PCR of a transcript overlapping the consecutive replication genes. RNA of strain Y27 was isolated and reverse-transcribed into cDNA. The cDNA, RNA and Y27 genomic DNA were used as templates for PCR amplification and their products were electrophoresed in 1.5% agarose gel at 20 V/cm for 1 h.

Figure 2

Characterization of the binding reaction of Rep1A protein with iteron DNA by EMSA and footprinting. (a). Iteron of pWTY27. Possible iteron sequences from 338 to 606 bp on pWTY27 and AT-rich regions are shown. DR: direct repeat; IR: inverted repeat. The RepA binding sequences determined by DNA footprinting are boxed. The binding sequences of RepA protein are indicated by shading. (b). Detection of the binding activity of RepA protein with the iteron by EMSA. The DNA probe for each lane was 2 ng and the unlabeled probe was also used as specific competitor. The DNA-protein complex is indicated. (c). Determination of the binding sequence by DNA footprinting. The γ[³²p]ATP-radiolabelled primer was sequenced and electrophoresed (lanes G, A, T and C) as a control. The amounts of RepA protein used in lanes 1–5 were 0.17, 0.43, 0.85, 2.6 and 0 μg, respectively. Two sequences protected by RepA from digestion with DNaseI are shown and the RepA unbound sequences are underlined.

Figure 3

A plasmid containing the pWTY27 replication locus and pSLA2 telomeres propagated in linear mode inStreptomyces. Aliquots of genomic DNA were treated with E. coli exonuclease III and bacteriophage λ exonuclease and electrophoresed in 0.7% agarose gel at 1.3 V/cm for 12 h. Chromosomal (Chr) and linear plasmid (Lp) bands are indicated.

Figure 4

Identification of a pWTY27 locus for conjugal transfer inStreptomycescxx(a) and (b). Transfer frequencies of the plasmids in Streptomyces lividans are shown. Relevant genes are indicated by open arrowheads while tra is indicated by an arrowhead, and a possible clt by striped boxes.

Figure 5

Characterization of the binding reaction of TraA protein withcltDNA by EMSA and footprinting. (a). Characteristics of a clt sequence on pWTY27 for plasmid transfer. Possible DC (direct repeat) and IC (inverted repeat) sequences are shown. (b) as Figure 2 (b). (c) as Figure 2 (c). The amounts of TraA protein used in lanes 1–5 were 0, 0.6, 1.4, 2.8 and 4.2 μg, respectively. Two sequences protected by TraA from digestion with DNaseI are shown.

Figure 6

PCR amplifications of possible pWTY27repAandoriCfrom the genomic DNA of soil samples. Twelve soil samples (lanes 1–12) were collected, soil genomic DNA was isolated and nested PCR amplifications with primers of the pWTY27 repA, Y27 and A3(2) oriC were performed (Methods).

Figure 7

A model for interaction of the pWTY27 RepA and the iteron. The replication origin of plasmid pWTY27 contains multiple directed and inverted repeat sequences (DRs and IRs, Figure 2a). The IR2 is a long discontinous inverted-repeat sequence and may fold back itself during initiation of replication. Since there are six unbound sites (see Figure 2a) and RepA is a large protein (522 amino acids), we suggest that five RepA molecules (indicated by filled ovals) may bind to the folding-back IR2 region leaving six unbound sites (indicated by arrowheads).