BACKGROUND
streptomyces species are widely distributed in natural habitats, such as soils, lakes, plants and some extreme environments  <cit> .
they are gram-positive, mycelial bacteria with high g+c content  in their dna  <cit> .
more than  <dig> antibiotics and pharmacologically active metabolites  have been discovered in streptomyces species  <cit> .
streptomyces species usually harbor conjugative plasmids  <cit> .
modes of plasmid replication in streptomyces include rolling-circle    <cit> , and uni-directional or bi-directional theta types   <cit> .
some plasmids  replicate in chromosomally-integrating/autonomous forms  <cit> .
streptomyces rc plasmids are usually small , while theta-type plasmids are larger .
replication loci of streptomyces rc plasmids comprise a single rep gene, a dso  for initiation and termination of replication, and an sso  for conversion of the lagging strand into a double-stranded molecule  <cit> .
the replication locus of the theta-type scp <dig> comprises repi and repii genes and an adjacent non-coding sequence to which repi protein binds  <cit> .
pfp <dig> and pfp <dig> contain basic replication loci of rep and iteron types , to which rep proteins bind  <cit> .
conjugal transfer of streptomyces rc plasmid  needs a tra gene along with a clt  site  <cit> .
streptomyces tra genes encode a dna translocase resembling the chromosomal dna translocase ftsk of e. coli or spoiiie of b. subtilis <cit> , with double-stranded dna probably entering the recipient  <cit> .
the trab of psvh <dig> binds to the clt sequence as multimers on the mobilized plasmid and translocates unprocessed dna at the hyphal tip to a recipient cell  <cit> .
conjugal transfer of streptomyces theta-type plasmids  requires a major tra gene and two adjacent genes  <cit> .
in contrast to most bacteria, streptomyces species often harbor linear plasmids  <cit> .
unlike the terminal protein-capped linear replicons of adenoviruses that replicate by a mechanism of strand displacement  <cit> , streptomyces linear plasmids start replication from a centrally located ori locus  <cit>  and replication proceeds bi-directionally toward the telomeres  <cit> .
at least some streptomyces linear plasmids  can propagate in circular mode when the telomeres are deleted  <cit> , while some theta-type circular plasmids  can also propagate in linear mode when the telomeres from a linear plasmid are attached  <cit> .
RESULTS
identification of a widely distributed streptomyces species y <dig> and its indigenous plasmid pwty <dig> among endophytic streptomyces strains
during the course of investigating naturally circular plasmids, we detected  <dig> plasmids among ~ <dig> newly isolated actinomycete strains from plant samples of gingko, taxus and artemisia annua l in china.
interestingly,  <dig> of them  displayed similar sizes of ca.14-kb dna bands on agarose gel.
these plasmids were digested with ncoi and all showed five bands  on gel electrophoresis , suggesting that they were an identical plasmid .
the 16s rrna genes of the  <dig> strains were pcr-amplified and all showed the same sequence, resembling those of streptomyces species .
the chromosomal oric regions of these strains were also pcr-amplified with primers from the conserved dnaa and dnan genes and all these oric sequences were identical.
as shown in additional file 2: figure s <dig>  its 1136-bp non-coding sequence was predicted to contain  <dig> dnaa binding-boxes  of  <dig> bp , resembling that of typical streptomyces   <cit> .
the genomic dna of these strains was digested with sspi and electrophoresed in pulsed-field gel.
as shown in additional file 3: figure s <dig>  genomic bands of these strains were identical.
these results suggested that the  <dig> strains were identical .
sequencing and analysis of pwty27
the unique saci-treated pwty <dig> was cloned in an e. coli plasmid psp <dig> for shotgun cloning and sequencing .
the complete nucleotide sequence of pwty <dig> consisted of  <dig>  bp with  <dig> % gc content, resembling that of a typical streptomyces genome   <cit> .
fifteen open reading frames  were predicted by “frameplot  <dig> beta” ; seven of them resembled genes of characterized function, while eight were hypothetical or unknown genes.
these orfs were grouped into two large presumed transcriptional units .
interestingly, five orfs of pwty <dig> c resembled these of of psg <dig> of s. ghanaensis .
pwty <dig>  containing a domain  for dna segregation atpase ftsk/spoiiie resembled a major conjugation tra protein of streptomyces plasmid pjv <dig> .
like other streptomyces plasmids , pwty <dig> encodes genes showing similarity to transcriptional regulator kor , spd  and int  genes.
unexpectedly, pwty <dig>  resembled a chromosomally encoded phage head capsid in nocardia farcinica ifm  <dig>  suggesting the occurrence of a horizontal transfer event between plasmid and phage.
characterization of replication of pwty27
to identify a locus for plasmid replication, various pwty <dig> fragments were sub-cloned into an e. coli plasmid pfx <dig> containing a streptomyces apramycin resistance marker and were introduced by transformation into s. lividans zx <dig>  as shown in figure 1a, plasmids  containing pwty <dig> c, 2c and a 300-bp non-coding sequence  could replicate in s. lividans zx <dig>  but deletion of pwty <dig> c  or pwty <dig> c  or the ncs  abolished propagation in s. lividans zx <dig>  adding the 300-bp ncs , but not a 149-bp ncs , to pwt <dig> restored its replication activity.
co-transcription of pwty <dig> c and 2c was confirmed by pcr amplification of their co-transcribed rna products into cdna .
these results indicated that a basic locus for pwty <dig> replication was pwty <dig> c , pwty <dig> c  and a 300-bp  ncs.
pwt <dig> was introduced by conjugation from e. coli et <dig>  into  <dig> randomly-selected endophytic streptomyces strains , and apramycin resistant transconjugants were obtained from eight of them, indicating a wide host range for this plasmid.
repa protein binds specifically to intact ir <dig> of the iteron sequence in vitro
the pwty <dig> repb was predicted to be a dna primase/polymerase and repa a hypothetical protein.
the 300-bp ncs was predicted as an iteron containing five direct repeats of  <dig> bp , five direct repeats of  <dig> bp  and three pairs of inverted repeats .
to see if there was an interaction between the repa protein and this iteron sequence, electrophoretic mobility shift assays for dna-protein complex formation were employed.
the 6his-tagged repa protein was incubated with a atp-labeled iteron dna, and then electrophoresed and autoradiographed.
as shown in figure 2b, the “shifted” dna bands were visualized by adding repa protein, indicating that the repa protein could bind to the dna probe to form a dna-protein complex.
formation of this complex was inhibited by adding a 15-fold excess of unlabeled probe but was not affected by adding even a 1000-fold excess of polydidc dna as a non-specific competitor, indicating that the binding reaction of the repa protein with iteron dna was highly specific.
to precisely determine the binding sequence of the repa protein and iteron dna, a “footprinting” assay was employed.
as shown in figure 2c, two sequences  protected from digestion with dnasei were visualized on adding repa protein.
these sequences  covered intact ir <dig>  of the iteron .
a plasmid containing the replication locus of pwty <dig> propagates in linear mode when the telomeres of a linear plasmid are attached
the replication locus of pwty <dig> comprised rep and an iteron, resembling those of bi-directionally replicating streptomyces plasmids   <cit> .
to see if pwty <dig> could also replicate in linear mode when the telomeres of a linear plasmid were attached, we constructed pwt <dig> , containing the replication locus of pwty <dig>  and two 381-bp functional telomeres of linear plasmid psla <dig>  <cit> .
drai-linearized pwt <dig> dna from e. coli was introduced by transformation into s. lividans zx <dig>  transformants were obtained at a frequency of  <dig> × 103/μg dna.
genomic dna was isolated, and a ~ <dig> -kb plasmid dna band was detected on an agarose gel.
as shown in figure  <dig>  this band was resistant to treatment by λ exonuclease but sensitive to e. coli exonuclease iii, suggesting that it was a double-stranded linear dna with free 3′ but blocked 5′ ends.
identification of a tra gene and its adjacent essential sequence for plasmid transfer
pwty <dig>  resembled the major conjugation protein tra of streptomyces plasmid pjv <dig>  <cit> .
as shown in figure 4a, plasmids  containing pwty <dig>  and its adjacent 159-bp sequence  could transfer at high frequencies.
deletion of pwty <dig>   abolished transfer of the plasmid.
complete  or partial deletion  of the 159-bp sequence decreased transfer frequencies ca.
1000- and 10-fold, respectively.
thus, a basic locus for pwty <dig> transfer comprised pwty <dig>   and its adjacent ~159-bp sequence.
to precisely determine the essential segment of the short sequence for plasmid transfer, various fragments were pcr-amplified and then cloned into pwt <dig> containing intact traa but not the 159-bp sequence.
as shown in figure 4b, a plasmid  containing a 175-bp fragment  could transfer at a high frequency.
deletions of  <dig> bp within traa  decreased transfer frequency ca.
1000-fold.
deletions of  <dig> bp  and  <dig> bp  of the clt decreased transfer frequencies ca.
10- and 1000-fold, respectively.
these results suggested that the essential region for plasmid transfer was ca.
<dig> bp covering  <dig> bp within traa and its adjacent  <dig> bp , while the  <dig> bp  next to it also played a role in plasmid transfer.
traa protein binds specifically to the clt sequence in vitro
two trans-membrane domains  in the 688-aa traa protein were predicted .
a truncated traa  lacking the trans-membrane domains could be expressed in e. coli as soluble protein.
the 175-bp clt sequence  contained four direct repeats  and two inverted repeats  .
to see if there was an interaction between traa protein and the clt sequence, a “band-shift” assay for dna-protein complex formation was employed.
as shown in figure 5b, traa protein could bind to the dna probe to form a dna-protein complex.
formation of this complex was inhibited by adding 1– <dig> fold excess of unlabeled probe but was not affected by adding a 30-fold  excess of polydidc dna as a non-specific competitor, indicating that the binding reaction of the traa protein with the clt dna was highly specific.
2
.
as figure
2
.
the amounts of traa protein used in lanes 1– <dig> were  <dig>   <dig> ,  <dig> ,  <dig>  and  <dig>  μg, respectively.
two sequences protected by traa from digestion with dnasei are shown.
a “footprinting” assay was employed to precisely determine the binding sequence of traa protein and clt dna.
as shown in figure 5c, two sequences  protected from digestion with dnase i were visualized on adding traa protein.
one sequence  covered all the four dc <dig> and one dc <dig> and most of ic <dig>  and another  covered two dc <dig> and part of ic <dig> of the clt .
the pwty <dig> sequence contained four dr <dig> within the clt while carrying fifteen dc <dig> distributing randomly in the plasmid, suggesting an essential role of the dc <dig> for plasmid transfer.
detection of a variety of repa of pwty <dig> and oric sequences of y <dig> among soil samples
by detecting the indigenous plasmid pwty <dig>  we have identified a widely distributed streptomyces strain y <dig> among plant samples.
to see if this species along with the plasmid could also reside in soil, we collected  <dig> soil samples from  <dig> cities of nine provinces in china.
soil genomic dna was isolated and pcr-amplified with primers from the repa of pwty <dig> and the oric of y <dig>  as shown in figure  <dig>  pcr bands were visualized from five samples  for repa and also five samples  for oric, while no pcr bands were obtained for the oric of s. ceolicolor a <dig> from the twelve soil samples.
there was a correlation between the repa and the oric in four samples , while repa, but not oric, was detected from sample  <dig>  and oric, but not repa, from sample  <dig>  these pcr bands were sequenced, showing that the repa sequences of samples  <dig> and  <dig> were identical to that of pwty <dig>  while one point mutation  was found in that of sample  <dig>  one mutation  in sample  <dig>  and twelve point mutations in sample  <dig>  the oric sequences of samples  <dig>   <dig>   <dig> and  <dig> were identical to that of y <dig>  while there was one point mutation  in sample  <dig>  these results indicated that a number of point mutations for the repa and oric occurred from these soil samples.
discussion
more than  <dig> species or sub-species in the genus streptomyces have validly been designated and published  <cit> .
however, whether there was some predominant streptomces species in natural habitats was not clear.
from six isolates of an endophytic  streptomyces species across south australia, a  <dig> -bp plasmid pen <dig> was identified  <cit> .
here, we report identification of a  <dig> -bp plasmid pwty <dig> in an endophytic streptomyces species y <dig> from fourteen plant samples of gingko, taxus and artemisia annua l across china.
by integrating the egfp gene  in the y <dig> chromosome and then infecting leaves of ginkgo, however, we could not detect y <dig> strains growing inside the leaves .
by pcr amplification of soil genomic dna and sequencing, we found that four out of the  <dig> soil samples collected from  <dig> cities in china contained similar repa of pwty <dig> and oric of y <dig>  however, the absence of pwty27-repa and yt27-oric in certain soil samples can also be explained by the presence of a pcr-inhibitor  in the soil samples that gave negative results.
the sequence of pwty <dig> does not resemble that of pen <dig>  and the oric sequence of y <dig> is unique in the genbank database.
thus, we identified a widely distributed streptomyces species along with its indigenous plasmid from some plants and soils cross china by both culturing and nonculturing methods.
existence of a widely distributed species in natural habitats might reflect a versatile capacity to resist stresses.
the basic replication locus of pwty <dig> comprises repab genes and an iteron sequence, resembling that of streptomyces theta-type plasmids scp <dig>   <cit> , pfp <dig> and pfp <dig>   <cit> .
given the model of bi-directional replication of streptomyces linear replicons  <cit> , like scp <dig> and pfp <dig>  <cit> , the pwty2-rep locus with artificially attached telomeres from a streptomyces linear plasmid is also able to propagate in linear form, indicating that it replicates in a bi-directional mode.
the repi of scp <dig> binds to an upstream sequence of the repi gene  <cit> .
the repa proteins of pfp <dig> and pfp <dig> bind specifically to their iterons  <cit> .
the repa of pwty <dig> also binds highly specifically to the iteron in vitro, and further dna “footprinting” showed that the protein binds to intact ir <dig>  which overlaps with some dr <dig> and dr <dig>  but leaving some spacers, especially the “loop” of the ir <dig> unprotected from digestion with dnasei.
the long ir <dig> sequence may fold back to form hairpin structure.
in fact, dr <dig>  is almost the complementary sequence of dr <dig> , which means it is the same repeat but on the opposite strand.
these results suggest that repa may form multimers and recongnize a second structure  of the iteron dna .
conjugal transfer of streptomyces theta-type plasmids  requires a major tra and its adjacent genes  <cit> , while that of streptomyces rc-type plasmids  needs a tra gene and a clt site  <cit> .
the minimal pij <dig> clt-locus consists of a sequence ~ <dig> bp in size that includes an essential imperfect inverted repeat and three direct repeats  sequences and is located close to the korb gene  <cit> .
the pjv <dig> clt region contains nine direct repeats  and two pairs of imperfect inverted repeats  <cit> .
like these streptomyces rc-type plasmids, conjugal transfer of the theta-type pwty <dig> requires a major tra gene and its adjacent sequence.
such a clt locus in pwty <dig> has a 16-bp sequence within the traa gene.
the 175-bp clt sequence contains four direct repeats of dc <dig> /dc <dig>  and two inverted repeats .
thus, although the clt sequences of streptomyces conjugative plasmids are varied, they contain multiple direct repeats and/or inverted repeats.
reuther et al.
<cit>  report that trab protein of psvh <dig> binds to a 50-bp clt-like sequence containing a 14-bp direct repeat, producing a protein-dna complex too large to enter an agarose gel, indicating that multimers of trab are bound to the dna.
vogelmann et al.
<cit>  show that trab specifically recognizes repeated 8-bp motifs on psvh <dig> mediated by helix α <dig> of the c-terminal winged-helix-turn-helix domain of the protein, and trab assembles as a hexameric ring structure with a central  <dig> -nm channel and forms pores in lipid bilayers.
by removing the n-terminal trans-membrane domain, traa of pwty <dig> can be expressed in e. coli as a soluble protein.
traa recognizes and binds specifically to two regions, one  containing all the four dc <dig> and one dc <dig> and most part of ic <dig> and another  covering two dc <dig> and part of ic <dig> of the clt, suggesting that formation of a high-ordered protein-dna complex.
CONCLUSIONS
in this work, a widely distributed streptomyces strain y <dig> along with its indigenous plasmid pwty <dig> from plants and soil samples cross china are identified by both culturing and nonculturing methods.
the complete nucleotide sequence of pwty <dig> consists of  <dig>  bp.
a minimal locus for plasmid replication comprises repab genes and an adjacent iteron sequence.
repa protein binds specifically in vitro to a long inverted-repeat  of the iteron sequence.
plasmid containing the replication locus and two telomeres from streptomyces linear plasmid can propagate in linear mode, indicating a bi-directional replication mode for pwty <dig>  as for rolling-circle plasmids, a single traa gene and a clt sequence on pwty <dig> are required for plasmid transfer.
we find that traa binds specifically to the two regions of the clt sequence, one containing all the four dc <dig> of  <dig> bp  and one dc <dig>  and most of ic <dig>  and another covering two dc <dig> and part of ic <dig>  suggesting formation of a high-ordered dna-protein complex.

 streptomyces species are widely distributed in natural habitats, such as soils, lakes, plants and some extreme environments. they are gram-positive, mycelial bacteria with high g+c content in their dna. more than antibiotics and pharmacologically active metabolites have been discovered in streptomyces species. streptomyces species usually harbor conjugative plasmids. modes of plasmid replication in streptomyces include rolling-circle, and uni-directional or bi-directional theta types. some plasmids replicate in chromosomally-integrating/autonomous forms. streptomyces rc plasmids are usually small, while theta-type plasmids are larger. replication loci of streptomyces rc plasmids comprise a single rep gene, a dso for initiation and termination of replication, and an sso for conversion of the lagging strand into a double-stranded molecule. the replication locus of the theta-type scp comprises repi and repii genes and an adjacent non-coding sequence to which repi protein binds. pfp and pfp contain basic replication loci of rep and iteron types, to which rep proteins bind. conjugal transfer of streptomyces rc plasmid needs a tra gene along with a clt site. streptomyces tra genes encode a dna translocase resembling the chromosomal dna translocase ftsk of e. coli or spoiiie of b. subtilis, with double-stranded dna probably entering the recipient. the trab of psvh binds to the clt sequence as multimers on the mobilized plasmid and translocates unprocessed dna at the hyphal tip to a recipient cell. conjugal transfer of streptomyces theta-type plasmids requires a major tra gene and two adjacent genes. in contrast to most bacteria, streptomyces species often harbor linear plasmids. unlike the terminal protein-capped linear replicons of adenoviruses that replicate by a mechanism of strand displacement, streptomyces linear plasmids start replication from a centrally located ori locus and replication proceeds bi-directionally toward the telomeres. at least some streptomyces linear plasmids can propagate in circular mode when the telomeres are deleted, while some theta-type circular plasmids can also propagate in linear mode when the telomeres from a linear plasmid are attached.  identification of a widely distributed streptomyces species y and its indigenous plasmid pwty among endophytic streptomyces strains during the course of investigating naturally circular plasmids, we detected plasmids among ~ newly isolated actinomycete strains from plant samples of gingko, taxus and artemisia annua l in china. interestingly, of them displayed similar sizes of ca.14-kb dna bands on agarose gel. these plasmids were digested with ncoi and all showed five bands on gel electrophoresis, suggesting that they were an identical plasmid. the 16s rrna genes of the strains were pcr-amplified and all showed the same sequence, resembling those of streptomyces species. the chromosomal oric regions of these strains were also pcr-amplified with primers from the conserved dnaa and dnan genes and all these oric sequences were identical. as shown in additional file 2: figure s its 1136-bp non-coding sequence was predicted to contain dnaa binding-boxes of bp, resembling that of typical streptomyces. the genomic dna of these strains was digested with sspi and electrophoresed in pulsed-field gel. as shown in additional file 3: figure s genomic bands of these strains were identical. these results suggested that the strains were identical. sequencing and analysis of pwty27 the unique saci-treated pwty was cloned in an e. coli plasmid psp for shotgun cloning and sequencing. the complete nucleotide sequence of pwty consisted of bp with % gc content, resembling that of a typical streptomyces genome. fifteen open reading frames were predicted by “frameplot beta”; seven of them resembled genes of characterized function, while eight were hypothetical or unknown genes. these orfs were grouped into two large presumed transcriptional units. interestingly, five orfs of pwty c resembled these of of psg of s. ghanaensis. pwty containing a domain for dna segregation atpase ftsk/spoiiie resembled a major conjugation tra protein of streptomyces plasmid pjv. like other streptomyces plasmids, pwty encodes genes showing similarity to transcriptional regulator kor, spd and int genes. unexpectedly, pwty resembled a chromosomally encoded phage head capsid in nocardia farcinica ifm suggesting the occurrence of a horizontal transfer event between plasmid and phage. characterization of replication of pwty27 to identify a locus for plasmid replication, various pwty fragments were sub-cloned into an e. coli plasmid pfx containing a streptomyces apramycin resistance marker and were introduced by transformation into s. lividans zx as shown in figure 1a, plasmids containing pwty c, 2c and a 300-bp non-coding sequence could replicate in s. lividans zx but deletion of pwty c or pwty c or the ncs abolished propagation in s. lividans zx adding the 300-bp ncs, but not a 149-bp ncs, to pwt restored its replication activity. co-transcription of pwty c and 2c was confirmed by pcr amplification of their co-transcribed rna products into cdna. these results indicated that a basic locus for pwty replication was pwty c, pwty c and a 300-bp ncs. pwt was introduced by conjugation from e. coli et into randomly-selected endophytic streptomyces strains, and apramycin resistant transconjugants were obtained from eight of them, indicating a wide host range for this plasmid. repa protein binds specifically to intact ir of the iteron sequence in vitro the pwty repb was predicted to be a dna primase/polymerase and repa a hypothetical protein. the 300-bp ncs was predicted as an iteron containing five direct repeats of bp, five direct repeats of bp and three pairs of inverted repeats. to see if there was an interaction between the repa protein and this iteron sequence, electrophoretic mobility shift assays for dna-protein complex formation were employed. the 6his-tagged repa protein was incubated with a atp-labeled iteron dna, and then electrophoresed and autoradiographed. as shown in figure 2b, the “shifted” dna bands were visualized by adding repa protein, indicating that the repa protein could bind to the dna probe to form a dna-protein complex. formation of this complex was inhibited by adding a 15-fold excess of unlabeled probe but was not affected by adding even a 1000-fold excess of polydidc dna as a non-specific competitor, indicating that the binding reaction of the repa protein with iteron dna was highly specific. to precisely determine the binding sequence of the repa protein and iteron dna, a “footprinting” assay was employed. as shown in figure 2c, two sequences protected from digestion with dnasei were visualized on adding repa protein. these sequences covered intact ir of the iteron. a plasmid containing the replication locus of pwty propagates in linear mode when the telomeres of a linear plasmid are attached the replication locus of pwty comprised rep and an iteron, resembling those of bi-directionally replicating streptomyces plasmids. to see if pwty could also replicate in linear mode when the telomeres of a linear plasmid were attached, we constructed pwt, containing the replication locus of pwty and two 381-bp functional telomeres of linear plasmid psla. drai-linearized pwt dna from e. coli was introduced by transformation into s. lividans zx transformants were obtained at a frequency of × 103/μg dna. genomic dna was isolated, and a ~ -kb plasmid dna band was detected on an agarose gel. as shown in figure this band was resistant to treatment by λ exonuclease but sensitive to e. coli exonuclease iii, suggesting that it was a double-stranded linear dna with free 3′ but blocked 5′ ends. identification of a tra gene and its adjacent essential sequence for plasmid transfer pwty resembled the major conjugation protein tra of streptomyces plasmid pjv. as shown in figure 4a, plasmids containing pwty and its adjacent 159-bp sequence could transfer at high frequencies. deletion of pwty abolished transfer of the plasmid. complete or partial deletion of the 159-bp sequence decreased transfer frequencies ca. 1000- and 10-fold, respectively. thus, a basic locus for pwty transfer comprised pwty and its adjacent ~159-bp sequence. to precisely determine the essential segment of the short sequence for plasmid transfer, various fragments were pcr-amplified and then cloned into pwt containing intact traa but not the 159-bp sequence. as shown in figure 4b, a plasmid containing a 175-bp fragment could transfer at a high frequency. deletions of bp within traa decreased transfer frequency ca. 1000-fold. deletions of bp and bp of the clt decreased transfer frequencies ca. 10- and 1000-fold, respectively. these results suggested that the essential region for plasmid transfer was ca. bp covering bp within traa and its adjacent bp, while the bp next to it also played a role in plasmid transfer. traa protein binds specifically to the clt sequence in vitro two trans-membrane domains in the 688-aa traa protein were predicted. a truncated traa lacking the trans-membrane domains could be expressed in e. coli as soluble protein. the 175-bp clt sequence contained four direct repeats and two inverted repeats. to see if there was an interaction between traa protein and the clt sequence, a “band-shift” assay for dna-protein complex formation was employed. as shown in figure 5b, traa protein could bind to the dna probe to form a dna-protein complex. formation of this complex was inhibited by adding 1– fold excess of unlabeled probe but was not affected by adding a 30-fold excess of polydidc dna as a non-specific competitor, indicating that the binding reaction of the traa protein with the clt dna was highly specific. 2. as figure 2. the amounts of traa protein used in lanes 1– were,, and μg, respectively. two sequences protected by traa from digestion with dnasei are shown. a “footprinting” assay was employed to precisely determine the binding sequence of traa protein and clt dna. as shown in figure 5c, two sequences protected from digestion with dnase i were visualized on adding traa protein. one sequence covered all the four dc and one dc and most of ic and another covered two dc and part of ic of the clt. the pwty sequence contained four dr within the clt while carrying fifteen dc distributing randomly in the plasmid, suggesting an essential role of the dc for plasmid transfer. detection of a variety of repa of pwty and oric sequences of y among soil samples by detecting the indigenous plasmid pwty we have identified a widely distributed streptomyces strain y among plant samples. to see if this species along with the plasmid could also reside in soil, we collected soil samples from cities of nine provinces in china. soil genomic dna was isolated and pcr-amplified with primers from the repa of pwty and the oric of y as shown in figure pcr bands were visualized from five samples for repa and also five samples for oric, while no pcr bands were obtained for the oric of s. ceolicolor a from the twelve soil samples. there was a correlation between the repa and the oric in four samples, while repa, but not oric, was detected from sample and oric, but not repa, from sample these pcr bands were sequenced, showing that the repa sequences of samples and were identical to that of pwty while one point mutation was found in that of sample one mutation in sample and twelve point mutations in sample the oric sequences of samples and were identical to that of y while there was one point mutation in sample these results indicated that a number of point mutations for the repa and oric occurred from these soil samples. discussion more than species or sub-species in the genus streptomyces have validly been designated and published. however, whether there was some predominant streptomces species in natural habitats was not clear. from six isolates of an endophytic streptomyces species across south australia, a -bp plasmid pen was identified. here, we report identification of a -bp plasmid pwty in an endophytic streptomyces species y from fourteen plant samples of gingko, taxus and artemisia annua l across china. by integrating the egfp gene in the y chromosome and then infecting leaves of ginkgo, however, we could not detect y strains growing inside the leaves. by pcr amplification of soil genomic dna and sequencing, we found that four out of the soil samples collected from cities in china contained similar repa of pwty and oric of y however, the absence of pwty27-repa and yt27-oric in certain soil samples can also be explained by the presence of a pcr-inhibitor in the soil samples that gave negative results. the sequence of pwty does not resemble that of pen and the oric sequence of y is unique in the genbank database. thus, we identified a widely distributed streptomyces species along with its indigenous plasmid from some plants and soils cross china by both culturing and nonculturing methods. existence of a widely distributed species in natural habitats might reflect a versatile capacity to resist stresses. the basic replication locus of pwty comprises repab genes and an iteron sequence, resembling that of streptomyces theta-type plasmids scp, pfp and pfp. given the model of bi-directional replication of streptomyces linear replicons, like scp and pfp, the pwty2-rep locus with artificially attached telomeres from a streptomyces linear plasmid is also able to propagate in linear form, indicating that it replicates in a bi-directional mode. the repi of scp binds to an upstream sequence of the repi gene. the repa proteins of pfp and pfp bind specifically to their iterons. the repa of pwty also binds highly specifically to the iteron in vitro, and further dna “footprinting” showed that the protein binds to intact ir which overlaps with some dr and dr but leaving some spacers, especially the “loop” of the ir unprotected from digestion with dnasei. the long ir sequence may fold back to form hairpin structure. in fact, dr is almost the complementary sequence of dr, which means it is the same repeat but on the opposite strand. these results suggest that repa may form multimers and recongnize a second structure of the iteron dna. conjugal transfer of streptomyces theta-type plasmids requires a major tra and its adjacent genes, while that of streptomyces rc-type plasmids needs a tra gene and a clt site. the minimal pij clt-locus consists of a sequence ~ bp in size that includes an essential imperfect inverted repeat and three direct repeats sequences and is located close to the korb gene. the pjv clt region contains nine direct repeats and two pairs of imperfect inverted repeats. like these streptomyces rc-type plasmids, conjugal transfer of the theta-type pwty requires a major tra gene and its adjacent sequence. such a clt locus in pwty has a 16-bp sequence within the traa gene. the 175-bp clt sequence contains four direct repeats of dc /dc and two inverted repeats. thus, although the clt sequences of streptomyces conjugative plasmids are varied, they contain multiple direct repeats and/or inverted repeats. reuther et al. report that trab protein of psvh binds to a 50-bp clt-like sequence containing a 14-bp direct repeat, producing a protein-dna complex too large to enter an agarose gel, indicating that multimers of trab are bound to the dna. vogelmann et al. show that trab specifically recognizes repeated 8-bp motifs on psvh mediated by helix α of the c-terminal winged-helix-turn-helix domain of the protein, and trab assembles as a hexameric ring structure with a central -nm channel and forms pores in lipid bilayers. by removing the n-terminal trans-membrane domain, traa of pwty can be expressed in e. coli as a soluble protein. traa recognizes and binds specifically to two regions, one containing all the four dc and one dc and most part of ic and another covering two dc and part of ic of the clt, suggesting that formation of a high-ordered protein-dna complex.  in this work, a widely distributed streptomyces strain y along with its indigenous plasmid pwty from plants and soil samples cross china are identified by both culturing and nonculturing methods. the complete nucleotide sequence of pwty consists of bp. a minimal locus for plasmid replication comprises repab genes and an adjacent iteron sequence. repa protein binds specifically in vitro to a long inverted-repeat of the iteron sequence. plasmid containing the replication locus and two telomeres from streptomyces linear plasmid can propagate in linear mode, indicating a bi-directional replication mode for pwty as for rolling-circle plasmids, a single traa gene and a clt sequence on pwty are required for plasmid transfer. we find that traa binds specifically to the two regions of the clt sequence, one containing all the four dc of bp and one dc and most of ic and another covering two dc and part of ic suggesting formation of a high-ordered dna-protein complex. 
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 pwty as for rolling-circle plasmids, a single traa gene and a clt sequence on pwty were required for plasmid transfer. streptomyces species are widely distributed in natural habitats, such as soils, lakes, plants and some extreme environments. traa recognized and bound specifically to the two regions of the clt sequence, one containing all the four dc of bp and one dc and most of ic and another covering two dc and part of ic suggesting formation of a high-ordered dna-protein complex. replication loci of several streptomyces theta-type plasmids have been reported, but are not characterized in details. a basic locus for plasmid replication comprised repab genes and an adjacent iteron sequence, to a long inverted-repeat of which the repa protein bound specifically in vitro, suggesting that repa may recognize a second structure of the iteron dna. the complete nucleotide sequence of pwty consisted of bp. we report the detection of a widely distributed streptomyces strain y and its indigenous plasmid pwty from fourteen plants and four soil samples cross china by both culturing and nonculturing methods.
 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.  we report the detection of a widely distributed streptomyces strain y and its indigenous plasmid pwty from fourteen plants and four soil samples cross china by both culturing and nonculturing methods. the complete nucleotide sequence of pwty consisted of bp. a basic locus for plasmid replication comprised repab genes and an adjacent iteron sequence, to a long inverted-repeat of which the repa protein bound specifically in vitro, suggesting that repa may recognize a second structure 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 pwty as for rolling-circle plasmids, a single traa gene and a clt sequence on pwty were required for plasmid transfer. traa recognized and bound specifically to the two regions of the clt sequence, one containing all the four dc of bp and one dc and most of ic and another covering two dc and part of ic suggesting formation of a high-ordered dna-protein complex.  this work isolates a widespread streptomyces strain y and sequences its indigenous theta-type plasmid pwty27; identifies the replication and conjugation loci of pwty and; characterizes the binding sequences of the repa and traa proteins. streptomycesplasmidreplicationconjugation 