The complete nucleotide sequence of the multicopy Streptomyces plasmid pUlOl has been determined and correlated with previously published genetic data. The circular DNA molecule is 8,830 nucleotides in length and has a G+C composition of 72.98%. The use of a computer program, FRAME, enabled identification in the sequence of seven open reading frames, four of which, tra (621 amino acids [aa]), spdA (146 aa), spdB (274 aa), and kilB (177 aa), appear to be genes involved in plasmid transfer. At least two of the above genes are predicted to be transcribed by known promoters that are regulated in trans by the products of the korA (241 aa) and korB (80 aa) loci on the plasmid. (14); thus, the organization of DNA sequences required for autonomous replication and for the control of gene expression may well be different from that found in procaryotic organisms such as Escherichia coli, which has an average G+C composition of 50%.pIJlOl is a circular multicopy Streptomyces plasmid of 8.9 kilobases (kb). Originally found in S. lii'idans ISP 5434, it has a broad host range, and a number of widely used cloning vectors have been derived from it (16,19,21). More is presently known about the basic biology of pIJlOl than is known for any other Streptomyces plasmid. Its essential replication functions have been localized to a 2.1-kb DNA segment (21), and other loci affecting plasmid maintenance and plasmid transfer have been described (16,20,21 DNA isolations and manipulations. Standard cloning techniques and plasmid DNA isolation protocols were used for both E. coli (22) and S. liv'idans (16). Occasionally, restriction endonuclease-generated DNA fragments to be sequenced were isolated from low-melting-point agarose gels as described previously (20). Single-stranded M13 DNA sequencing templates were prepared essentially as described by Messing (23) (Boehringer Mannheim) were added to this mixture, and 2.5 p.l of the resulting solution was placed on the lip of each of four tubes containing 2 [L1 of the appropriate dNTP°mixes. The dNTP°mixes contained 10 mM Tris hydrochloride (pH 7.5), 20 mM MgCl2, and deoxynucleoside triphosphate (dNTP) and dideoxy-NTP (ddNTP) nucleotides as follows. dATP°was 100 nM dGTP, 100 nM dCTP, 100 nM dTTP, and 75 nM ddATP; dGTP°was 2.5 nM dGTP, 100 nM dCTP, 100 nM dTTP, and 38 nM ddGTP; dCTP°was 100 nM dGTP, 2.5 nM dCTP, 100 nM dTTP, and 20 nM ddCTP; dTTP°was 100 nM dGTP, 100 nM dCTP, 1.25 nM dTTP, and 225 nM ddTTP. The solutions were mixed to start the reaction by centrifugation, and
The 8.9-kilobase Streptomyces plasmid plJl0 is self-transmissible at high frequency into recipient strains. By genetic analysis of the transfer region of the plasmid, we identified six plasmid-encoded loci involved in gene transfer and the associated pocking phenomenon. Two loci, kilA and kilB, could not be cloned into Streptomyces lividans on a minimal p1U101-based replicon unless suitable kil-override (kor) genes were present, either in cis or in trans. korA could control the lethal effects of both kilA and kilB, whereas korB could control only the effects of kilB. kilB mutants were defective in their pocking reaction; kilA mutants produced no visible pocks whatsoever. Mutations in two other loci, tra and spd, produced no pocks and defective pocks, respectively. These results suggest that kil, kilB, tra, and spd are intimately involved in plasmid transfer and that the actions of kilA and kilB are regulated by the products of the korA and korB genes. Currently, little is known about the mechanisms by which plasmids are transferred in Streptomyces spp. or about the interrelationship between plasmid transfer and the complex life cycle that these organisms exhibit. We report here the results of initial studies aimed at identifying and elucidating the function of genes involved in the transfer of pIJlOl, a small multicopy conjugative Streptomyces plasmid from which a number of commonly used cloning vectors have been derived (15). Our studies identified several plasmidencoded genes that appear to be involved in plasmid transfer and the associated pocking phenomenon. Certain of these genes specify functions lethal to either the host cell or the plasmid (kil genes), while others (kor genes) encode products that control expression of the Kil phenotype. MATERIALS AND METHODSBacterial strains and plasmids. Escherichia coli MC1061 hsdR hsdM+ araDl39 A(ara leu)7697 AlacX74 galU galK rpsL F' Alac-150 proAB Kanr (5) was used as the E. coli host * Corresponding author. throughout this study. Streptomyces lividans TK64 pro-2 str-6 SLP2-SLP3-(11) was used as the Streptomyces host. The E. coli vector pUC19 (24) and the Streptomyces plasmids pIJlOl, pIJ303, pIJ350 (15), and pFM4 (16) have been described previously. All other plasmids used were constructed during this study and are described in the text.Media. E. coli MC1061 was grown at 37°C in LB broth (17) or on LB agar (1.5%). S. lividans was usually grown at 34°C, either in LB broth or on R2YE (6) containing 0.5% yeast extract. Pocks were found to be more distinct when strains were grown on R2YE containing 0.1% yeast extract. Streptomyces protoplasts were prepared from cultures grown in YEME plus 34% sucrose (6).pUC19 derivatives were selected in E. coli by using ampicillin at 60 ,ug/ml (solid media) or 100 ,ug/ml (liquid culture). Streptomyces plasmids were selected by using thiostrepton (5 ,ug/ml in LB; 50 pLg/ml in R2YE) or hygromycin (50 ,ug/ml in LB; 200 ,ug/ml in R2YE). linkers, and filling in of 5' protruding ends of restriction endonuclease-generated fragments by usin...
Four regulated promoters that direct the transcription of genes (i.e., korA, tra, kilB, and korB) involved in the transfer of the Streptomyces plasmid pUJlOl were isolated following the in vitro fusion of plasmid DNA fragments to a promoterless gene encoding the S. lividans extracellular enzyme I-galactosidase. Introduction of pLUlOl into cells carrying each of these promoter-lac fusions resulted in decreased lac expression. The sites of initiation of transcription by the promoter sequences were identified by primer extension experiments, and the DNA sequences specifically required for promoter activity and regulation by pU101-encoded functions were determined by deletion analysis. The data obtained indicate that the korB locus encodes a repressor that regulates its own transcription, as well as transcription of the kill promoter; korA and tra are transcribed from overlapping divergent promoters that are coregulated by the korA gene product. Common DNA sequence domains within coregulated promoters allowed the identification of putative binding sites for each of the kor gene products.
We describe the cloning and analysis of two overlapping DNA fragments from Streptomyces coelicolor that cause aerial mycelium to appear more rapidly than usual when introduced into Streptomyces lividans on a low-copy-number plasmid vector. Colonies of S. lividans TK64 harboring either clone produce visible aerial mycelia after only 48 h of growth, rather than the usual 72 to 96 h. From deletion and sequence analysis, this rapid aerial mycelium (Ram) phenotype appears to be due to a cluster of three genes that we have designated ramA, ramB, and ramR. Both ramA and ramB potentially encode 65-kDa proteins with homology to ATP-dependent membrane-translocating proteins. A chromosomal ramB disruption mutant of S. lividans was found to be severely defective in aerial mycelium formation. ramR could encode a 21-kDa protein with significant homology to the UhpA subset of bacterial two-component response regulator proteins. The overall organization and potential proteins encoded by the cloned DNA suggest that this is the S. coelicolor homolog of the amf gene cluster that has been shown to be important for aerial mycelium formation in Streptomyces griseus. However, despite the fact that the two regions probably have identical functions, there is relatively poor homology between the two gene clusters at the DNA sequence level.
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