Plasmid transfer in Streptomyces lividans: identification of a kil-kor system associated with the transfer region of pIJ101

Abstract

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...

Regulation and function of the Streptomyces plasmid pSN22 genes involved in pock formation and inviability

“…Analysis of the 8.9-kb multicopy plasmid pIJ101 identified four plasmid genes, tra (transfer), spdA and spdB (plasmid spread), and the repressor gene korA (kil override), which are involved in conjugative plasmid transfer and pock formation (10,11,13,23). tra is essential for conjugative plasmid transfer, mobilization of chromosomal markers (fertility), and pock formation, which is the result of extensive plasmid transfer.…”

“…Plasmids in Streptomyces species have been investigated for conjugation and pock formation (1,10,13,14,29), replication (5,16,19), incompatibility (5,27), site-specific recombination (2,6,17,18), and involvement in antibiotic production (3,31). Analysis of the 8.9-kb multicopy plasmid pIJ101 identified four plasmid genes, tra (transfer), spdA and spdB (plasmid spread), and the repressor gene korA (kil override), which are involved in conjugative plasmid transfer and pock formation (10,11,13,23).…”

Conjugative Transfer of Streptomyces Plasmid pSN22.

“…but not in the cytoplasmic or cell-surface fractions 34 ). It is notewonhy that The transfer ability of pESS500mB is retained but the efficiency is reduced 10 a level of the spdB phenOlype, suggest ing that TraB is involved in imramycelialtransfer as well as illlermycelialtransfer and that the N-terminal modification of TraB might inhibit the SpdB function 14 ) . The spdB locus coasists of four ORFs, encoding prOleins which show a high hydropathy plOI, implying that they are localized in the cytoplasmic membrane, just like TraB.…”

Linear plasmids and their 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). aaaa 2 Copurification of DNA polymerase I (PolA) and a topoisomerase I with the Tap protein suggests a role in replication.…”