Mutational analysis of the tra locus of the broad-host-range Streptomyces plasmid pIJ101

The tra gene of Streptomyces lividans plasmid pIJ101 encodes a 621-amino-acid protein that can mediate both plasmid transfer and the interbacterial transfer of chromosomal genes (i.e., chromosome-mobilizing ability [Cma]) during mating. Here we report the results of in-frame insertional mutagenesis studies aimed at defining regions of Tra required for these functions. While hexameric linker insertions throughout the tra gene affected plasmid and chromosomal gene transfer, insertions in a 200-amino-acid region of the Tra protein that contains presumed nucleotide-binding motifs and that is widely conserved among a functionally diverse family of bacterial and plasmid proteins (K. J. Begg, S. J. Dewar, and W. D. Donachie, J. Bacteriol. 177:6211-6222, 1995) had especially prominent effects on both functions. Insertions near the N terminus of Tra reduced Cma for either circular or linear host chromosomes to a much greater extent than pIJ101 plasmid transfer. Our results suggest that Cma involves Tra functions incremental to those needed for plasmid DNA transfer.     

An in vivo assay for conjugation-mediated recombination yields novel results for Streptomyces plasmid pIJ101

Efficient transmission of circular plasmids in Streptomyces spp. proceeds by an uncharacterized mechanism that requires a cis-acting locus of transfer (clt) and often only a single plasmid-encoded protein. For circular plasmids from other bacteria, site- and strand-specific nicking takes place at the cis-acting oriT locus via the plasmid-encoded relaxase protein prior to single-strand transfer. Using an assay originally designed to demonstrate that conjugative transfer of plasmids containing tandem oriT loci results in the formation of a single composite oriT locus, we show here that an analogous construct involving the pIJ101 clt locus apparently does not undergo such a conjugation-mediated event during plasmid transfer. Our results, which imply that streptomycete plasmids are transferred by a functionally distinct mechanism compared to oriT-containing plasmids, are complementary to other recent evidences that support a novel double-stranded model for streptomycete circular plasmid transfer.     

Unraveling the essential role in conjugation of the Tra protein of Streptomyces lividans plasmid pIJ101

Plasmid pIJ101 from Streptomyces lividans encodes a single gene, tra, that is essential for both plasmid transfer and mobilization of chromosomes during mating. The tra gene product (Tra) is a membrane protein, a portion of which shows similarity to transfer proteins of other streptomycete plasmids as well as additional bacterial chromosome partitioning proteins. This paper reviews past and present work that has focused on elucidating the precise role of the Tra protein of pIJ101 in conjugation in Streptomyces.     

Temperature-sensitive mutants of the Streptomyces plasmid pIJ702

DNA from the Streptomyces plasmid pIJ702 was mutagenized in vitro using hydroxylamine and transformed into Streptomyces lividans. One plasmid with temperature-sensitive replication (pMT660) and one plasmid with a temperature-sensitive tyrosinase (pMT661) were isolated. The plasmid pMT661 contains a novel PstI restriction endonuclease site within the tyrosinase gene.     

Novel post-replicative DNA modification in Streptomyces: analysis of the preferred modification site of plasmid pIJ101.

Both Streptomyces lividans and Streptomyces avermitilis have the ability to site specifically modify their DNA, rendering it susceptible to in vitro Tris-dependent double-strand cleavage. We have cloned a 160 bp fragment containing the preferred modification site of plasmid pIJ101 and, employing an in vitro primer extension assay, determined that the modifications occur at guanine residues on either strand separated by 3 bp. These guanines are located within a 6 bp palindromic 'core' sequence. A cloned copy of a 35 bp region of the plasmid containing this core sequence was not recognized by the modifying activity in vivo. To further investigate the nature of the site specificity a set of deletion mutants of the 160 bp sequence were analysed. This revealed that a substantial portion of this sequence is essential for authentic modification. The essential region contains three 13 bp direct repeats, the central one containing the core sequence, while the left-hand and right-hand copies overlap two potential stem-loop structures. Deletion of either left- or right-hand repeat structures abolishes modification within the core sequence, although the left-hand deletion resulted in modification at a secondary site within the right-hand direct repeat. These data support a post-replicative mechanism of modification, underlined by the observation that the modifications are not detected in single-stranded plasmid replication intermediates.     

pIJ101, a multi-copy broad host-range Streptomyces plasmid: functional analysis and development of DNA cloning vectors

Streptomyces lividans ISP 5434 contains four small high copy number plasmids: pIJ101 (8.9 kb), pIJ102 (4.0 kb), pIJ103 (3.9 kb) and pIJ104 (4.9 kb). The three smaller species appear to be naturally occurring deletion variants of pIJ101. pIJ101 and its in vivo and in vitro derivatives were studied after transformation into S. lividans 66. pIJ101 was found to be self-transmissible by conjugation, to elicit "lethal zygosis" and to promote chromosomal recombination at high frequency in both S. lividans 66 and S. coelicolor A3(2). A restriction endonuclease cleavage map of pIJ101 was constructed for 11 endonucleases; sites for five others were lacking. Many variants of pIJ101 were constructed in vitro by inserting DNA fragments determining resistance to neomycin, thiostrepton or viomycin, and having BamHI termini, into MboI or BclI sites on the plasmid, sometimes with deletion of segments of plasmid DNA. The physical maps of these plasmids were related to their phenotypes in respect of lethal zygosis and transfer properties. In vivo recombination tests between pairs of variant plasmids were also done. These physical and genetic studies indicated that determinants of conjugal transfer occupy less than 2.1 kb of the plasmid. A second segment is required for spread of the plasmid within a plasmid-free culture to produce the normal lethal zygosis phenotype: insertion of foreign DNA in this region caused a marked reduction in the diameter of lethal zygosis zones. The minimum replicon was deduced to be 2.1 kb or less in size; adjacent to this region is a 0.5 kb segment which may be required for stable inheritance of the plasmid. The copy number of several derivatives of pIJ101 in S. lividans 66 was between 40 and 300 per chromosome and appeared to vary with the age or physiological state of the culture. pIJ101 derivatives have a wide host range within the genus Streptomyces: 13 out of 18 strains, of diverse species, were successfully transformed. Knowledge of dispensable DNA segments and the availability of restriction sites for the insertion of DNA, deduced from the properties of plasmids carrying the E. coli plasmid pACYC184 introduced at various sites, was used in the construction of several derivatives of pIJ101 suitable as DNA cloning vectors. These were mostly designed to be non-conjugative and to carry pairs of resistance genes for selection. They include a bifunctional shuttle vector for E. coli and Streptomyces; a Streptomyces viomycin resistance gene of this plasmid is expressed in both hosts.     

Cloning and expression of a Streptomyces cholesterol oxidase gene in Streptomyces lividans with plasmid pIJ702

The cholesterol oxidase gene (cho) of Streptomyces sp. was cloned into Streptomyces lividans with the vector pIJ702. Deletion analysis of the recombinant plasmid showed that entire coding sequence of the cho gene was located within a 2.5-kilobase segment of the chromosomal DNA obtained from the cholesterol oxidase-producing strain. When cloned cells of S. lividans were grown in an appropriate medium, the cells produced severalfold more cholesterol oxidase extracellularly than did the producing strain.     

Complete nucleotide sequence of the Streptomyces lividans plasmid pIJ101 and correlation of the sequence with genetic properties.

The complete nucleotide sequence of the multicopy Streptomyces plasmid pIJ101 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. The segment of the plasmid capable of autonomous replication contains one large open reading frame (rep; 450 aa) and a noncoding region presumed to be the origin of replication. Four other small (less than 90 aa) open reading frames are also present on the plasmid, although no function can be attributed to them. The sequence of the pIJ101 replication segment present in several widely used cloning vectors (e.g., pIJ350 and pIJ702) has also been determined, so that the complete nucleotide sequences of these vectors are now known.     

Cloning and expression of a Streptomyces cholesterol oxidase gene in Streptomyces lividans with plasmid pIJ702.

The cholesterol oxidase gene (cho) of Streptomyces sp. was cloned into Streptomyces lividans with the vector pIJ702. Deletion analysis of the recombinant plasmid showed that entire coding sequence of the cho gene was located within a 2.5-kilobase segment of the chromosomal DNA obtained from the cholesterol oxidase-producing strain. When cloned cells of S. lividans were grown in an appropriate medium, the cells produced severalfold more cholesterol oxidase extracellularly than did the producing strain.     

Transfer of the plJ101 plasmid in Streptomyces lividans requires a cis-acting function dispensable for chromosomal gene transfer

The tra gene of Streptomyces lividans plasmid plJ101 is required for both plasmid DNA transfer and plJ101-induced mobilization of chromosomal genes during mating. We show that a chromosomally inserted copy of tra mediates transfer of chromosomal DNA at high frequency but promotes efficient transfer of plasmids only when they contain a previously unknown locus, here named clt. Insertional mutation or deletion of clt from plJ101 reduced plasmid transfer mediated by either plasmid-borne or chromosomally located tra by at least three orders of magnitude, abolished the transfer-associated pocking phenomenon, and interfered with the ability of tra⁺ plasmids to promote transfer of chromosomal DNA. Our results indicate that plasmid transfer in S. lividans involves a cis-acting function dispensable for chromosomal gene transfer and imply that either the S. lividans chromosome encodes its own clt-like function or, alternatively, that transfer of plasmid and chromosomal DNA occurs by different mechanisms.     

Complementation of Conjugation Functions of Streptomyces lividans Plasmid pIJ101 by the Related Streptomyces Plasmid pSB24.2

A database search revealed extensive sequence similarity between Streptomyces lividans plasmid pIJ101 and Streptomyces plasmid pSB24. 2, which is a deletion derivative of Streptomyces cyanogenus plasmid pSB24.1. The high degree of relatedness between the two plasmids allowed the construction of a genetic map of pSB24.2, consisting of putative transfer and replication loci. Two pSB24.2 loci, namely, the cis-acting locus for transfer (clt) and the transfer-associated korB gene, were shown to be capable of complementing the pIJ101 clt and korB functions, respectively, a result that is consistent with the notion that pIJ101 and the parental plasmid pSB24.1 encode highly similar, if not identical, conjugation systems.     

Mutations that affect regulation of the korB gene of Streptomyces lividans plasmid plJ101 alter plasmid transmission

Using the catechol dehydrogenase gene as a reporter, we isolated random mutations in the plJ101 korB gene operator/promoter (OP) region that affect korB expression and regulation. We mapped these mutations to inverted repeat sequences within the promoter and studied their effects on binding of the KorB repressor protein to the OP, on expression of the korB gene, and on plasmid transmission during mating. Additionally, we investigated the biological effects of KorB binding to a locus (sti, for st rong I ncompatibility) adjacent to the korB OP and implicated in plJ101 replication. Our results identify sites that influence the synthesis and autoregulation of KorB; they also show that interaction of KorB with sti affects repression of korB and transmission of plasmids to spores of recipients.