Streptomyces Plasmid Research Articles

Development of pLR591, a Streptomyces-Escherichia coli positive selection shuttle vector.

The Escherichia coli positive selection vector pEcoR251 was ligated with the broad host range, high copy number Streptomyces plasmid pIJ702 to produce pLR591, a Streptomyces-E. coli positive selection shuttle vector. The EcoRI and thiostrepton resistance genes of pLR591 were expressed in E. coli and Streptomyces lividans respectively. The positive selection shuttle vector pLR591 facilitates the construction in E. coli of genomic libraries which can be screened in Streptomyces strains.

Isolation and nucleotide sequencing of an aminocyclitol acetyltransferase gene from Streptomyces rimosus forma paromomycinus.

A gene (aacC7) encoding an aminocyclitol 3-N-acetyltransferase type VII [AAC(3)-VII] from Streptomyces rimosus forma paramomycinus NRRL 2455 was cloned in the Streptomyces plasmid pIJ702 and expressed in Streptomyces lividans 1326. Subcloning experiments located the aacC7 structural gene on a 1.05-kilobase DNA sequence. The direction of transcription of aacC7 was determined by using riboprobes synthesized in vitro from a DNA fragment internal to the gene. A DNA segment encoding the AAC(3)-VII activity and comprising 1,495 base pairs was sequenced. The aacC7 gene was located in an open reading frame of 864 base pairs that encoded a polypeptide of Mr 31,070, consistent with the Mr (32,000) of the AAC(3)-VII enzyme as determined by physicochemical methods. High-resolution S1 nuclease mapping suggested that transcription starts at or near the A residue of the ATG initiator codon. A DNA fragment from the 5' region of aacC7 had promoter activity in the promoter-probe plasmid pIJ486. The -10 and -35 regions of this fragment showed limited sequence resemblance to other Streptomyces promoters. The primary structure of the AAC(3)-VII enzyme showed strong homology with those of the AAC(3)-III and AAC(3)-IV enzymes encoded by plasmids in gram-negative bacterial genera. Upstream of the aacC7 gene was an open reading frame of 357 nucleotides which did not appear to be involved in controlling the expression of the aacC7 gene.

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.

Transposon Tn4556 of Streptomyces fradiae: nucleotide sequence of the ends and the target sites.

A transposon, Tn4556, has recently been isolated from Streptomyces fradiae (S.-T. Chung, J. Bacteriol. 169:4436-4441, 1987). The ends of Tn4556 were found to contain inverted repeats of 38 base pairs with 70% sequence identity with the ends of Tn3. Insertion of Tn4556 into a Streptomyces plasmid resulted in a 5-base-pair duplication of the target site.

Genetic transformation of Micromonospora rosaria by the Streptomyces plasmid pIJ702.

Genetic transformation of Micromonospora rosaria by the Streptomyces plasmid pIJ702.

Cloning of whiG, a gene critical for sporulation of Streptomyces coelicolor A3(2).

In whiG mutants of Streptomyces coelicolor A3(2), aerial hyphae do not show any sign of sporulation. A library of S. coelicolor DNA was prepared in a phi C31 temperate phage vector (KC516), and one recombinant phage (KC750) that could restore the wild-type phenotype to a collection of whiG mutants when integrated into their genomes was found. Subcloning experiments with low- and high-copy-number Streptomyces plasmid vectors allowed partial localization of whiG in the cloned DNA and revealed that hypersporulation was associated with the presence of extra copies of whiG.

The Streptomyces ghanaensis low copy plasmid pSG2 and its use for vector construction.

A plasmid, pSG2, was isolated from Streptomyces ghanaensis and characterized by electron microscopy, buoyant density measurement, and restriction enzyme analysis. The length of 13.8 kb, single restriction sites for HindIII, EcoRV and PvuII and the possibility of deleting non-essential regions of the plasmid made pSG2 a suitable basic replicon for vector development. pSG2 has a copy number of about four. Plasmid pSG2 was fused to a pACYC184 derivative modified to harbour a thiostrepton resistance gene. The resulting plasmid, designated pSW1, is a 16.6 kb shuttle plasmid which replicates in Escherichia coli and in several Streptomyces strains, including S. ghanaensis, S. lividans and S. viridochromogenes. Replacement of a BglII-fragment of plasmid pSG2 by a fragment encoding thiostrepton resistance resulted in a low copy 12.2 kb Streptomyces plasmid. This plasmid, designated pSW2, is a Streptomyces broad host range plasmid.

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

The 8.9-kilobase Streptomyces plasmid pIJ101 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 pIJ101-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 kilA, 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.

Giant linear plasmids in Streptomyces which code for antibiotic biosynthesis genes.

A number of examples of circular plasmids with specific functions are known in both prokaryotes and eukaryotes. Several linear plasmids have also been identified, but these are all relatively small: large linear plasmids cannot be separated from chromosomal DNA by conventional techniques. There are several cases where the genetic evidence suggests that a character is encoded by a plasmid but no plasmid can be physically detected. This has been the case for antibiotic synthesis genes in Streptomyces; in particular a plasmid SCP1 in Streptomyces coelicolor has been shown to be involved in methylenomycin production by genetic evidence. We report here the application of orthogonal-field-alternation gel electrophoresis to the isolation of linear plasmids from Streptomyces. We have discovered a large linear plasmid of around 520 kilobases in Streptomyces lasaliensis and subsequently similar giant linear plasmids in other Streptomyces strains. We have confirmed that genes for methylenomycin biosynthesis are located on a series of giant linear plasmids in S. coelicolor. These observations may bear on the genetic variability and unstable genetic character of Streptomyces species.

Efficient transformation of Amycolatopsis orientalis (Nocardia orientalis) protoplasts by Streptomyces plasmids.

Conditions for efficient transformation of Amycolatopsis orientalis (Nocardia orientalis) protoplasts by Streptomyces plasmid cloning vectors were identified. Three streptomycete plasmid origins of replication function in A. orientalis, as do the apramycin resistance gene from Escherichia coli, the thiostrepton resistance gene from Streptomyces azureus, and the tyrosinase gene from Streptomyces antibioticus. A. orientalis appears to express some restriction and modification, because highest transformation frequencies (10(6)/micrograms of DNA) were obtained when plasmid pIJ702 was modified by passage in A. orientalis.

Characterization of the incompatibility region of Streptomyces plasmid pSL1

The incompatibility region of the Streptomyces plasmid pSL1 was identified as a 240-bp segment, though some other function from the vector plasmid was also necessary. A 540-bp fragment including the 240-bp region was enough for full expression of incompatibility. Inserted mutation analysis led to a more detailed location of the region essential for replication.

Identification of the minimal replication region of the multicopy Streptomyces plasmid pSL1

The 1.52-kb minimal replication origin of the 3.9-kb Streptomyces plasmid pSL1 was determined using a bifunctional derivative, pMCP44, of pSL1. Plasmids with linker insertions into the pSL1 part of pMCP44 were isolated from Escherichia coli. The sites of insertion were determined by restriction enzyme analysis and the ability of the mutant plasmids to replicate in S. lividans 66 was determined. All except one of the inserts in the 1.52-kb essential region inactivated replication. A 104-bp segment from this region could function as a replication origin in the presence of a helper plasmid containing a nonoverlapping pSL1 fragment. The sequence of this 104-bp fragment shows similarities to those of known plasmid replication origins.

9] Plasmid and phage vectors for gene cloning and analysis in Streptomyces

This chapter presents a discussion on plasmid and phage vectors for gene cloning and an analysis in Streptomyces . The chapter presents a detailed bench-level account of typical shotgun cloning experiments using a plasmid and a phage vector and catalogs and discusses a representative selection of all the tried and tested vectors. The chapter includes some promoter-probe vectors and general cloning vectors. Because commercially important Streptomyces antibiotics are produced by derivatives of different soil isolates, a wide range of hosts is used in genetic studies. However, the strain of Streptomyces coelicolor A3(2) that produces four antibiotics is preeminent as a model system. The chapter discusses Streptomyces plasmids, the isolation of chromosomal DNA from S. coelicolor A3(2), minilysate procedure for the analysis of Streptomyces plasmids and others.

Optimization of gene expression in Streptomyces lividans by a transcription terminator.

The ability of an inverted repeat sequence (IRS) from the 3' end of the aph gene from Streptomyces fradiae to induce transcription termination in vivo has been examined. As a model system, a DNA fragment encoding the human interferon alpha 2 inserted in the Streptomyces plasmid pIJ702 was used. When the IRS was inserted downstream from this sequence and transcription assayed in Streptomyces lividans, highly efficient (approximately 90%) transcription termination was observed occurring immediately after the 3' terminus of the dyad. In contrast, gene constructions lacking the IRS transcribed longer mRNAs. Moreover, the IRS gave rise to increased amounts of the hIFN alpha 2 suggesting that the putative stem-loop structure stabilised the transcript.

Two promoters from the Streptomyces plasmid pIJ101 and their expression in Escherichia coli

An RNA polymerase-binding restriction fragment from the small, high-copy-number Streptomyces plasmid pIJ101 has been shown to have promoter activity in vivo using a promoter-probe vector. The nucleotide sequence of the promoter (the pIJ101B promoter) and the approximate position of the transcription start point as identified by in vitro run-off transcription are presented. Both the pIJ101B promoter and the previously characterised pIJ101A promoter were found to promote transcription in Escherichia coli. The transcription start point in E. coli for the pIJ101A promoter has been determined using high-resolution S1 mapping. Initiation occurs at the same point or within 1 or 2 nucleotides of the transcription start point previously identified in Streptomyces lividans, indicating that the same transcriptional signals are recognised in both genera. The data support the idea that one type of RNA polymerase holoenzyme in Streptomyces recognises a class of promoters similar to the major consensus promoters of E. coli, and that the manner of promoter recognition is similar in both genera.

Development of a plasmid-cloning system for Streptomyces viridochromogenes Tü494.

A plasmid-cloning system was developed for Streptomyces viridochromogenes Tü494, a producer of the tripeptide antibiotic phosphinothricyl-alanyl-alanine (PTT). Parameters affecting protoplast formation and transformability of S. viridochromogenes were investigated in detail. A procedure giving rise to transformation efficiencies of 10(4)-10(5) transformants per microgram DNA was worked out. Several Streptomyces plasmid vectors such as pIJ350, pIJ61, pEB2, pGM4 and pSW2 were tested in S. viridochromogenes. Some of these vectors (pGM4, pEB2) showed a high copy number, whereas the copy number of others (pIJ350, pIJ61 and pSW2) was markedly lower. Under non-selective conditions some of the vectors (pIJ350, pEB2) were not stably maintained, in contrast to the vectors pGM4 and pSW2 which did not require any selection pressure for stable maintenance. Therefore, the plasmid vectors pGM4 and pSW2 derived from endogenous replicons of Streptomyces ghanaensis strains represent appropriate plasmid vehicles for cloning experiments in S. viridochromogenes.

Structural stability of heterologous genes cloned in Streptomyces plasmid pIJ702.

A recombinant plasmid pWCL1 containing Streptomyces plasmid pIJ702, E. coli plasmid pUC12, and hepatitis B viral surface antigen (HBsAg) gene was stably maintained in E. coli, but exhibited structural instability in S. lividans 1326. The deletions were found ranging from 2.75 to 5.65 kilobases (kb) and most of them occurred within the melanin (mel) gene of pIJ702, resulting in the loss of part of the mel gene sequence plus the insert. The removal of the pUC12 sequence from pWCL1 eliminated the instability. However, pUC12 alone inserted in either orientation on pIJ702 also caused the deletion in S. lividans 1326. The results indicated that the structural instability of hybrid plasmid of pIJ702 depended on the interaction between the mel sequence and the inserted sequence.

A 2.6 kb DNA sequence of Streptomyces coelicolor A3(2) which functions as a transposable element

Streptomyces coelicolor A3(2) contains CCC DNA molecules, 2.6 kb in size, with an average copy number of less than one per ten chromosomes. Southern hybridisation revealed, in addition, two linear, integrated copies (A and B) of this "mini-circle" sequence per chromosome. The two integrated copies have similar (if not identical) ends and are present in the same locations in various S. coelicolor A3(2) derivatives. The mini-circle sequence is absent from S. lividans 66 and S. violaceolatus ISP5438 and from several Streptomyces species less closely related to S. coelicolor A3(2). None of a variety of Streptomyces plasmids tested contained homology to the mini-circle sequence. When a 1.8 kb fragment of the mini-circle lacking the ends of the integrated copies was inserted into KC515 (a derivative of the temperate phage phi C31 which is unable to lysogenise host strains by the natural route because the phage attachment site has been deleted) the resulting phage lysogenized S. coelicolor A3(2) (integrating into the genome of this host by homologous recombination with resident minicircle sequences) but not S. lividans or a variety of other phi C31 hosts. In contrast, a KC515 derivative (KC591) carrying the entire 2.6 kb mini-circle sequence linearized at its single BclI site (and therefore containing the integration site of the free mini-circle) lysogenized not only S. coelicolor A3(2) but also S. lividans 66 and most other strains normally lysogenized by phi C31. The KC591 lysogens of the eight Streptomyces species tested contained a linear, integrated prophage with termini apparently identical to those of the linear mini-circle copies of S. coelicolor.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of a Streptomyces plasmid promoter in Escherichia coli

A 166-bp DNA fragment from the Streptomyces multicopy plasmid pIJ 101 with in vivo promoter activity both in Streptomyces lividans and in Escherichia coli was isolated. The start point of the RNA transcribed from this fragment, determined by high resolution S1 nuclease mapping, was the same in S. lividans and in E. coli. This suggests that the E. coli RNA polymerase recognizes the same sequence determinants and chooses the point of initiation of RNA synthesis in the same way as the corresponding S. lividans enzyme. The putative promoter sequence shows good homology to the E. coli promoter consensus sequence in the ‘−35’ region but poor homology in the ‘−10’ region.

Cloning and expression in biologically active form of the gene for human interferon α2 in Streptomyces lividans

A fragment of human DNA encoding the mature form of interferon α2(hIFN-α2), and carrying both an in-phase ATG initiation codon and the ribosome binding site (RBS)of the Escherichia colimembrane lipoprotein gene( lpp)was fused to the aminoglycoside phosphotransferase gene( aph) promoter ( aphP) from Streptomyces fradiae. When this construction was inserted, in the two possible orientations, in the Streptomyces plasmid pIJ702, plasmids pNIS19 and pNIS91 were obtained. A 20-kDa polypeptide that immunoreacted with an hIFN-α2 monoclonal antibody was expressed in S.lividans clones carrying these plasmids. Moreover, these clones contained an intracellular antiviral activity similar to that of hIFN-a2. When plasmids pNIS19 and pNIS91 were deprived of the aphpno expression of activity was found. Therefore,it is concluded that the hIFN gene can be efficiently expressed in Streptomyces as directed by the aph gene promoter.