Plasmid SCP1 Research Articles

High-frequency homologous plasmid-plasmid recombination coupled with conjugation of plasmid SCP2* in Streptomyces.

Non-transmissible derivatives of the Streptomyces multi-copy plasmid plJ101 were mobilized, by cointegrate formation, at frequencies approaching 100% (measured per recipient) by derivatives of the conjugative, low-copy-number Streptomyces coelicolor A3(2) plasmid SCP2*. Efficient co-integrate formation required that the plasmids shared at least 112 bp sequence identity, and it occurred only during conjugation. An SCP2* plasmid gene is involved in the process. Co-integrates were presumably formed in the donor cells and transported to the recipient cells. This is a new phenomenon, not known in other bacteria.

Analysis of the transfer region of the Streptomyces plasmid SCP2.

pIJ903, a bifunctional derivative of the 31.4 kb low-copy-number, conjugative Streptomyces plasmid SCP2*, was mutagenized in Streptomyces lividans using Tn4560. Mutant plasmids differing in their transfer frequencies, chromosome mobilization abilities, pock formation, and complementation properties were isolated. The mutations defined five transfer-related genes, traA, traB, traC, traD and spd, clustered in a region of 9 kb. The deduced sequences of the putative TraA and TraB proteins showed no overall similarity to known protein sequences, but the phenotype of traA mutant plasmids and sequence motifs in the putative TraA protein suggested that it might be a DNA helicase.

Structural analysis of the giant linear plasmid SCP1 in various Streptomyces coelicolor strains

Summary: The methylenomycin biosynthetic gene cluster was previously shown to be present on the 350 kb giant linear plasmid SCP1. The structures of SCP1 in various Streptomyces coelicolor A3(2) strains were analysed by pulsed field gel electrophoresis (PFGE). S. coelicolor A3(2) strains 1147, M138 and M146, previously genetically characterized as SCP1+, contained the free form of SCP1. S. coelicolor 1984 and 2106, earlier characterized as SCP1′-cysB and a stable cysD donor respectively, were found to contain very large linear plasmids, 550 kb and 1700 kb in size. Strain JCM4979, another SCP1+ derivative of A3(2), had a series of SCP1-related linear plasmids of 390–630 kb. The integrated states of SCP1 in S. coelicolor SCP1-NF strains, an NF-like strain and a stable pabA donor were confirmed by PFGE, which revealed a lack of the intact SCP1 termini. In addition, a 70 kb linear plasmid was found in Streptomyces violaceoruber SANK95570, another methylenomycin producer. However, the plasmid was shown by hybridization to a methylenomycin resistance gene not to carry the methylenomycin gene cluster of this strain.

Physical and Genetic Characterization of Chromosomal Copies of the Streptomyces coelicolor Mini-circle

The two linear, integrated mini-circle copies of Streptomyces coelicolor A3(2) were cloned in Escherichia coli and their positions on the S. coelicolor genetic map were determined. Mini-circle copy A is close to the argA locus, 20 kb upstream of the gyl operon. Mini-circle copy B is close to the cysD locus and is absent from S. coelicolor J1501, which has suffered a chromosomal deletion including mini-circle copy B and possibly associated with the pgl mutation. The mini-circle copies were not involved in any of the several previously identified physical interactions between the plasmid SCP1 and the S. coelicolor chromosome. A new insertion sequence was identified close to the right end of mini-circle copy B in S. coelicolor M145. The free mini-circle of S. coelicolor, when inserted into an attP-deleted derivative of phage ϕC31, actively integrated this phage into the chromosomes of S. coelicolor and S. lividans at preferred and secondary sites. The resulting prophages were stably inherited and remained physically intact. No precise excision of prophages from S. lividans lysogens carrying insertions at preferred or secondary integration sites was detected: instead, free phages were generated by imprecise excisions. These phages allowed the in vivo cloning of segments (> 3 kb in length) of the chromosomal DNA flanking preferred and secondary integration sites. Attempts to delete the preferred integration site by double homologous recombination with a clone carrying flanking DNA sequences and an antibiotic resistance gene were unsuccessful.

A vector for studying plasmid stability functions in Streptomyces.

SummaryWe constructed a cloning vector (pMT603) based on the low copy number plasmid SCP2*. pMT6O3 is unstable because it lacks the SCP2* stability region and carries the selectable marker thiostrepton-resistance and a tyrosinase gene which results in melanin production. This allows easy testing of plasmid stability and we demonstrated its usefulness by cloning a plasmid stability function.

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.

Nucleotide sequence analysis reveals similarities between proteins determining methylenomycin A resistance in Streptomyces and tetracycline resistance in eubacteria

Previous studies had localised the gene ( mmr) for resistance to methylenomycin A (Mm) to a 2.5-kb PstI fragment in the middle of a cluster of Mm biosynthetic genes from the Streptomyces coelicolor plasmid SCP1. In this paper, the gene has been more precisely located by sub-cloning, and the nucleotide sequence of the whole fragment has been determined. The predicted mmr-specified protein ( M r 49238) would be hydrophobic, with some homology at the amino acid level to tetracycline-resistance proteins from both Gram-positive and Gram-negative bacteria. Comparisons of hydropathy plots of the amino acid sequences reinforces the idea that the proteins are similar. It is suggested that Mm resistance may be conferred by a membrane protein, perhaps controlling efflux of the antibiotic. No significant homology was detected by hybridisation analysis between mmr and a cloned oxytetracycline (OTc)-resistance gene ( tetB) of the OTc producer Streptomyces rimosus, and no cross-resistance was conferred by these genes. Sequences on both sides of mmr appear to encode proteins. The direction of translation in each case would be opposite to that of mmr translation. This suggests that mmr is transcribed as a monocistronic mRNA from a bidirectional promoter. An extensive inverted repeat sequence between the stop codons of mmr and the converging gene may function as a bidirectional transcription terminator.

SCP1-dependent Weigle activation of the VP5 phage in Streptomyces coelicolor A3(2)

The kinetics of induction of the UV-irradiated bacteriophage VP5 (Weigle reactivation) in Streptomyces coelicolor A3(2) strains with and without plasmid was investigated. Chloramphenicol (CAF) inhibits Weigle reactivation (WR) in UF strains (SCP1 absent) but not in SCP1 + strains of IF fertility (free plasmid). CAF, moreover, inhibits protein synthesis in non-irradiated UF and IF strains. In UV-irradiated IF strains, on the other hand, protein synthesis takes place irrespective of CAF. Weigle reactivation appears to require protein synthesis: the SCP1 plasmid, by protecting synthesis from CAF inhibition in UV-irradiated strains, allows WR. The proteins synthesized after UV induction during the pre-incubation period were investigated and the results suggest that a new UV-induced protein, coded by a gene localized on the plasmid, interacts with the cellular SOS system.

The minimal replicon of a Streptomycete plasmid produces an ultrahigh level of plasmid DNA

A functional map of Streptomyces coelicolor plasmid SCP2 ∗ was deduced from derivatives constructed by in vitro deletions. Functions were analyzed on bifunctional shuttle plasmids that contained pBR322 for selection and replication in Escherichia coli and fragments of SCP2 ∗ for replication in Streptomyces griseofuscus C581 and strains of Streptomyces lividans. The aph gene for neomycin resistance from Streptomyces fradiae and the tsr gene for thiostrepton resistance from Streptomyces azureus were incorporated as selectable antibiotic resistance markers in streptomycetes. An 11.8-kb sequence bounded by EcoRI and KpnI restriction sites contains the information for self-transfer and normal replication of the plasmid. A 5.9-kb EcoRI- SalI fragment contains all of the information for normal replication. Partial digestion generated a 2.2-kb Sau3A fragment that is sufficient for replication but it produces ten times higher plasmid copy number than the basic replicon. pHJL400 and PHJL401 are useful shuttle vectors containing the moderate-copy-number streptomycete plasmid combined with the E. coli plasmid pUC19. A 1.4-kb BclI- Sau3A fragment with an additional internal BclI site contains the minimal replicon but it produces 1000 times higher plasmid copy number than the basic replicon. pHJL302 is a useful shuttle vector containing the ultrahigh-copy-number streptomycete plasmid combined with the E. coli plasmid pUC 19.

Identification of a DNA sequence associated with plasmid integration in Streptomyces coelicolor A3(2).

We identified a DNA element of length about 1 kb that is present in two copies in the chromosome of Streptomyces coelicolor A3(2) and is also present on the plasmid SCP1 which has been carefully defined genetically, but never isolated as extrachromosomal DNA. A copy of the element is close (within 5 kb) of a gene coding for an extracellular agarase in the chromosome of S. coelicolor A3(2) and in an NF strain, in which SCP1 has integrated into the chromosome, the agarase gene has been deleted. The element has properties reminiscent of Insertion Sequences in Escherichia coli, but it is not yet know if it can transpose.

Resistance, regulatory and production genes for the antibiotic methylenomycin are clustered.

At least 17 kb of DNA from the large unisolatable Streptomyces coelicolor A3(2) plasmid SCP1 are concerned with methylenomycin biosynthesis. Mutational cloning analysis, using insert-directed integration of att site deleted phage vectors into an SCP1-containing host, provided evidence of two large transcription units, of at least 6.6 kb and 9.5 kb. At the leftmost apparent end of the larger (left-hand) transcription unit is a region apparently involved in negative regulation of methylenomycin biosynthesis: when fragments from this region were used to direct phage integration, marked overproduction of methylenomycin resulted. The methylenomycin resistance determinant is located at the rightmost end of this same transcription unit. Hybridisation analysis with 13 kb of the cloned mmy region showed that it was closely similar to a segment of pSV1, a plasmid that specifies methylenomycin biosynthesis in S. violaceus-ruber SANK 95570.

The Streptomyces plasmid SCP2∗: its functional analysis and development into useful cloning vectors

Detailed restriction maps of the plasmid SCP2* and its deletion derivative pSCP103 were constructed. DNA fragments carrying hygromycin (Hyg), thiostrepton (Thio) or viomycin-resistance (Vio11) determinants were inserted into pSCP103, and various segments were deleted from the resulting plasmids. Changes in plasmid phenotypes associated with these insertions and deletions allowed the localisation and characterisation of plasmid replication, stability, transfer and fertility functions. Several useful cloning vectors were constructed. They are able to maintain large (> 30 kb) DNA inserts, with stable inheritance at a low copy number (1–2 per chromosome) and without structural rearrangements, in Streptomyces hosts. The vectors have a broad host range in the genus Streptomyces. One of them (pIJ903) is a shuttle vector for Streptomyces and Escherichia coli.

Shuttle vectors for cloning recombinant DNA in Escherichia coli and Streptomyces griseofuscus C581

The replicon of the Streptomyces plasmid SCP2 was located on a 5.9-kilobase EcoRI-SalI restriction fragment. The SCP2 replicon was combined with Escherichia coli plasmid pBR322 and genes specifying neomycin resistance and thiostrepton resistance in streptomycetes to construct shuttle vectors that are useful for cloning in E. coli and streptomycetes.

The restriction mapping of c gene deletions in Streptomyces bacteriophage φ C31 and their use in cloning vector development

In addition to 20 previously mapped restriction sites in the DNA of φ C31, we have determined eight sites for SphI, four for Eco RV, and two for SstII; there are none for BglII or SstI. Nine sites were in a 12-kb segment of DNA containing no previously mapped sites. Deletions causing clear-plaque morphology were located in this part of the DNA, in a 3-kb interval between an EcoRV and an SphI site at the centre of the DNA molecule. One of the deletions (ΔC3) was obtained in a previously described φ C31 c +:: vph (viomycin phosphotransferase) derivative containing two PstI sites separated by 3.9-kb of inessential DNA. After in vitro PstI treatment, plaque-forming phages lacking the 3.9-kb fragment were obtained from the c + phage but not from its ΔC3 derivative. Thus a 36.2-kb genome, but not one of 34.4 kb, was able to give infectious virions. PstI-generated DNA fragments of up to 8 kb can be inserted in vitro into the ΔC3 derivative with retention of the vph selective marker. With the insertion of a 6.03-kb Pst I fragment of plasmid SCP2, the latter phage became a potential vector (with loss of vph) for BamHI-generated DNA fragments of up to 9kb. In the course of this work, several ClaI sites in φ C31:: pBR322 bifunctional replicons were shown to be lost when the DNA was propagated in a dam + Escherichia coli strain. This will allow the use of such replicons for the cloning of ClaI-generated DNA fragments of up to 6.7 kb.

Determination of methylenomycin A synthesis by the pSV1 plasmid from Streptomyces violaceus-ruber SANK 95570.

A plasmid (pSV1) of 110 x 10(6) daltons from a methylenomycin A producing strain of Streptomyces violaceus-ruber was detected on an isolated from agarose gels. Elimination of this plasmid by protoplasting and regeneration resulted in the simultaneous loss of methylenomycin A production and resistance. pSV1 hybridized with pBR322 containing a cloned fragment of 1.7 x 10(6) daltons from S. coelicolor A3(2) which codes for methylenomycin A resistance. The pSV1 plasmid could be transferred to S. lividans by conjugation and by transformation and plasmid DNA identical in size to pSV1 could be isolated from the recipient strains. These experiments show that pSV1 codes for methylenomycin A production and resistance, in close analogy to the SCP1 plasmid from S. coelicolor A3(2).

Plasmid-dependent co-mutation in Streptomyces coelicolor A3(2)

A spontaneous chromosomalmutation(plc A(-)) in Streptomyces coelicolor A3(2) made the inborn plasmid SCP1 susceptible to curing by UV irradiation. Lack of the SCP1 plasmid (in SCP1(-) strains) prevented the occurrence of the co-mutation process after MNNG treatment, although it left the susceptibility to the lethal effect of the mutagen virtually unaffected. SCP1(-) strains were, however, ultrasensitive to the lethal effect of UV. Curing a plc A(-) strain of its SCPI plasmid made it refractory to co-mutation by MNNG and sensitive to the lethal effect of UV; reinfected by the plasmid, the strain resumed both the co-mutation proficiency and the UV-resistance. The occurrence on the SCP1 plasmid of a gene comparable to the uvrE gene of E. coli (Nevers and Spatz 1975) was assumed.

Genetic Studies of the Fertility Plasmid SCP2 and its SCP2* Variants in Streptomyces coelicolor A3(2)

The plasmid SCP2, initially discovered through the occurrence of a high fertility variant, SCP2*, is a self-transmissible fertility factor capable of promoting chromosomal recombination within Streptomyces coelicolor A3(2). Further high fertility variants of SCP2, similar to SCP2*, were isolated from amongst the recombinants produced in matings involving SCP2, and their genetic properties were compared. SCP2 and its derivatives elicit lethal zygosis on transfer into an SCP2- recipient; this plasmid-determined phenotype allowed the isolation of SCP2- strains and the detection of the interspecific transfer of SCP2* by mating from S. coelicolor to Streptomyces parvulus and Streptomyces lividans, whereupon it underwent stable maintenance. The transfer genes of SCP2 and SCP2*, which are not normally fully expressed, were shown to undergo transient derepression on entry into an SCP2- strain. An ‘entry disadvantage’ system determined by SCP2 and SCP2* was recognized.

A Chromosomal Locus Controlling Extracellular Agarase Production by Streptomyces coelicolor A 3(2), and its Inactivation by Chromosomal Integration of Plasmid SCP1

The properties of various mutants showed that, in the presence of an inorganic nitrogen source, agar utilization by Streptomyces coelicolor requires the action of at least three groups of enzymes: a diffusible or cell-free extracellular agarase, some further degradative step(s), and the enzymes of galactose metabolism. Two unselected mutations (dag), both leading to loss of diffusible agarase and an undefined second agar utilization function, were detected among a small number of S. coelicolor derivatives from a culture collection, and mapped to the 9 o’clock ‘silent’ region of the genetic map. One of these mutations (dagA1) had apparently resulted, directly or indirectly, from the insertion of the plasmid SCP1 in this region during formation of the NF fertility type. The other (dag-2) was 100% linked to dagA1 and the integrated SCP1, but only 98% linked to the site of insertion of SCP1 in an ‘NF-like’ but Dag+ strain. SCP1-chromosome interactions in other regions of the genetic map had no effect on the agar-solubilizing phenotype, nor had three other unstable genetic phenomena in S. coelicolor.

Fertility properties and regulation of antimicrobial substance production by plasmid SCP2 of Streptomyces coelicolor.

Streptomyces coelicolor A3(2) possesses two plasmids (SCP1 and SCP2) that act as sex factors. The plasmid deoxyribonucleic acid isolated from S. coelicolor A3(2) SCP1- strains A617 and A585 had the same molecular weight and endonuclease cleavage pattern as the SCP2 plasmid. The plasmidless strain S18 SCP2- was isolated from the A617 X A585 cross. SCP2 plasmid-containing strains acted as donors of chromosomal markers, whereas the plasmidless strain acted as recipient. The transfer of SCP2+ donor strain markers into the SCP2- recipient occurred at high frequencies (approximately 75%), was unidirectional, was initiated from a fixed region of the chromosome, and had the SCP2 fertility factor transferred first. The introduction of the SCP2 plasmid into a recipient strain greatly reduced the recombination frequency. These fertility properties differed from those previously reported, thereby suggesting that the SCP2 plasmid examined in this investigation may be an additional variant to those described in the literature. The SCP2 plasmid also regulated production of three antibacterial substances and conveyed resistance for S. coelicolor A3(2) strains against growth inhibition by one of them.

Genetic determination of methylenomycin synthesis by the SCP1 plasmid of Streptomyces coelicolor A3(2).

Evidence is presented that genes determining the pathway of methylenomycin A synthesis are carried on the SCP1 plasmid. All 16 mutations (mmy) leading to lack of antibiotic synthesis were SCP1-linked. Phenotypic classification, by co-synthesis and other criteria, suggested that they fell into at least five classes. When the wild-type SCP1 plasmid was transferred to Streptomyces lividans or Streptomyces parvulus, material that was chromatographically and biologically indistinguishable from methylenomycin A was produced. Recombination between some pairs of mmy mutations was detected. In crosses of mmy mutants of NF (integrated SCP1 donor) strains with SCP1-, a very high frequency of chromosomal recombination occurred; thus methylenomycin production appears not to be an important cause of the ultra-fertility normally associated with NF X SCP1- crosses.