Post-transformational rearrangement of an in vitro reconstructed group-A streptococcal erythromycin resistance plasmid

Cleavage of the group-A streptococcal macrolide, lincosamide, and streptogramin B (MLS) resistance plasmid pSM19035 yields 2 fragments [13 and 4 megadaltons (MD)] with EcoRI, and 15 fragments with HindIII, 12 of which are 6 pairs of identical fragments derived from the inverted repeats that comprise about 80% of the pSM19035 genome. The large EcoRI fragment was isolated, ligated, and used to transform the Challis strain of Streptococcus sanguis to erythromycin resistance. Plasmids (pDB101, pDB102, and pDB103) isolated from three different transformants had lower molecular masses than the original large EcoRI fragment. HindIII digestion of these molecules and subsequent analysis of fragment radioactivity distributions indicated the loss of plasmid segments of various sizes. The deletions, all of which occurred in the palindrome, did not affect the level and the inducible nature of pSM19035-determined antibiotic resistance. Only pDB101 retained the unique EcoRI cleavage site. The results of this analysis allowed the construction of an EcoRI and HindIII cleavage-site map of pSM19035 and promise to simplify future studies of genetic functions specified by streptococcal MLS resistance plasmids.     

Involvement of the ftsA gene product in late stages of the Escherichia coli cell cycle.

The erythromycin resistance determinant of plasmid pDB102, a derivative of plasmid pSM19035, was cloned into the single HindIII site of the 3.6-megadalton cryptic Streptococcus mutans plasmid pVA318 and introduced into Streptococcus sanguis strain Challis by transformation. Plasmid pDB201, which was isolated from one of the transformants, consisted of the vector plasmid and the 1.15-megadalton HindIII fragment D of pSM19035. HindIII fragment D contained within it one of the two unique "spacer" sequences of pSM19035. Electron micrographs of self-annealed molecules of the recombinant plasmid revealed classical stem-loop structures, and the resistance determinant of pSM19035 appeared as a transposon-like structure. No differences were observed in either the type or the level of erythromycin resistance by pSM19035 or pDB201. The availability of a cloned erythromycin resistance determinant should be useful for future comparative studies of macrolide, lincosamide, and streptogramin B resistance plasmids in streptococci.     

Double or triple sets of replication functions as inverted and direct repeats on in vitro reconstructed streptococcal MLS resistance plasmids

In vitro rearrangement of plasmid pDB102 together with comparative studies of other streptococcal plasmids allowed the localization of replication and copy control functions on sequences which were present on pDB102 and its naturally occurring ancestor pSM19035 as duplicates in inverted orientation. Evidence is presented that neither the presence of duplicate replication regions nor their arrangement in inverted orientation was essential for plasmid survival. Among the in vitro reconstructed plasmids were several that stably carried two or three sets of replication and copy control functions either as inverted or direct repeats or both. A copy control mutation is described which led to a tenfold increase of copy number over that of the naturally occurring plasmid pSM19035.     

Functional Analysis of Three Plasmids from Lactobacillus plantarum

Lactobacillus plantarum WCFS1 harbors three plasmids, pWCFS101, pWCFS102, and pWCFS103, with sizes of 1,917, 2,365, and 36,069 bp, respectively. The two smaller plasmids are of unknown function and contain replication genes that are likely to function via the rolling-circle replication mechanism. The host range of the pWCFS101 replicon includes Lactobacillus species and Lactococcus lactis, while that of the pWCFS102 replicon also includes Carnobacterium maltaromaticum and Bacillus subtilis. The larger plasmid is predicted to replicate via the theta-type mechanism. The host range of its replicon seems restricted to L. plantarum. Cloning vectors were constructed based on the replicons of all three plasmids. Plasmid pWCFS103 was demonstrated to be a conjugative plasmid, as it could be transferred to L. plantarum NC8. It confers arsenate and arsenite resistance, which can be used as selective markers.     

Recombination between the linear plasmid pPZG101 and the linear chromosome of Streptomyces rimosus can lead to exchange of ends

The 387 kb linear plasmid pPZG101 of Streptomyces rimosus R6 can integrate into the chromosome or form a prime plasmid carrying the oxytetracycline biosynthesis cluster. The integration of plasmid pPZG101 into the linear chromosome of S. rimosus R6-501 in mutant MV25 was shown to be due to a single cross-over at a 4 bp common sequence. pPZG101 had integrated into a 250 kb DNA sequence that was reiterated at a low level. This sequence includes the oxytetracycline biosynthesis cluster, so that homologous recombination generated a mixed population carrying different copy numbers of the region. The 1 Mb linear plasmid pPZG103 in mutant MV17 had also arisen from a cross-over between pPZG101 and the chromosome, so that one end of pPZG103 consists of c . 850 kb of chromosomal sequence including the oxytetracycline biosynthesis cluster. The plasmid pPZG101 was shown to consist of a unique central region of about 30 kb flanked by terminal inverted repeats of about 180 kb. Analysis of a presumed ancestor plasmid pPZG102 suggested that the long terminal repeats had arisen by a recombination event during the strain development programme.     

In vivo linearization and autonomous replication of plasmids containing human telomeric DNA in Aspergillus nidulans

Plasmids containing two inverted 0.6-kb stretches of human telomeric repeats transform Aspergillus nidulans at frequencies characteristic of autonomously replicating vectors. Transformation frequency is not affected when the plasmids are linearized in vitro prior to transformation by cutting between the inverted repeats. Southern analysis reveals the presence of a homogeneous pool of linear plasmid molecules in mycelium of transformants. Addition of the AMA1 plasmid replicator to the telomere-containing plasmids has only a minor effect on transformation. The phenotypic stability of the transformants is low. However, unlike conventional replicative transformants containing AMA1-bearing plasmids, these transformants are prone to spontaneous stabilization which occurs predominantly by conversion of the mutant chromosomal allele of the marker gene to the plasmid-borne allele. The data strongly suggest that telomeric DNA can act as a plasmid replicator. An alternative interpretation is that autonomous replication of linear DNA fragments, in contrast to covalently closed supercoiled molecules, does not require any special replicator sequences.     

Sequence organization and expression of a yeast plasmid DNA.

Saccharomyces cerevisiae strain A364A D5 contains circular double-stranded DNA molecules of 6230 +/- 30 base pairs (2mu DNA) which are present in 68 copies per cell and make up 2.4% of the haploid genome. About 0.4% of non-poly A containing yeast RNA hybridizes to the yeast DNA circles. When denatured and then self-annealed, the DNA molecules assume a characteristic "dumbbell" shape in the electron microscope indicating that each circle possesses a non-tandem inverted repeat sequence of 630 +/- 10 base pairs. Eco-RI digestion of purified 2mu DNA yields 4 fragments on an agarose gel whose combined molecular mass is twice that of the monomer circle, suggesting that there are 2 populations of circles, each of the same molecular weight. Representatives of each population have been separated by cloning in Escherichia coli via the bacterial plasmid pSC101. Heteroduplex analysis of the cloned circles show that the 2 different populations arise because of intramolecular recombination between the inverted repeat sequences. Acrylamide gel patterns of polypeptides synthesized in bacterial mini-cells containing the hybrid plasmids between 2mu DNA and pSC101 are significantly different than the pattern obtained from mini-cells containing pSC101 alone.     

A novel type of transposon generated by insertion element Is102 present in a psc101 derivative

We describe a novel type of transposon in the tetracycline resistance plasmid pYM103, a derivative of pSC101 carrying a single copy of an insertion element IS102. The new transposons we found were identified as DNA segments, approximately 6 kb (Tn1021) and 10 kb (Tn1022) in length, able to mediate the cointegration of pYM1O3 with plasmid Col E1. The resulting cointegrate contains either of these pYM1O3 segments duplicated in a direct orientation at the junctions of the parent plasmids. A direct duplication of a 9 bp sequence at the target site in Col E1 is found at the junctions for cointegration. Both transposons have IS1O2 at one end and also contain different lengths of the pYM103 DNA adjacent to IS102, including the tetracycline resistance gene. Each transposon contains terminal inverted repeats of a short nucleotide sequence. These results and the fact that IS102 can itself mediate plasmid cointegration, giving rise to a duplication of a 9 bp target sequence, indicate that IS102 is responsible for generation of Tn1021 and Tn1022. They are quite different from the common IS-associated transposons, which are always flanked by two copies of an IS element, and may be similar to transposons such as those of the Tn3 family and phage Mu.     

Characterization of the ColE1-Km plasmids pCR1 and pCR11 by electron microscope and restriction endonuclease mapping.

The ColE1-Km plasmids pCR1 and pCR11 have been characterized by electron microscope techniques. Their sizes have been determined to be 13.1 and 9.2 kb, respectively, and heteroduplex studies show that the plasmids differ in the presence of a 3.9 kb deletion in the ColE1 region of pCR11. Both contain a nontandem inverted duplication of a 1.06 kb sequence. The single HindIII site, 3.5 kb from the EcoR1 site, lies in the 0.97 kb of DNA between the inverted repeat sequences. Since DNA insertions at the HindIII site destroy kanamycin resistance, it can be concluded that the kanamycin phosphotransferase gene is contained in this region. Electron microscopy of self-annealed plasmids treated with restriction endonucleases shows that each inverted duplication sequence contains one HindII site and at least two SmaI sites.     

In vivo linearization and autonomous replication of plasmids containing human telomeric DNA in Aspergillus nidulans

Plasmids containing two inverted 0.6-kb stretches of human telomeric repeats transform Aspergillus nidulans at frequencies characteristic of autonomously replicating vectors. Transformation frequency is not affected when the plasmids are linearized in vitro prior to transformation by cutting between the inverted repeats. Southern analysis reveals the presence of a homogeneous pool of linear plasmid molecules in mycelium of transformants. Addition of the AMA1 plasmid replicator to the telomere-containing plasmids has only a minor effect on transformation. The phenotypic stability of the transformants is low. However, unlike conventional replicative transformants containing AMA1-bearing plasmids, these transformants are prone to spontaneous stabilization which occurs predominantly by conversion of the mutant chromosomal allele of the marker gene to the plasmid-borne allele. The data strongly suggest that telomeric DNA can act as a plasmid replicator. An alternative interpretation is that autonomous replication of linear DNA fragments, in contrast to covalently closed supercoiled molecules, does not require any special replicator sequences. Received: 13 January 1998 / Accepted: 10 June 1998     

The genetic organization and evolution of the broad host range mercury resistance plasmid pSB102 isolated from a microbial population residing in the rhizosphere of alfalfa

Employing the biparental exogenous plasmid isolation method, conjugative plasmids conferring mercury resistance were isolated from the microbial community of the rhizosphere of field grown alfalfa plants. Five different plasmids were identified, designated pSB101–pSB105. One of the plasmids, pSB102, displayed broad host range (bhr) properties for plasmid replication and transfer unrelated to the known incompatibility (Inc) groups of bhr plasmids IncP-1, IncW, IncN and IncA/C. Nucleotide sequence analysis of plasmid pSB102 revealed a size of 55 578 bp. The transfer region of pSB102 was predicted on the basis of sequence similarity to those of other plasmids and included a putative mating pair formation apparatus most closely related to the type IV secretion system encoded on the chromosome of the mammalian pathogen Brucella sp. The region encoding replication and maintenance functions comprised genes exhibiting different degrees of similarity to RepA, KorA, IncC and KorB of bhr plasmids pSa (IncW), pM3 (IncP-9), R751 (IncP-1β) and RK2 (IncP-1α), respectively. The mercury resistance determinants were located on a transposable element of the Tn5053 family designated Tn5718. No putative functions could be assigned to a quarter of the coding capacity of pSB102 on the basis of comparisons with database entries. The genetic organization of the pSB102 transfer region revealed striking similarities to plasmid pXF51 of the plant pathogen Xylella fastidiosa.     

Plasmid deletion formation between short direct repeats in Bacillus subtilis is stimulated by single-stranded rolling-circle replication intermediates

Summary The effects of the rolling-circle mode of replication and the generation of single-stranded DNA (ss DNA) on plasmid deletion formation between short direct repeats in Bacillus subtilis were studied. Deletion units consisting of direct repeats (9, 18, or 27 bp) that do or do not flank inverted repeats (300 bp) were introduced into various plasmid replicons that generate different amounts of ss DNA (from 0% to 40% of the total plasmid DNA). With ss DNA-generating rolling-circle-type plasmids, deletion frequencies between the direct repeats were 3- to 13-fold higher than in plasmids not generating ss DNA. When the direct repeats flanked inverted repeats the deletion frequencies in ss DNA-generating plasmids were increased by as much as 20- to 140-fold. These results support models for deletion formation based on template-switching errors during complementary strand synthesis of rolling-circle-type plasmids. The structural instability (deletion formation between short direct repeats) of the ss DNA-generating plasmid pTA1060 in B. subtilis was very low in the presence of a functional initiation site for complementary strand synthesis (minus origin). This observation suggests that it will be possible to develop stable host-vector cloning systems for B. subtilis.     

Physical mapping of plasmid pDB101: A potential vector plasmid for molecular cloning in streptococci

A physical map of the streptococcal macrolides, lincomycin, and streptogramin B (MLS) resistance plasmid pDB101 was constructed using six different restriction endonucleases. Ten recognition sites were found for HindIII, seven for HindII, eight for HaeII, and one each for EcoRI, HpaII, and KpnI. The localization of the restriction cleavage sites was determined by double and triple digestions of the plasmid DNA or sequential digestions of partial cleavage products and isolated restriction fragments, and all sites were aligned with a single EcoRI reference site. Plasmid pDB101 meets all requirements essential for a potential molecular cloning vehicle in streptococci; i.e., single restriction sites, a MLS selection marker, and a multiple plasmid copy number. The vector plasmid described here makes it possible to clone selectively any fragment of DNA cleaved with EcoRI, HpaII, or KpnI, or since the sites are close to each other in map position, any combination of two of these restriction enzymes.     

Efficient transposition of IS204-derived plasmids in Streptomyces coelicolor

In order to study functional gene expression in Streptomyces coelicolor, a mini-transposon encoding the apramycin resistance gene aac(3)IV within its inverted repeat (IR) boundaries was constructed based on IS204, which was previously identified in the genome of Nocardia asteroides YP21. The mini-transposon and IS204 transposase gene were then put on a kanamycin-resistant conjugative plasmid pDZY101 that can only replicate in Escherichia coli. After mating with S. coelicolor A3(2) M145, resistant colonies arose efficiently on both apramycin and kanamycin plates. Plasmid rescue indicated that entire plasmids were inserted into the M145 genome with cleavage at an inverted repeat junction formed by the right inverted repeat (IRR) and the last 18 bp of the transposase gene, while the left inverted repeat (IRL) was untouched. Southern blot analysis of the mutants using an aac(3)IV gene probe showed that transposition of plasmid pDZY101 was genetically stable, with a single-copy insertion within the S. coelicolor M145 genome. Several mutagenesis libraries of S. coelicolor M145 were constructed using plasmid pDZY101 derivatives and the transposon insertion site was determined. The correlation between novel mutant phenotypes and previously uncharacterized genes was established and these transposon locations were widely scattered around the genome.     

Homologous recombination in a mammalian plasmid

Summary Bovine papillomavirus (BPV) shuttle vectors replicate as a circular plasmid in mouse cell nuclei without impairing host cell viability. We used these vectors to analyze homologous recombination in mammalian cells. When several BPV-based plasmids carrying direct repeats were introduced into C127 cells, we detected many recombinant plasmid molecules that have lost the sequence between the repeats. Many recombinant type molecules as well as parental type molecules were detected in all the cell clones isolated for analysis. Sequencing after rescue of the plasmid inEscherichia coli showed that most of the recombinants were from accurate homologous recombination. When the repeats on the plasmid were in inverted orientation, no crossing-over type products were detected. We discuss possible mechanisms that explain these features.     

Sequence relationships between plasmids associated with conventional MLS resistance and zonal lincomycin resistance in Streptococcus pyogenes

Summary By using electron microscopy of self-annealed DNA and restriction enzyme analysis, we have compared the physical maps of two group A streptococcal plasmids associated with conventional MLS resistance (pEL1; 20 Md) and zonal lincomycin resistance (pSM10419; 15 Md). Of their monomeric molecules, about 40% and 60%, respectively, are occupied by identical non-tandem inverted repeats containing sequences specifying putative replication functions. Sequence homology also exists between their resistance determinants which are located in unique DNA. Moreover, homology between additional regions of unknown function is so extensive and restriction fragment arrangement so similar that, formally, pSM10419 can be considered a deletion variant of pEL1. The results suggest that MLS and zonal lincomycin resistance have the same biochemical basis (i.e. methylation of 23S ribosomal RNA) and differ only quantitatively in the inducible control systems.     

Abnormal Reaction Product from Condensation of Phloroaceto-phenone Dimethylether with p-Methoxybenzaldehyde

We identified and analyzed a DNA region that is required for the stable maintenance of plasmids in the genus Sphingomonas. This DNA fragment, a 244?bp, is localized in the upstream region of the repA gene of low-copy-number small plasmid pYAN-1 (4896?bp) of Sphingobium yanoikuyae. It has four inverted repeats and one direct repeat for possible secondary structures. We were able to stabilize not only another unstable plasmid, pYAN-2, in the genus Sphingomonas, but also the unstable plasmid pSC101 without par locus in Escherichia coli. The copy-number levels between the unstable plasmid and the parental plasmid were similar, and these results suggest that the stabilization of unstable plasmids by this DNA region of pYAN-1 was not due to an increase in plasmid copy number. We concluded that the stabilization of the plasmid was due to a plasmid partition mechanism encoded by a DNA fragment of pYAN-1.     

Analysis of chromosomal integration and deletions of yeast plasmids.

Plasmid DNAs from six strains of Saccharomyces cerevisiae were compared. Three different plasmids were found, designated Scp 1, Scp 2 and Scp 3, with monomer lengths of 6.19, 6.06 and 5.97 kilobases as referenced to sequenced phiX174 DNA. DNA from each of the plasmids was inserted into a lambda vector DNA. Hybrid phage containing inserted DNA of the desired size were enriched by genetic selection and their DNAs analysed by rapid techniques. All three plasmids share the same organization, two unique sequences separated by two inverted repeats, and share basically the same DNA sequences. Scp 2 and Scp 3 differ from Scp 1 by missing a unique HpaI site and by having small overlapping deletions in the same region. The HpaI site in Scp 1 is, therefore, in a nonessential region and suitable for insertion of foreign DNA in the potential use of the yeast plasmid as a vector. Hybridization of labelled cloned plasmid DNA to restriction fragments of linear yeast DNA separated on agarose gels showed that the plasmid DNA was not stably integrated into the yeast chromosomal DNA.     

Sequences of Two Related Multiple Antibiotic Resistance Virulence Plasmids Sharing a Unique IS26-Related Molecular Signature Isolated from Different Escherichia coli Pathotypes from Different Hosts

Enterohemorrhagic Escherichia coli (EHEC) and atypical enteropathogenic E. coli (aEPEC) are important zoonotic pathogens that increasingly are becoming resistant to multiple antibiotics. Here we describe two plasmids, pO26-CRL₁₂₅ (125 kb) from a human O26:H- EHEC, and pO111-CRL₁₁₅ (115kb) from a bovine O111 aEPEC, that impart resistance to ampicillin, kanamycin, neomycin, streptomycin, sulfathiazole, trimethoprim and tetracycline and both contain atypical class 1 integrons with an identical IS26-mediated deletion in their 3´-conserved segment. Complete sequence analysis showed that pO26-CRL₁₂₅ and pO111-CRL₁₁₅ are essentially identical except for a 9.7 kb fragment, present in the backbone of pO26-CRL₁₂₅ but absent in pO111-CRL₁₁₅, and several indels. The 9.7 kb fragment encodes IncI-associated genes involved in plasmid stability during conjugation, a putative transposase gene and three imperfect repeats. Contiguous sequence identical to regions within these pO26-CRL₁₂₅ imperfect repeats was identified in pO111-CRL₁₁₅ precisely where the 9.7 kb fragment is missing, suggesting it may be mobile. Sequences shared between the plasmids include a complete IncZ replicon, a unique toxin/antitoxin system, IncI stability and maintenance genes, a novel putative serine protease autotransporter, and an IncI1 transfer system including a unique shufflon. Both plasmids carry a derivate Tn21 transposon with an atypical class 1 integron comprising a dfrA5 gene cassette encoding resistance to trimethoprim, and 24 bp of the 3´-conserved segment followed by Tn6026, which encodes resistance to ampicillin, kanymycin, neomycin, streptomycin and sulfathiazole. The Tn21-derivative transposon is linked to a truncated Tn1721, encoding resistance to tetracycline, via a region containing the IncP-1α oriV. Absence of the 5 bp direct repeats flanking Tn3-family transposons, indicates that homologous recombination events played a key role in the formation of this complex antibiotic resistance gene locus. Comparative sequence analysis of these closely related plasmids reveals aspects of plasmid evolution in pathogenic E. coli from different hosts.