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PubMed1986

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 pUC19.

DNA Replication↗

PubMedDate not supplied

Characterization of non-virulence plasmids with homology to the virulence plasmid of Salmonella dublin.

Six wild-type (wt) strains of Salmonella typhimurium, one wt strain of S. heidelberg and 12 wt strains of Escherichia coli were isolated based on both hybridization to a 6-kb HindIII fragment of the non-virulence coding part of the S. dublin serovar-specific virulence plasmid and the absence of hybridization to the virulence genes (spv genes) of the same plasmid. Such hybridization was shown to be caused by resident plasmids in all strains and to involve the same region of 30 to 37 kb of consecutive HindIII fragments on the S. dublin virulence plasmid, suggesting a common origin of this plasmid DNA. Nine of the plasmids were selected for detailed characterization and were shown not to be of the same plasmid species. They varied in size between 44 and 88 kb, they showed incompatibility with the plasmid K-MP10, or belonged to incompatibility group X, and with the exception of five plasmids from E. coli, they showed different HindIII restriction profile patterns.

DNA, Bacterial↗

PubMed2006

Mobilization of the gonococcal 5.2 kb beta-lactamase plasmid pSJ5.2 into Escherichia coli by cointegration with several gram-conjugative plasmids.

We report the mobilization by cointegration of the gonococcal 5.2 kb beta-lactamase plasmid pSJ5.2 in an Escherichia coli background. Transfer of pSJ5.2 was measured by filter mating assays with five different conjugative plasmids from Enterobacteriaceae and the gonococcal 41 kb tet(M). Plasmid pSJ5.2 was mobilized to E. coli at frequencies of 1.7x10(-6), 9.3x10(-8) and 2.7x10(-5) by the tet(M), R64 drd-33 and N3 conjugative plasmids, respectively. Mobilization of pSJ5.2 by the 41 kb tet(M) conjugative plasmid resulted in stable Amp(R) E. coli transconjugants consisting of pSJ5.2 plasmid with an insertion located in the 2.4 kb BamHI-BamHI fragment. Mobilization of pSJ5.2 by R64drd-33 and N3 conjugative plasmids involved stable cointegrates as detected by Southern Blot with a DIG-labelled PstI-digested pSJ5.2 probe. Restriction analysis of the R64::pSJ5.2 and N3::pSJ5.2 cointegrates and Southern Blot with the pSJ5.2 probe showed that cointegrates formed by deletion of DNA regions within the 1.8 kb BamHI-HindIII fragment of pSJ5.2. The plasmid thus appears to use multiple recombination mechanisms for cointegration with different conjugative plasmids. The complete nucleotide sequence of pSJ5.2 was determined, and will be a useful tool to further investigate the molecular mechanisms leading to its cointegrative transfer.

Conjugation, Genetic↗

PubMed2003

The patchwork nature of rolling-circle plasmids: comparison of six plasmids from two distinct Bacillus thuringiensis serotypes.

Bacillus thuringiensis, the entomopathogenic bacteria from the Bacillus cereus group, harbors numerous extrachromosomal molecules whose sizes vary from 2 to more than 200kb. Apart from the genes coding for the biopesticide delta-endotoxins located on large plasmids, little information has been obtained on these plasmids and their contribution to the biology of their host. In this paper, we embarked on a detailed comparison of six small rolling-circle replicating (RCR) plasmids originating from two major B. thuringiensis strains. The complete nucleotide sequences of plasmid pGI1, pGI2, pGI3, pTX14-1, pTX14-2, and pTX14-3 have been obtained and compared. Replication functions, comprising, for each plasmid, the gene encoding the Rep-protein, double-strand origin of replication (dso), single-strand origin of replication (sso), have been identified and analyzed. Two new families, or homology groups, of RCR plasmids originated from the studies of these plasmids (Group VI based on pGI3 and Group VII based on pTX14-3). On five of the six plasmids, loci involved in conjugative mobilization (Mob-genes and origin of transfer (oriT)) were identified. Plasmids pTX14-1, pTX14-2, and pTX14-3 each harbor an ORF encoding a polypeptide containing a central domain with repetitive elements similar to eukaryotic collagen (Gly-X-Y triplets). These genes were termed bcol for Bacillus-collagen-like genes.

Amino Acid Sequence↗

PubMedDate not supplied

Effects of plasmid copy number and runaway plasmid replication on overproduction and excretion of beta-lactamase from Escherichia coli.

Runaway plasmid replication can be used to increase target gene dosage and thereby overproduce proteins within the bacterium Escherichia coli. However, the presence of excessive plasmid DNA often alters normal cell functions. High copy number plasmids with strong promoters place a severe metabolic burden on the cell, causing a decreased specific growth rate and changes in cell physiology. Induction of beta-lactamase synthesis from the tac promoter on plasmid pKN causes runaway plasmid replication and excretion of beta-lactamase. Runaway plasmid replication results from readthrough of tac promoter transcripts into the replication region of the plasmid. Both high plasmid copy numbers and a strong promoter (tac) are necessary to achieve the level of overproduction necessary for excretion of beta-lactamase, but high-level target protein synthesis is detrimental to the cell. A derivative of pKN which is more easily regulated was constructed by adding the lacI gene to the plasmid.

DNA Replication↗

PubMedDate not supplied

Transduction efficiency of pantropic retroviral vectors is controlled by the envelope plasmid to vector plasmid ratio.

Pantropic retroviral vectors pseudotyped with vesicular stomatitis virus envelope G protein (VSV-G) are typically produced by transient transfection of the VSV-G expression plasmid because constitutive expression of VSV-G is cytotoxic. To produce pantropic vectors, the VSV-G expression plasmid and the vector plasmid are cotransfected into a packaging cell line, such as 293-gag-pol. Typically, the ratio of VSV-G plasmid to the vector plasmid ranges from 0.33 to 1.0. However, it is not clear that this range is optimal for vector production. In this study we have systematically examined the effect of the ratio of VSV-G plasmid (pVSV-G) to vector plasmid on vector production. For this, 293-gag-pol stable packaging cells were cotransfected with pVSV-G and an enhanced green fluorescent protein- (EGFP-) expressing retroviral vector plasmid (pLTR-EGFP) by use of lipofectamine. Vector was collected following transfection and used to transduce three target cell lines, namely, 3T3 fibroblasts, telomerase-immortalized human diploid fibroblasts (HDF), and the human hepatoma cell line HuH7. Transduction efficiency was evaluated for vectors produced at different pVSV-G:pLTR-EGFP ratios such that the total amount of plasmid transfected into 293-gag-pol cells was kept constant. Our results indicate that transduction efficiency is greatest when the pVSV-G:pLTR-EGFP ratio is substantially below 1.0. For 3T3 and HDF cells, the maximum transduction efficiency was obtained when a ratio of pVSV-G:pLTR-EGFP ranging from 0.053 to 0.2 was used for transfection. The relative magnitude of this effect was greater for lower transduction efficiencies in control cultures. For HuH7 cells, the beneficial effects were smaller than those observed when HDF or 3T3 cells were used. The difference in transduction efficiency for vector produced under various pVSV-G:pLTR-EGFP ratios was not due to differences in the proliferation of packaging cells or target cells. Further characterization showed that the amount of vector RNA relative to p30gag decreased as the ratio of pVSV-G:pLTR-EGFP increased. These results indicate that transduction efficiency increases with increasing levels of vector RNA as long as a minimally sufficient level of pantropic envelope protein is expressed.

3T3 Cells↗

PubMed1981

Replication of plasmid pT181 DNA in vitro: requirement for a plasmid-encoded product.

PT181 is a naturally occurring 4.5-kilobase Staphylococcus aureus plasmid encoding resistance to tetracycline. The plasmid has a copy number of about 20 per cell; a mutant, cop-608, that has a copy number of 800-1000 has been isolated. A cell-free extract has been developed that carries out complete replication of this plasmid. Extracts made from a strain containing the mutant have much greater replication activity than do extracts of strains containing pT181. In an extract from which endogenous DNA has been removed, DNA synthesis is dependent upon the addition of exogenous plasmid DNA. The replication system is specific for pT181 and related plasmids but it is inactive with other S. aureus plasmids. Furthermore, pT181 DNA does not replicate in extracts made from plasmid-negative strains or strains containing other plasmids. The results suggest that a specific plasmid-encoded substance is required for the replication of pT181 DNA.

Cell-Free System↗

PubMed1986

Unique type of plasmid maintenance function: postsegregational killing of plasmid-free cells.

The stability locus parB+ of plasmid R1 has been found to specify a unique type of plasmid maintenance function. Two genes, hok (host killing) and sok (suppressor of killing), are required for the stabilizing activity. The hok gene encodes a highly toxic gene product, whose overexpression causes a rapid killing and a concomitant dramatic change in morphology of the host cell. The other gene, sok, was found to encode a product that counteracts the hok gene-mediated killing. The parB+ region was inserted in a plasmid with a temperature-sensitive replication system. At nonpermissive temperature, the parB+ plasmid was maintained in the population for a significantly longer period than the corresponding parB- plasmid. Coupled to this extended maintenance, a large fraction of the population was shown to be nonviable plasmid-free cells with the characteristic hok-induced change in morphology. Based on these findings, we propose that the parB+ locus mediates plasmid stability by killing cells that have lost the parB+ plasmid during the preceding cell division, thereby ensuring that a growing bacterial culture predominantly consists of plasmid-containing cells.

Cell Division↗

PubMed1998

Dictyostelium discoideum nuclear plasmid Ddp5 is a chimera related to the Ddp1 and Ddp2 plasmid families.

The 14,955-bp Dictyostelium discoideum nuclear plasmid Ddp5 contains six transcribed open reading frames. One of these is related to the rep gene of the Ddp2 plasmid, and the other five are related to genes present on the Ddp1 plasmid. The absence of a homolog of the Ddp1 G1 gene, coupled with the presence of the Ddp2 rep gene homolog and of a 1.6-kb inverted repeat analogous to the inverted repeats on members of the Ddp2 plasmid family, suggests that Ddp5 uses Ddp2-like replication and copy number control mechanisms and that it should be assigned to the Ddp2 plasmid family. Ddp5 carries genes homologous to the D1/D3 and D2 genes of the Ddp1 plasmid as well as the Ddp1 G2/G3/D4, G5/D6, and G6/G4/D5 genes. The products of the Ddp5 G2-like, G5-like, and G6-like genes are likely to be transcription factors regulating the expression of themselves and of the other Ddp5 genes. The D1-like and D2-like genes may confer a selective advantage to plasmid-bearing cells, because they can be deleted from plasmid-based shuttle vectors with no apparent effect on vector maintenance. Updated sequence information for the Ddp1 G5/D6, D1/D3, and D2 genes as well as the Dmp1 and Dmp2 G5-like genes is presented. The locations of introns in the G5-like and D1-like genes of Ddp5 and in the homologous genes of the Ddp1, Dmp1, and Dmp2 plasmids were identified. These introns all have GU at the 5' intron border and AG at the 3' intron border, are short (59 to 71 nucleotides), and are AT-rich. A conserved HHCC domain was identified in the G5 proteins; this is a putative zinc binding domain and may be involved in protein-DNA interaction.

Amino Acid Sequence↗

PubMed1984

The effect of plasmid R391 and other IncJ plasmids on the survival of Escherichia coli after UV irradiation.

The presence of the IncJ plasmids R391, R997, R705, R706, R748 and R749 was shown to sensitize Escherichia coli AB1157 and both its uvrA and lexA derivatives to UV irradiation. No alteration in post-irradiation survival was observed in a recA mutant containing these plasmids, compared with the non-plasmid-containing recA strain. Analysis of recombination frequency in Hfr crosses to recA+ cells containing plasmid R391 indicated a reduction in recombination frequency compared with that obtained in similar crosses to a non-plasmid-containing strain. This effect was not due to plasmid-encoded restriction or entry exclusion systems and therefore must be considered as a real block in recombination. When cells containing plasmid R391 were irradiated and allowed to photoreactivate, an increase in survival was observed which was comparable to that observed in the non-plasmid-containing derivative. This indicated that post-irradiation processing of UV-induced damage, or lack of such processing, by mechanisms other than photoreactivation was responsible for the UV sensitivity associated with plasmid R391.

Escherichia coli↗

PubMed1995

The DNA and RNA polymerase genes of yeast plasmid pGKL2 are essential loci for plasmid integrity and maintenance.

Novel recombinant plasmids derived from the Kluyveromyces lactis killer plasmid k2 have been constructed to study plasmid biology and gene function. In vivo recombination between native resident k2 and suitable disruption vectors, employing the KITRP1 gene fused to a plasmid promoter as selection marker, yielded ORF2 and ORF6 deletion plasmids at high frequencies. As judged from Southern hybridization and plasmid restriction mapping analyses, these novel hybrids, termed rk2/2 and rk2/6, respectively, carry deletions in their putative DNA (ORF2) and RNA (ORF6) polymerase structural genes with central regions replaced by the input marker DNA. Long-term selection for TRP1 over 350 generations of growth did not favour maintenance of hybrids over wild-type k2. Thus, neither rk2/2 nor rk2/6 was fully functional and able to displace parental k2, indicating that both target genes are essential for plasmid integrity or maintenance. Recombinant plasmids were reduced in copy number relative to k2 with rk2/2 more drastically affected than rk2/6 implying a direct involvement of the ORF2 product in plasmid replication and an indirect maintenance function for the ORF6 gene product.

Base Sequence↗

PubMed1991

The 106-kilobase plasmid of Salmonella braenderup and the 100-kilobase plasmid of Salmonella typhimurium are not necessary for the pathogenicity in experimental models.

Among 1.041 clinical isolates (77 serovars) of Salmonella which had been derived from cases with acute enterocolitis, 601 (58%) contained one or more plasmids. Large serovar-specific plasmids were seen in 95 of 307 isolates (31%) of Salmonella typhimurium, in 34 of 34 isolates (100%) of Salmonella enteritidis and in 36 of 38 isolates (94.7%) of Salmonella braenderup: the sizes of which were 100, 60 and 106 kilobases (kb), respectively. In order to determine the role of these plasmids in pathogenicity for enterocolitis, the plasmids were eliminated from some strains of S. braenderup and S. typhimurium and the pathogenicity of the plasmid-less strains was compared with that of the parent strains by invasiveness to HeLa cells, fluid accumulation in the rabbit ligated ileal loop, lesion of mucosal tissue and the Sereny test. The virulence of all the plasmid-less strains was as strong as that of the plasmid-bearing strains in these pathogenicity assay systems. We therefore concluded that the 106-kb plasmid of S. braenderup and the 100-kb plasmid of S. typhimurium are not necessary for their pathogenicity in the experimental models: invasiveness to HeLa cells, fluid accumulation in the rabbit ligated ileal loop, and Sereny test.

Animals↗

PubMed1998

Small cryptic plasmids of Streptococcus pneumoniae belong to the pC194/pUB110 family of rolling circle plasmids.

The DNA sequences of two related plasmids pPR1 and pPR3 described previously in Streptococcus pneumoniae isolates from Germany and Spain were now determined. Both plasmids belong to a family of rolling circle (RC) plasmids found in a variety of bacteria. Their GC content with 32% is lower than that of the S. pneumoniae chromosomal DNA. The plasmid pPR3 has a molecular size of 3160 bp with four putative open reading frames, whereas pPR1 contained a deletion of 313 bp that included the 5'-part of ORF2 and upstream regions and differed by three bp from pPR3. The predicted protein of ORF1 showed high similarity to replication proteins of RC plasmids with 74% identical amino acids to RepA of Streptococcus thermophilus plasmids. Sequences similar to the plus origin of replication of ssDNA plasmids were present in both plasmids. They also contained a 152-bp region with over 83% identity to the minus origin of replication of the Streptococcus agalacticae plasmid pMV158.

Base Sequence↗

PubMed2006

Increased abundance of IncP-1beta plasmids and mercury resistance genes in mercury-polluted river sediments: first discovery of IncP-1beta plasmids with a complex mer transposon as the sole accessory element.

Although it is generally assumed that mobile genetic elements facilitate the adaptation of microbial communities to environmental stresses, environmental data supporting this assumption are rare. In this study, river sediment samples taken from two mercury-polluted (A and B) and two nonpolluted or less-polluted (C and D) areas of the river Nura (Kazakhstan) were analyzed by PCR for the presence and abundance of mercury resistance genes and of broad-host-range plasmids. PCR-based detection revealed that mercury pollution corresponded to an increased abundance of mercury resistance genes and of IncP-1beta replicon-specific sequences detected in total community DNA. The isolation of IncP-1beta plasmids from contaminated sediments was attempted in order to determine whether they carry mercury resistance genes and thus contribute to an adaptation of bacterial populations to Hg pollution. We failed to detect IncP-1beta plasmids in the genomic DNA of the cultured Hg-resistant bacterial isolates. However, without selection for mercury resistance, three different IncP-1beta plasmids (pTP6, pTP7, and pTP8) were captured directly from contaminated sediment slurry in Cupriavidus necator JMP228 based on their ability to mobilize the IncQ plasmid pIE723. These plasmids hybridized with the merRTDeltaP probe and conferred Hg resistance to their host. A broad host range and high stability under conditions of nonselective growth were observed for pTP6 and pTP7. The full sequence of plasmid pTP6 was determined and revealed a backbone almost identical to that of the IncP-1beta plasmids R751 and pB8. However, this is the first example of an IncP-1beta plasmid which had acquired only a mercury resistance transposon but no antibiotic resistance or biodegradation genes. This transposon carries a rather complex set of mer genes and is inserted between Tra1 and Tra2.

Bacteria↗

PubMed1982

Restriction endonuclease analysis of the lactose plasmid in Streptococcus lactis ML3 and two recombinant lactose plasmids.

We investigated the molecular relationship between the 60-megadalton (Mdal) recombinant lactose plasmids in ML 3 x LM2301 lactose-positive (Lac+) transconjugants and the genetic material of Streptococcus lactis ML3. Lactose metabolism is linked to the 33-Mdal plasmid pSK08 in ML3, and the recipient LM2301 is cured of plasmid DNA. The plasmids were analyzed with a series of restriction enzymes. We found that the 60-Mdal plasmids of Lac+ transconjugants contained pSK08 DNA, but were not simply dimers of pSK08. The 60-Mdal plasmids contained a segment of DNA not apparent in pSK08. The restriction patterns of the 60-Mdal plasmid in a Lac+ nonclumping transconjugant and that in a Lac+ clumping transconjugant were different. This suggested that there was a molecular differences between these two recombinant plasmids. We conclude that the segment of DNA in the 60-Mdal plasmids that was not present in pSK08 was the proposed transfer factor responsible for cell aggregation and high-frequency conjugation.

Base Sequence↗

PubMed2003

Plasmid introduction in metal-stressed, subsurface-derived microcosms: plasmid fate and community response.

The nonconjugal IncQ plasmids pMOL187 and pMOL222, which contain the metal resistance-encoding genes czc and ncc, were introduced by using Escherichia coli as a transitory delivery strain into microcosms containing subsurface-derived parent materials. The microcosms were semicontinuously dosed with an artificial groundwater to set a low-carbon flux and a target metal stress (0, 10, 100, and 1,000 micro M CdCl(2)), permitting long-term community monitoring. The broad-host-range IncPalpha plasmid RP4 was also transitorily introduced into a subset of microcosms. No novel community phenotype was detected after plasmid delivery, due to the high background resistances to Cd and Ni. At fixed Cd doses, however, small but consistent increases in Cd(r) or Ni(r) density were measured due to the introduction of a single pMOL plasmid, and this effect was enhanced by the joint introduction of RP4; the effects were most significant at the highest Cd doses. The pMOL plasmids introduced could, however, be monitored via czc- and ncc-targeted infinite-dilution PCR (ID-PCR) methods, because these genes were absent from the indigenous community: long-term presence of czc (after 14 or 27 weeks) was contingent on the joint introduction of RP4, although RP4 cointroduction was not yet required to ensure retention of ncc after 8 weeks. Plasmids isolated from Ni(r) transconjugants further confirmed the presence and retention of a pMOL222-sized plasmid. ID-PCR targeting the RP4-specific trafA gene revealed retention of RP4 for at least 8 weeks. Our findings confirm plasmid transfer and long-term retention in low-carbon-flux, metal-stressed subsurface communities but indicate that the subsurface community examined has limited mobilization potential for the IncQ plasmids employed.

Bacteria↗

PubMed1981

Shigella sonnei plasmids: evidence that a large plasmid is necessary for virulence.

Virulent form I Shigella sonnei strains contain a 120-megadalton plasmid that is absent in their form II derivatives, which are always avirulent and devoid of O side chains. In the present study, 165 biochemical and antibiotic traits were assessed, but no experimentally useful phenotype could be associated with this large form I plasmid. Therefore, the form I plasmids of several S. sonnei strains were tagged with the antibiotic resistance transposons Tn3, Tn5, or Tn10. Transposon-tagged form I plasmids were not self-transmissible, but could be mobilized by the plasmid R386. Form II S. sonnei transconjugants for the form I plasmid acquired both virulence and the ability to synthesize form I antigen, establishing that these properties are plasmid mediated. Further studies indicate that this 120-megadalton form I plasmid is physically unstable in any of several host bacteria and suggest that it is a member of the FI incompatibility group. Also, two commonly observed, small plasmids of S. sonnei, of 3.2 and 3.9 megadaltons, were shown to encode either colicin E1 production or resistance to streptomycin and sulfonamide, respectively.

Anti-Bacterial Agents↗

PubMed2001

Complete DNA sequence of Yersinia enterocolitica serotype 0:8 low-calcium-response plasmid reveals a new virulence plasmid-associated replicon.

The complete nucleotide sequence and organization of the Yersinia enterocolitica serotype 0:8 low-calcium-response (LCR) plasmid, pYVe8081, were determined. The 67,720-bp plasmid encoded all the genes known to be part of the LCR stimulon except for ylpA. Eight of 13 intact open reading frames of unknown function identified in pYVe8081 had homologues in Yersinia pestis plasmid pCD1 or in Y. enterocolitica serotype 0:9 plasmid pYVe227. A region of approximately 17 kbp showed no DNA identity to pCD1 or pYVe227 and contained six potential new genes, a possible new replicon, and two intact insertion sequence (IS) elements. One intact IS element, ISYen1, was a new IS belonging to the IS256 family. Several vestigial IS elements appeared different from the IS distribution seen in the other LCR plasmids. The RepA proteins encoded by Y. enterocolitica serotype 0:8 pYVeWA and pYVe8081 were identical. The putative pYVe8081 replicon showed significant homology to the IncL/M replicon of pMU407.1 but was only distantly related to the replicons of pCD1 and pYVe227. In contrast, the putative partitioning genes of pYVe8081 showed 97% DNA identity to the spy/sopABC loci of pCD1 and pYVe227. Sequence analysis suggests that Yersinia LCR plasmids are from a common ancestor but that Y. enterocolitica serotype 0:8 plasmid replicons may have evolved independently via cointegrate formation following a transposition event. The change in replicon structure is predicted to change the incompatibility properties of Y. enterocolitica serotype 0:8 plasmids from those of Y. enterocolitica serotype 0:9 and Y. pestis LCR plasmids.

Amino Acid Sequence↗