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PubMed1998

Bacterial fitness and plasmid loss: the importance of culture conditions and plasmid size.

Several pBluescript-derived plasmids of various sizes were constructed to study the effects of multicopy plasmid size on bacterial fitness and plasmid loss. Transformed and untransformed bacterial clones were grown in media with or without ampicillin. Bacterial fitness (measured by growth rate), plasmid presence or absence, and plasmid copy number were assessed during successive subculturings. In selective media (minimal medium or Luria Broth plus ampicillin), the clone transformed with the largest plasmid (pBluescript with a 9000-bp insert) had a significantly longer lag phase than all other clones. In nonselective media the rate of plasmid loss during successive subculturings was greatest in the clone with the largest insert. The clone with the largest insert displayed a lower plasmid copy number than clones with a small insert or no insert at all. Plasmid loss in the form of segregational instability and plasmid copy number reduction in nonselective environments are important to the understanding of the evolution of the bacteria-plasmid associations and the appreciation of the potential for altering the genetic properties of a clone maintained or subcultured on a standard medium.

Ampicillin↗

PubMed1990

Plasmid profiles of Shigella dysenteriae type 1 isolates from Ethiopia with special reference to R-plasmids.

Plasmid profiles of 80 Shigella dysenteriae type 1 (Shiga's bacillus) strains, collected between 1974 and 1985 in Ethiopia, were examined. Strains with the dominant antibiotic-resistance (R-) type--resistance to ampicillin (A), chloramphenicol (C), streptomycin (S), sulphadiazine (Su) and tetracycline (T)--showed two distinct plasmid profiles (PP). Six plasmid bands were demonstrated in "Ethiopian strains" with PP-1A isolated between 1974 and 1982. In mating experiments with these strains, Escherichia coli K12 recipients showed plasmids pYH10a (72 Mda, atypical Inc FIme, coding for ACSSuT resistance). Eight plasmid bands were demonstrated in strains with PP-2A. These strains were first isolated in 1980 and carried plasmid pYH11a (40 Mda, Inc X, coding for ACT resistance). Strains with PP-2A were identical with a "Zairian strain" described elsewhere. Strains with R-type ACT were "Zairian strains" lacking the 5.1- and 4.2-Mda plasmids. Those with R-type CSSuT were temporally clustered in 1978-1980 and carried plasmid pYH12 (58 Mda, Inc B, coding for the same R-type). A trimethoprim-resistant strain (Gimira strain) had a pattern of small plasmids similar to those of the "Zairian strain" and is probably a subclone of the latter. The fact that a limited number of plasmid profiles have remained constant over many years shows the limitation of plasmid profile analysis as an epidemiological tool. However, when the usual profile is known for a given area, identification of a distinctly different pattern becomes easy and epidemiologically useful.

DNA Replication↗

PubMed1986

Transposon Mutagenesis and Excision of R' Plasmids by Conjugative, Chimeric Plasmid pUW942 in Extra-Slow-Growing Rhizobium japonicum Strains.

Transposons Tn501 (specifying mercury resistance) and Tn7 (specifying resistance to trimethoprim and streptomycin) were introduced into extra-slow-growing Rhizobium japonicum by conjugal transfer of the 82 kilobase chimeric plasmid pUW942. Mercury-resistant transconjugants were obtained at a frequency of 10 to 10. The transfer frequency of streptomycin resistance was lower than that of mercury resistance, and Tn7 was relatively unstable. pUW942 was not maintained as an autonomously replicating plasmid in R. japonicum strains. However, some of the Hg transconjugants from the RJ19FY, RJ17W, and RJ12S strains acquired antibiotic markers of the vector plasmid pUW942. Southern hybridization of plasmid and chromosomal DNA of R. japonicum strains with P-labeled pUW942 and pAS8Rep-1, the same plasmid as pUW942 except that it does not contain Tn501, revealed the formation of cointegrates between pUW942 and the chromosome of R. japonicum. More transconjugants with only Tn501 insertions in plasmids or the chromosome were obtained in crosses with strains RJ19FY and RJ17W than with RJ12S. These retained stable Hg both in plant nodules and under nonselective in vitro growth conditions. One of the RJ19FY and two of the RJ12S Hg transconjugants with vector plasmid-chromosome cointegrates conjugally transferred plasmids of 82, 84 or 86, and 90 kilobases, respectively, into plasmidless Escherichia coli C. These plasmids strongly hybridized to pUW942 and EcoRI digests of total DNA of each respective R. japonicum strain but not to indigenous plasmid DNA of the R. japonicum strains. These R' plasmids consisted of pUW942-specific EcoRI fragments and an additional one or two new fragments derived from the R. japonicum chromosome.

Journal Article↗

PubMed1980

Plasmid replication functions: two distinct segments of plasmid R1, RepA and RepD, express incompatibility and are capable of autonomous replication.

The genetic determinants for replication and incompatibility of plasmid R1 were investigated by gene cloning methods, and three types of R1 miniplasmid derivatives were generated. The first, exemplified by plasmid pKT300, consisted of a single BglII endonuclease-generated deoxyribonucleic acid fragment derived from the R1 region that is located between the determinants for conjugal transfer and antibiotic resistance. Two types of miniplasmids could be formed from PstI endonuclease-generated fragments of pKT300. One of these, which is equivalent to miniplasmids previously generated from plasmids R1-19 and R1-19B2, consisted of two adjacent PstI fragments that encode the RepA replication system of plasmid R1. The other type contained a segment of R1, designated the RepD replication region, that is adjacent to the RepA region and that has not been identified previously as having the capacity for autonomous replication. Plasmid R1, therefore, contained two distinct deoxyribonucleic acid segments capable of autonomous replication. The RepA-RepD miniplasmid pKT300 had a copy number about eightfold higher than that of R1 and hence lacked a determinant for the regulation of plasmid copy number. Like R1, it was maintained stably in dividing bacteria. RepA miniplasmids had copy numbers which were two- to fourfold higher than that of R1 (i.e., which were lower than that of pKT300) and were maintained slightly less stably than those of pKT300 and R1. The RepD miniplasmid was not maintained stably in dividing bacteria. Previous experiments have shown that incompatibility of IncFII group plasmids is specified by a plasmid copy control gene. Despite the fact that RepA miniplasmids of R1 were defective in copy control, they nevertheless expressed incompatibility. This suggests that two genes are responsible for plasmid copy control, one that specifies incompatibility and is located on RepA miniplasmids and another that is located outside of, but adjacent to, the RepA replication region. Hybrid plasmids composed of pBR322 and one PstI fragment from the RepA region, P-8, exhibited incompatibility towards R2 and RepA miniplasmids but not the RepD miniplasmid, whereas hybrids composed of pBR322 and the PstI fragment of the RepD region, P-3, exhibited incompatibility towards R1 and the RepD miniplasmid but not RepA miniplasmids. These results indicate that the two replication systems are functionally distinct and that, although the RepA system is the principal replication system of R1, the RepD system also plays a role in the maintenance of this plasmid.

Cloning, Molecular↗

PubMed1985

[Plasmids for naphthalene biodegradation incompatible with IncP-2 and IncP-7 group plasmids].

Fourteen conjugative naphthalene degradative plasmids have been classified by incompatibility. It is shown that the plasmids of IncP-9 group are characterized by the minor entry exclusion, with respect to the R plasmids belonging to IncP-2 or IncP-7 groups. On the other hand, the naphthalene degradative plasmids of incompatibility group P-7 exhibit a markedly pronounced entry exclusion, with respect to the R plasmids of the same incompatibility groups. Two naphthalene degradative plasmids reveal incompatibility with the reference plasmids of two Inc groups (P-2 and P-7). These plasmids control also resistance of bacterial cells to potassium tellurite, which is characteristic of the IncP-2 plasmids. Two other naphthalene degradative plasmids are capable of stable coexistence with the IncP-2, P-7 or P-9 reference plasmids.

Biodegradation, Environmental↗

PubMed1982

Use of a known plasmid and transposon as tracers for the incompatibility classification of a cryptic plasmid.

We have developed a novel genetic technique by which an unknown plasmid can be classified by the use of plasmid of known incompatibility group. In phenocopy state, cells harboring plasmids of known incompatibility group behave as normal recipients and receive plasmids belonging to the same incompatibility group at a high frequency. This work provides additional evidence that plasmids in 'phenocopy' hosts do not replicate and therefore fail to demonstrate their incompatibility barrier to the incoming plasmids. A cryptic plasmid, now called pWS7, has been identified by this method in a pili, fla female strain of E. coli K12. Genetic analysis shows the plasmid pWS7 is in fact, a sex-factor which is curable with acridine orange. It belongs to the Inc F1 group. Physical analysis confirms its size to be 124 Kb. The plasmid has been labelled genetically with a transposon Tn903 in a recA host and further characterized by heteroduplex analysis. A DNA sequence homology between pWS7 and F'lac plasmid extends only in F-regions, 2.8F-94.5F. The pili, fla host strain of pWS7 shows a high frequency of transformation for recombinant DNA and rapid propagation for a male-specific RNA phage, R17.

Acridine Orange↗

PubMed1980

Partitioning of plasmid R1 in Escherichia coli. I. Kinetics of loss of plasmid derivatives deleted of the par region.

The stability of inheritance of plasmid R1drd-19 was tested. The copy number of the plasmid was determined in two different ways: As the ratio between covalently closed circular DNA and chromosomal DNA, and by quantitative determination of single-cell resistance to ampicillin. In the latter case, strains carrying the R1 ampicillin transposon Tn3 on prophage lambda was used as standard. The values were transformed to copy number per cell by using the Cooper-Helmstetter model for chromosome replication as well as by determination of chromosomal DNA per cell by the diphenylamine method. The copy number was found to be five to six per cell (or about four per newborn cell). Nevertheless, plasmid R1drd-19 was found to be completely stably inherited. This stability was shown not to be due to retransfer of the plasmid by the R1 conjugation system, since transfer-negative derivatives of the plasmid were also completely stably inherited. Smaller derivatives of plasmid R1drd-19 were found to be lost at a frequency of about 1.5% per cell generation. The copy-number control was not affected in these miniplasmids, since their copy numbers were the same as that of the full size plasmid. Quantitatively, the instability of the miniplasmids was in accord with random partitioning. It is, therefore, suggested that the plasmid R1drd-19 carries genetic information for partitioning (par) of plasmid copies at cell division, and that the par mechanism is distinct from the copy number control (cop) system. Finally, the par gene maps on the resistance transfer part of the plasmid, but far away from the origin of replication and the so-called basic replicon; this is in accord with the approximate location of the repB gene (Yoshikawa, 1974, J. Bacteriol., 118, 1123-1131).

Cell Division↗

PubMed1985

A plasmid vector allowing positive selection of recombinant plasmids in Streptococcus pneumoniae.

A new plasmid, pSP2, was constructed as a cloning vector for use in Streptococcus pneumoniae. It allows direct selection of recombinant plasmids, even for DNA fragments not homologous to the S. pneumoniae chromosome, as based on the failure to maintain long inverted repeats (LIRs) hyphen-free in bacterial plasmids. Plasmid pSP2 contains a 1.4-kb BamHI fragment ("hyphen") flanked by 1.9-kb LIRs. The removal of the 1.4-kb BamHI fragment followed by ligation creates a plasmid containing a 1.9-kb insert-free LIR; plasmids with such non-hyphenated LIRs were not established when transferred into S. pneumoniae. Replacement of the original 1.4-kb insert by other restriction fragments restored plasmid viability. Investigation of plasmid transfer by transformation suggests that intrastrand synapsis between the LIRs could occur, thus facilitating plasmid establishment (a process we call self-facilitation). Such an intrastrand synapsis could also account for rare occurrences of insert-inversion noticed upon transfer as well as for the formation of palindrome-deleted derivatives at low frequency. Plasmid pSP2 carries two selectable genes, tet and ermC, and can be used for cloning of fragments produced by a variety of restriction enzymes (BamHI, Bg/II, Bc/I or Sau3A, and Sa/I or XhoI).

Chromosome Mapping↗

PubMed1992

Macroevolution of plasmids: a model for plasmid speciation.

A new evolutionary model for diversification in plasmid incompatibility groups (plasmid speciation) is suggested. The model is based on the formation of plasmid cointegrates from two compatible plasmids. The existence of plasmid cointegrates is well known, however, their potential key role in plasmid macroevolution has not yet been recognized. In a hypothesis presented here, one of the rep genes is supposed to be relaxed from selection in plasmid cointegrates and thus becomes free to accumulate mutations. These mutations can lead to a change in incompatibility specificity. Evidence supporting this hypothesis comes from the common occurrence of multi-replicon plasmids in nature as well as from experimental studies on plasmid cointegrate formation. A more speculative extension of this model hypothesizes an evolutionary scenario for origin of the eubacterial single-replicon genome and the eukaryotic multi-replicon genome, as well as the place of plasmids and viruses in this picture.

Bacteria↗

PubMed1998

Characterization of plasmid regions of foodborne Yersinia enterocolitica biogroup 1A strains hybridizing to the Yersinia enterocolitica virulence plasmid.

The aim of our study was to find out if plasmids of foodborne Yersinia enterocolitica biogroup 1A strains harbour genes related to the virulence genes located on the virulence plasmid pYV of Yersinia enterocolitica. The foodborne strains were isolated from pork, as pigs are considered as an important reservoir for enteropathogenic Y. enterocolitica 0:3 and 0:9 strains. The plasmids of the foodborne strains were characterized by restriction enzyme analysis and hybridized to the virulence plasmid pYV of pathogenic Y. enterocolitica strains (0:3 biogroup 4; 0:9 biogroup 2). In several cases the plasmids of the foodborne strains showed homologies to parts of the pYV plasmid. Analysis of the hybridizing regions revealed that genes involved in replication, sequences of transposable elements and an endonuclease gene caused the observed hybridization to the virulence plasmid. In cause of the study also a remnant of a Tn3-like transposon was shown to be present adjacent to the yadA gene on the pYV plasmid. Although there is evidence that at least some strains of Y. enterocolitica biogroup 1A might possess pathogenic properties none of the well known plasmid encoded virulence genes were present on the plasmids of the investigated foodborne biogroup 1A strains.

Animals↗

PubMed1986

Plasmid-mediated resistance to beta-lactam antibiotics in gram-negative bacteria: the role of in-vivo recyclization reactions in plasmid evolution.

Over 20 different plasmid-encoded beta-lactamases have so far been discovered. This paper considers genetic mechanisms by which beta-lactamase genes encoded by plasmids are disseminated across generic boundaries. Particular emphasis is placed on the evolution of plasmids carrying all or part of Tn3 and encoding TEM-1 beta-lactamase in Haemophilus and Neisseria species. Examples of the acquisition of broad host range plasmids carrying Tn3 sequences and of rescue of transposon sequences to indigenous plasmids or to the host chromosome have been found in these two genera. Studies on nonconjugative beta-lactamase plasmids in Neisseria and Haemophilus are consistent with the evolution of a family of plasmids originating from the insertion of Tn3 into an indigenous progenitor plasmid. A series of subsequent insertional and deletional events, most probably occurring as a consequence of genetic transfer, have given rise to the existing group of small, closely related ampicillin-resistance plasmids found currently in these genera. A general model for deletional and other rearrangements caused by recombinational recyclization during the evolution of resistance plasmids is described.

Anti-Bacterial Agents↗

PubMed2006

PlasmID: a centralized repository for plasmid clone information and distribution.

The Plasmid Information Database (PlasmID; http://plasmid.hms.harvard.edu) was developed as a community-based resource portal to facilitate search and request of plasmid clones shared with the Dana-Farber/Harvard Cancer Center (DF/HCC) DNA Resource Core. PlasmID serves as a central data repository and enables researchers to search the collection online using common gene names and identifiers, keywords, vector features, author names and PubMed IDs. As of October 2006, the repository contains >46 000 plasmids in 98 different vectors, including cloned cDNA and genomic fragments from 26 different species. Moreover, the clones include plasmid vectors useful for routine and cutting-edge techniques; functionally related sets of human cDNA clones; and genome-scale gene collections for Saccharomyces cerevisiae, Pseudomonas aeruginosa, Yersinia pestis, Francisella tularensis, Bacillus anthracis and Vibrio cholerae. Information about the plasmids has been fully annotated in adherence with a high-quality standard, and clone samples are stored as glycerol stocks in a state-of-the-art automated -80 degrees C freezer storage system. Clone replication and distribution is highly automated to minimize human error. Infor-mation about vectors and plasmid clones, including downloadable maps and sequence data, is freely available online. Researchers interested in requesting clone samples or sharing their own plasmids with the repository can visit the PlasmID website for more information.

Biological Specimen Banks↗

PubMed2006

Biodistribution of DNA plasmid vaccines against HIV-1, Ebola, Severe Acute Respiratory Syndrome, or West Nile virus is similar, without integration, despite differing plasmid backbones or gene inserts.

The Vaccine Research Center has developed a number of vaccine candidates for different diseases/infectious agents (HIV-1, Severe Acute Respiratory Syndrome virus, West Nile virus, and Ebola virus, plus a plasmid cytokine adjuvant-IL-2/Ig) based on a DNA plasmid vaccine platform. To support the clinical development of each of these vaccine candidates, preclinical studies have been performed in mice or rabbits to determine where in the body these plasmid vaccines would biodistribute and how rapidly they would clear. In the course of these studies, it has been observed that regardless of the gene insert (expressing the vaccine immunogen or cytokine adjuvant) and regardless of the promoter used to drive expression of the gene insert in the plasmid backbone, the plasmid vaccines do not biodistribute widely and remain essentially in the site of injection, in the muscle and overlying subcutis. Even though approximately 10(14) molecules are inoculated in the studies in rabbits, by day 8 or 9 ( approximately 1 week postinoculation), already all but on the order of 10(4)-10(6) molecules per microgram of DNA extracted from tissue have been cleared at the injection site. Over the course of 2 months, the plasmid clears from the site of injection with only a small percentage of animals (generally 10-20%) retaining a small number of copies (generally around 100 copies) in the muscle at the injection site. This pattern of biodistribution (confined to the injection site) and clearance (within 2 months) is consistent regardless of differences in the promoter in the plasmid backbone or differences in the gene insert being expressed by the plasmid vaccine. In addition, integration has not been observed with plasmid vaccine candidates inoculated i.m. by Biojector 2000 or by needle and syringe. These data build on the repeated-dose toxicology studies performed (see companion article, Sheets et al., 2006) to demonstrate the safety and suitability for investigational human use of DNA plasmid vaccine candidates for a variety of infectious disease prevention indications.

Acquired Immunodeficiency Syndrome↗

PubMed2000

A second T-region of the soybean-supervirulent chrysopine-type Ti plasmid pTiChry5, and construction of a fully disarmed vir helper plasmid.

Agrobacterium tumefaciens Chry5, which is particularly virulent on soybeans, induces tumors that produce a family of Amadori-type opines that includes deoxyfructosyl glutamine (Dfg) and its lactone, chrysopine (Chy). Cosmid clones mapping to the right of the known oncogenic T-region of pTiChry5 conferred Amadori opine production on tumors induced by the nopaline strain C58. Sequence analysis of DNA held in common among these cosmids identified two 25-bp, direct repeats flanking an 8.5-kb segment of pTiChry5. These probable border sequences are closely related to those of other known T-regions and define a second T-region of pTiChry5, called T-right (TR), that confers production of the Amadoriopines. The oncogenic T-left region (TL) was located precisely by identifying and sequencing the likely border repeats defining this segment. The two T-regions are separated by approximately 15 kb of plasmid DNA. Based on these results, we predicted that pKYRT1, a vir helper plasmid derived from pTiChry5, still contains all of TR and the leftmost 9 kb of TL. Consistent with this hypothesis, transgenic Arabidopsis thaliana plants selected for with a marker encoded by a binary plasmid following transformation with KYRT1 co-inherited production of the Amadori opines at high frequency. All opine-positive transgenic plants also contained TR-DNA, while those plants that lacked TR-DNA failed to produce the opines. Moreover, A. thaliana infected with KYRT1 in which an nptII gene driven by the 35S promoter of Cauliflower mosaic virus was inserted directly into the vir helper plasmid yielded kanamycin-resistant transformants at a low but detectable frequency. These results demonstrate that pKYRT1 is not disarmed, and can transfer Ti plasmid DNA to plants. A new vir helper plasmid was constructed from pTiChry5 by two rounds of sacB-mediated selection for deletion events. This plasmid, called pKPSF2, lacks both of the known T-regions and their borders. pKPSF2 failed to transfer Ti plasmid DNA to plants, but mobilized the T-region of a binary plasmid at an efficiency indistinguishable from those of pKYRT1 and the nopaline-type vir helper plasmid pMP90.

Agrobacterium tumefaciens↗

PubMed1988

Physical and functional mapping of two cointegrate plasmids derived from RP4 and TOL plasmid pDK1.

Cointegrate plasmids were formed in vivo between the broad-host-range R-plasmid RP4 and two catabolic plasmids derived from Pseudomonas putida HS1. One of these was the wild-type plasmid pDK1 encoding the complete inducible toluene/xylene (TOL) catabolic pathway and one was pDKT1, a deletion derivative of pDK1 selected after growth of HS1 on benzoate and supporting growth on only toluene. The two plasmids formed, pDK2 and pDKT2 respectively, each consisted of a complete RP4 replicon in which was an insert of the parent plasmid DNA respectively 40 and 20 kbp in size. The detailed restriction maps of the two plasmids were determined and many of the catabolic genes were located by subcloning and enzyme assay of recombinant plasmids in Escherichia coli and Pseudomonas hosts. The insert in pDK2 contained both operons of the catabolic pathway, the 'upper pathway' operon (xylCAB) and the meta pathway operon (xylDLEGF(I,J,K)H), and a region identified as having the function of the regulator gene xylS. The insert in pDKT2 contained only the upper pathway operon and the regulatory region. Within each of the three coding regions there was great similarity with the same regions on TOL plasmids pWW0 and pWW53-4 apparent (a) by the same order of the genes, (b) by a similar pattern of restriction sites and (c) by hybridization studies. However, the order and orientations of the three coding regions differed from those previously described for both pWW0 and pWW53-4. The significance of these findings to the evolution of TOL plasmids is discussed.

Chromosome Mapping↗

PubMed1991

Characterization of a plasmid from Helicobacter pylori encoding a replication protein common to plasmids in gram-positive bacteria.

A 1.5 kb cryptic plasmid was isolated from Helicobacter pylori. Low-stringency hybridization analysis using this plasmid as a DNA probe revealed base sequence homology with other plasmids in this species. Nucleotide sequence analysis identified an open reading frame encoding a putative polypeptide of 25 kDa. This protein showed marked amino acid sequence similarity to replication-initiation proteins commonly found in small plasmids endogenous to Gram-positive bacteria which replicate by the 'rolling-circle' mechanism. Sequence motifs corresponding to the origins-of-replication consensus sequences were found on this cryptic plasmid. DNA and oligonucleotide probes to these plasmid replication sequences were used in hybridization analysis to identify similar sequences in other H. pylori plasmids. We believe this is the first plasmid isolated from a Gram-negative bacterium to show replication determinants characteristic of the 'rolling-circle' group of plasmids from Gram-positive bacteria. The cloned plasmid will be used to develop a shuttle-vector for H. pylori.

Amino Acid Sequence↗

PubMed1983

Transfer of plasmids from Escherichia coli to Pseudomonas aeruginosa: characterization of a Pseudomonas aeruginosa mutant with enhanced recipient ability for enterobacterial plasmids.

Pseudomonas aeruginosa strain GT424, derived by the transfer of plasmid R45 to strain GT1, was shown to have an Era+ phenotype (enhanced recipient ability) for enterobacterial plasmids from incompatibility (Inc) groups C, FI, FII, J, N, W, and X. Increase in transfer frequency ranged from 10 to 10(6)-fold. Plasmids of IncFII, IncJ, and IncX were found to be transmissible only to the Era+ strain. Plasmids of Inc groups A-C, M, and T transferred at low frequency (less than 10(-7)) to both GT1 and GT424 and did not respond to the Era+ character of the latter. Also not responsive to the Era character were plasmids R144drd3 (IncI alpha) and RP1 (IncP), which transferred to both GT424 and GT1 at intermediate (10(-4)) and high frequencies (10(-1)), respectively. All plasmid-mediated antibiotic resistances that could be measured as well as UV resistance and susceptibility were found to be expressed in P. aeruginosa. Plasmid-coded phage susceptibilities, however, generally were not phenotypically manifested in the P. aeruginosa strains. The physical structure of plasmids in P. aeruginosa could be classified into four major types. Some of the plasmids underwent additions or deletions and were unstable; the majority, however, appeared to integrate into the chromosome. Some members of IncN, IncC, IncJ, and IncP were found to be transmissible from P. aeruginosa back to Escherichia coli. This is the first report of the successful transfer to P. aeruginosa of plasmids from many of the Inc groups that have been defined in Enterobacteriaceae, namely, FI, I alpha, J, M, T, and X.

Conjugation, Genetic↗

PubMed1983

Characterization of virulence plasmids and plasmid-associated outer membrane proteins in Shigella flexneri, Shigella sonnei, and Escherichia coli.

The 140-megadalton plasmids of Shigella flexneri serotypes 1, 3, and 5, in addition to the 120-megadalton plasmid of Shigella sonnei, are associated with virulence. The present study showed that a 140-megadalton plasmid is also associated with virulence in Escherichia coli. When these plasmids were cleaved with EcoRI or BamHI restriction endonucleases, considerable homology was evident in plasmids from S. sonnei strains, whereas only a few common fragments were observed among the S. flexneri and enteroinvasive E. coli plasmids. Nitrocellulose filter hybridization demonstrated that, despite variations in restriction sites, all these plasmids shared a considerable complement of homologous sequences. Minicell-producing strains were obtained by N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. Transmission electron microscopy of infected HeLa cells showed that minicells from invasive strains retained the invasive phenotype. Sixteen polypeptides were labeled when S. flexneri 5 minicells were incubated with [35S]methionine. Fourteen of these plasmid-coded polypeptides were associated with the outer membrane in invasive strains of S. flexneri 5, and nine polypeptides of similar molecular weight were labeled in the outer membrane of invasive strains of S. flexneri 3, S. sonnei, and E. coli. Seven of the S. flexneri 5 polypeptides were not labeled in a noninvasive strain which had sustained a large deletion in the virulence-associated plasmid, and none were labeled in minicells which no longer harbored this plasmid.

Animals↗