Electrophoretic comparison of endonuclease-digested plasmids from Neisseria gonorrhoeae.

In order to associate virulence in Neisseria gonorrhoeae with an alteration of the nucleotide sequence of its small covalently closed plasmid, plasmid deoxyribonucleic acid was isolated from both virulent (T1) and avirulent (T3) morphological types for two strains. Electrophoretic and contour length measurements of intact plasmids indicated a homogeneous population with a molecular weight of approximately 2.6 x 10(6). Digestion with two restriction endonucleases. Hinf I and Hpa II, generated distinct fragment patterns which in each case were identical for T1 and T3 plasmid molecules from the same strain. The analysis suggests no sequence differences between the plasmids from virulent and avirulent types. For both strains, however, a deletion or addition of about 1.5% of the total deoxyribonucleic acid appeared in the Hpa II C digestion fragment when patterns for gonococci serially passaged 300 times were compared to those for bacteria freshly established from frozen stocks. The significance of the plasmid instability remains undetermined.     

Epstein-Barr virus shuttle vector for stable episomal replication of cDNA expression libraries in human cells.

Efficient transfection and expression of cDNA libraries in human cells has been achieved with an Epstein-Barr virus-based subcloning vector (EBO-pcD). The plasmid vector contains a resistance marker for hygromycin B to permit selection for transformed cells. The Epstein-Barr virus origin for plasmid replication (oriP) and the Epstein-Barr virus nuclear antigen gene have also been incorporated into the vector to ensure that the plasmids are maintained stably and extrachromosomally. Human lymphoblastoid cells can be stably transformed at high efficiency (10 to 15%) by such plasmids, thereby permitting the ready isolation of 10(6) to 10(7) independent transformants. Consequently, entire high-complexity EBO-pcD expression libraries can be introduced into these cells. Furthermore, since EBO-pcD plasmids are maintained as episomes at two to eight copies per cell, intact cDNA clones can be readily isolated from transformants and recovered by propagation in Escherichia coli. By using such vectors, human cells have been stably transformed with EBO-pcD-hprt to express hypoxanthine-guanine phosphoribosyltransferase and with EBO-pcD-Leu-2 to express the human T-cell surface marker Leu-2 (CD8). Reconstruction experiments with mixtures of EBO-pcD plasmids demonstrated that one clone of EBO-pcD-hprt per 10(6) total clones or one clone of EBO-pcD-Leu-2 per 2 x 10(4) total clones can be recovered intact from the transformed cells. The ability to directly select for expression of very rare EBO-pcD clones and to then recover these episomes should make it possible to clone certain genes where hybridization and immunological screening methods are not applicable but where a phenotype can be scored or selected in human cell lines.     

Plasmid profiles and randomly amplified polymorphic DNA analysis of Salmonella enterica serotype Enteritidis strains from outbreaks and sporadic cases in Turkey

The aim of the study was to investigate the characteristics of Salmonella serotype Enteritidis strains isolated from outbreaks and sporadic cases in Turkey by plasmid profiles and randomly amplified polymorphic DNA (RAPD) patterns. A total of 64 S. Enteritidis clinical strains were selected from the culture collection of the Enterobacteria Laboratory of Ankara University Medical School Department of Microbiology and Clinical Microbiology for molecular analysis using the plasmid profiles and RAPD method. Fifty-six isolates (88%) harbored one to four plasmids ranging in size from 2.5 to 100 kbp. 57 kbp plasmids were the most common plasmids, and forty-four strains (69%) carried 57 kbp plasmids alone or together with other plasmids. The outbreak strains carried the same plasmid profile: three plasmids sized 57, 40, 3.0 kbp. None of the strains analyzed displayed any RAPD bands with the primer OPB-17. By using primer p-1254, 42 strains (66%) were divided into fourteen RAPD patterns. Ten of the outbreak strains (77%) showed >80% similarity by cluster analysis program. Analysis of RAPD-PCR with primer p-1254 proved an easy, rapid and discriminative method complementing antibiogram and plasmid profiles in routine laboratories, and may contribute to the investigations of S. Enteritidis which still cause outbreaks in Turkey. This study presents the first report on S. Enteritidis isolates in Turkey investigated by plasmid profiles and RAPD methods.     

Use of interplasmid recombination to generate stable selectable markers for yeast transformation: application to studies of actin gene control

A plasmid recombination system has been developed that relies upon interplasmid exchanges for yeast cell viability. Two types of plasmids, one carrying the LEU2 allele inserted within yeast actin gene sequences and the other carrying 2-microns plasmid DNA and an intact actin gene, were constructed. Neither plasmid alone yielded transformants in the haploid Leu- strain AH22, but when cotransformed, a number of colonies were obtained. Southern blot analysis revealed that transformants arose because of recombination events within the homologous actin sequences that transferred the LEU2 gene to the actin gene on the 2-microns plasmid. The recombinant plasmids could be recovered, and sequence analysis of one recombination site revealed that the exchange event was faithful at the nucleotide level. The resulting recombinant plasmids carried a defective actin gene and presumably arose because of a double-crossover event. Deletion mutations that prevented actin gene expression on one donor plasmid enabled the recovery at a high frequency of transformants resulting primarily from single-crossover events between the two plasmids. This was presumably because such events no longer generated an intact actin gene on a multicopy plasmid. Infrequently a transformant from a plasmid with an intact gene was recovered, but in these cases the plasmid was not present in multiple copies. These cells exhibited a slower growth rate, and Northern blot analysis revealed an elevated level of actin mRNA.     

The large plasmids found in enterohemorrhagic and enteropathogenic Escherichia coli constitute a related series of transfer-defective Inc F-IIA replicons.

Forty-six of 52 (88.5%) enterohemorrhagic Escherichia coli (EHEC) strains screened carried a "common" plasmid of about 90 kb which encoded sequences homologous to the Inc F-IIA replicon. A similarly high incidence of Inc F-IIA plasmid-containing strains was observed in other groups of diarrheagenic E. coli, but not in random environmental coliform isolates. Enteropathogenic E. coli (EPEC) contain plasmids of similar properties and share a 23-kb DNA fragment with plasmids from EHEC. The common region encodes the F-IIA replication region and sequences homologous to the transfer operon of the Inc F-II plasmid R1. Sequence homology varied between plasmids isolated from different EHEC/EPEC strains with > 80% showing homology to the regions encoding the rep and par genes. Only 5% of plasmids from EHEC strains had intact sequences homologous to the DNA between these two regions, including the oriT site. Some plasmids with an apparently intact tra operon still failed to plaque F-pilus-specific phages. This is consistent with observations that the large plasmids of EHEC and EPEC are phenotypically nonconjugative. These results suggest that the large plasmids of EHEC/EPEC constitute a family of transfer-deficient Inc F-IIA plasmids with varying degrees of deletion in tra function. The evolutionary ramifications of this finding are considered.     

Amplification of the respiratory NADH dehydrogenase of Escherichia coli by gene cloning.

A relatively simple method has been used to clone the gene coding for the respiratory NADH dehydrogenase (NADH-ubiquinone oxidoreductase) of Escherichia coli from unfractionated chromosomal DNA. The restriction endonucleases EcoRI, BamI and HindIII were used to construct three hybrid plasmid pools from total E. coli DNA and the amplifiable plasmids pSF2124 and pGM706. Three different restriction endonucleases were used to increase the chances of cloning the ndh gene intact. Mobilization by the plasmid F was used to transfer the hybrid plasmids into ndh mutants and selection was made for Apr and complementation of ndh. DNA fragments complementing ndh were isolated from both the EcoRI and HindIII hybrid plasmid pools. The strain carrying the hybrid plasmid constructed with EcoRI produced about 8--10 times the normal level of the respiratory NADH dehydrogenase in the cytoplasmic membrane. Treating the cells with chloramphenicol to increase the plasmid copy number allowed the level of NADH dehydrogenase in the membrane to be increased to 50--60 times the level in the wild type. The results indicate the potential of gene cloning for the specific amplification of particular proteins prior to their purification.     

Trimethoprim resistance plasmids in Escherichia coli isolated from cases of diarrhoea in cattle, pigs and sheep

A total of 1572 isolates of Escherichia coli obtained from the faeces of young farm animals with diarrhoea over the period 1980–1983 were screened for resistance to trimethoprim (Tp). Resistance to Tp was detected in263/954 (28%) of bovine isolates,59/441 (13%) of porcine isolates and15/177 (9%) of ovine isolates. Seventy-five resistant isolates from separate outbreaks of infection on farms within a 25 mile radius of Nottingham were examined in detail. Sixty-eight (91%) of the 75 isolates were resistant to > 1024 mg Tp/1 and 34 (50%) of these 'highly resistant' isolates (45% of total resistant isolates) transferred their Tp resistance to E. coli K12. A further 13 (17%) isolates were demonstrated to carry non-self-transferable plasmids which were capable of being mobilized to E. coli K12 by the broad host range plasmid RP4. Thirty-one self-transferable Tp R plasmids were divided between the following incompatibility groups: IncB (14 plasmids), IncF_***H (4 plasmids), IncH₂ (1 plasmid), IncIaP (10 plasmids), IncIdT (1 plasmid) and IncP (1 plasmid). In terms of antibiotic resistance patterns and incompatibility properties, many of these plasmids closely resembled those isolated from human patients in the same area, suggesting that there may be a common pool of Tp R plasmids.     

The large plasmids found in enterohemorrhagic and enteropathogenic Escherichia coli constitute a related series of transfer-defective Inc F-IIA replicons

Forty-six of 52 (88.5%) enterohemorrhagic Escherichia coli (EHEC) strains screened carried a “common” plasmid of about 90 kb which encoded sequences homologous to the Inc F-IIA replicon. A similarly high incidence of Inc F-IIA plasmid-containing strains was observed in other groups of diarrheagenic E. coli, but not in random environmental coliform isolates. Enteropathogenic E. coli (EPEC) contain plasmids of similar properties and share a 23-kb DNA fragment with plasmids from EHEC. The common region encodes the F-IIA replication region and sequences homologous to the transfer operon of the Inc F-II plasmid R1. Sequence homology varied between plasmids isolated from different EHEC/EPEC strains with >80% showing homology to the regions encoding the rep and par genes. Only 5% of plasmids from EHEC strains had intact sequences homologous to the DNA between these two regions, including the oriT site. Some plasmids with an apparently intact tra operon still failed to plaque F-pilus-specific phages. This is consistent with observations that the large plasmids of EHEC and EPEC are phenotypically nonconjugative. These results suggest that the large plasmids of EHEC/EPEC constitute a family of transfer-deficient Inc F-IIA plasmids with varying degrees of deletion in tra function. The evolutionary ramifications of this finding are considered.     

Shuttle plasmids for Escherichia coli and Clostridium perfringens.

Small plasmids which replicate in both Escherichia coli and Clostridium perfringens were made by recombining E. coli plasmid pBR322 with three different small (less than 4 kilobases) plasmids native to C. perfringens. Subsequently, two homologous, though distinct, tetracycline resistance determinants (tet) from other C. perfringens plasmids were cloned into them. Both tet systems made E. coli resistant to at least 5 micrograms of tetracycline per ml when resident on the shuttle plasmids. The shuttle vectors have been used to transform L-phase variants and autoplasts of C. perfringens. In the latter case, the intact transforming plasmid could be isolated from walled cells after cell wall regeneration. Reciprocal transformation experiments in which plasmid DNAs derived from E. coli or C. perfringens were used suggest that restriction barriers exist between these two organisms. The plasmids contain restriction enzyme recognition sites in locations which are useful for cloning experiments.     

Regions on the F plasmid which affect plasmid maintenance and the ability to segregate into Escherichia coli minicells

Certain derivative mini-F plasmids were found to segregate into Escherichia coli minicells, in contrast to the intact mini-F plasmid which does not. Segregation was not related to the presence or absence of the normal origin of vegetative replication, but appeared to be affected by regions of F which encode replication, incompatibility, copy number control, and partitioning functions. Segregation of mini-F plasmids into minicells was not random; the plasmid concentration in minicells did not correlate with the plasmid concentration in cells. Genes, or gene products, of F from the region spanning the sequences 44.1–49.3F appeared to affect the ability of mini-F plasmids to segregate into minicells. Segregation of mini-F plasmids into minicells was not directly related to stable plasmid inheritance. These results argue for the sequestration of mini-F plasmids in host cells.     

Liposome fragment-mediated introduction of multiple plasmids into Bacillus subtilis

Transformation of microorganisms by plasmid introduction is one of the central techniques in modern biotechnology. However, applicable transformation methods for simultaneous introduction of multiple plasmids are still limiting. Here, we reported a liposome-mediated method that efficiently introduces multiple plasmids into B. subtilis. In this method, liposomes containing three kinds of plasmids were mixed with B. subtilis protoplasts in the presence of 36% polyethylene glycol (PEG), and the resultant protoplasts were grown in cell wall-regeneration media. We found that the rates of introduction of multiple plasmids were significantly increased in the presence of liposomes. We also found that an intact liposome structure was not required for introduction, and the presence of phosphatidylglycerol (PG) was important for efficient introduction of multiple plasmids. Therefore, the liposome- or liposome fragment-mediated transformation method reported here can advance studies utilizing multiple plasmids.     

Construction of isogenic gonococcal strains varying in the presence of a 4.2-kilobase cryptic plasmid.

A 4.2-kilobase (kb) cryptic plasmid is present in 96% of isolates of Neisseria gonorrhoeae. An inability to construct isogenic derivatives which vary in the presence of the 4.2-kb plasmid has prevented the study of its function. We report a method to deliver an intact 4.2-kb plasmid into plasmidless gonococcal strains. The method involved transformation with novel 15.7-kb hybrid penicillinase-producing (Pcr) plasmids, which were cointegrates containing two copies of the 4.2-kb plasmid arranged in tandem direct repeat plus one copy of the 7.2-kb Pcr plasmid pFA3. When the 15.7-kb hybrid Pcr plasmids were introduced into a gonococcal recipient lacking evident plasmids, they dissociated at a relatively high frequency into plasmids identical to their parents: the 4.2-kb cryptic plasmid and pFA10 (a stable 11.5-kb plasmid containing one copy of each of the 7.2-kb Pcr plasmid pFA3 and the 4.2-kb cryptic plasmid pFA1). Curing strains of their Pcr plasmids resulted in isogenic strains which varied only in the presence of the 4.2-kb plasmid. The presence of the autonomously replicating 4.2-kb plasmid did not affect a number of tested phenotypes, including auxotype, antibiotic sensitivity, and frequencies of variation of outer membrane protein II. The interpretation of the functional significance of the 4.2-kb plasmid was complicated, however, by the additional finding that each of three tested plasmid-free strains contained a chromosomal fragment of about 1.6 kb that hybridized under moderate stringency with a 1.65-kb HinfI fragment of the 4.2-kb plasmid.     

Study of the influence of plasmids on the arbitrary primer polymerase chain reaction fingerprint of Escherichia coli strains

Abstract To study the effect of plasmids on the arbitrary primer-polymerase chain reaction fingerprint of bacterial strains, the Escherichia coli strains DH5, Top10, and W3110 were transformed with plasmids of different sizes: respectively, pUC19, pCEP and two clinically important plasmids carrying resistance to several antibiotics. Total DNA, i.e. both chromosomal and plasmid DNA, was prepared from transformed cells by boiling the cell suspensions and by phenol-chloroform extraction; chromosomal DNA was prepared by the same methods from the non-transformed, plasmid-free strains; plasmid DNA of pUC19 was purchased; plasmid DNA of pCEP was purified from the transformed strains by caesium chloride density gradient centrifugation. Arbitrarily primed polymerase chain reaction was carried out for all of these preparations. Amplification carried out independently with three different primers resulted in similar patterns for the chromosomal preparations whether or not plasmid was present. Amplification of plasmid DNA gave different patterns, characterized by fragments larger than those obtained when total or chromosomal DNA were used as the target. These data illustrate that the plasmids studied here do not influence the chromosomal arbitrarily primed PCR fingerprint, although plasmids alone are amplified in the absence of chromosomal DNA. Experiments comparing different relative concentrations of plasmid and chromosomal DNA indicate that under natural conditions the amount of chromosomal DNA per cell is sufficient to inhibit observable amplification of the plasmid(s) present.     

Conjugal transferability of multiple resistance in Shigella strains

Shigella strains isolated in Japan between 1971 and 1979 were surveyed for drug resistance and distribution of R plasmids. Of 2,510 strains, 89.3% were resistant to either one or various combinations of four drugs, tetracycline, chloramphenicol, streptomycin, and sulfanilamide. About 66% of the Shigella isolates were quadruply resistant. The frequency of isolation of R plasmids from quadruply resistant Shigella strains was the highest when compared with other strains resistant to various combinations of the four drugs. The conjugal transferability of 204 quadruply resistant strains isolated between 1977 and 1979 was tested by various mixed-culture methods. Among the total strains examined, 70.6% carried transferable resistance when tested by the conventional broth culture method, 90.2% transferred their resistance when, in addition the replica-plating method was used and 97.5% could transfer their resistance when the membrane filter method was also used. Although the remaining five strains could not transfer their resistance by any of the mixed culture methods, the drug resistance of four of the five strains was mobilized by the concomitant presence of F-tet or T-kan plasmid. These results indicate that almost all of the quadruple resistance in Shigella isolates was mediated by plasmid.     

Co-Transduction of Plasmids Mediating Resistance to Tetracycline and Chloramphenicol in Staphylococcus aureus

Independent plasmids mediating resistance either to tetracycline (Tc) or chloramphenicol (Cm) were transduced successively into Staphylococcus aureus strain 8325. From this doubly resistant donor strain, Tc was co-transduced with a frequency of 40 to 50% when Cm was selected. Co-transduction of Cm was 5 to 10% with Tc selection. Plasmid elimination was infrequent and restricted to the Cm plasmid. A variant, doubly resistant strain gave 100% co-transduction of Tc and Cm and a high rate of joint elimination of both plasmid markers. Co-transduction of the plasmids from recombination-deficient donor strains was much reduced if the plasmids had been introduced separately into the donor strain, but occurred at the normal high rate if they had been introduced jointly. The plasmids were co-transformed at relatively low rates with closed circular deoxyribonucleic acid (DNA) from doubly resistant donors, but not with DNA from a mixed lysate of singly resistant strains. Our evidence favored a hypothesis of recombination-dependent, reversible linkage between the two plasmids as the basis for their co-transduction. Examination of plasmid DNA from the doubly resistant strains by ultracentrifugal and electron microscopic methods did not disclose any physical differences between singly and doubly resistant strains that might account for the observed co-transduction.     

Characterization of pGA1, a new plasmid from Corynebacterium glutamicum LP-6.

A new plasmid, pGA1, has been isolated from Corynebacterium glutamicum LP-6, and its detailed restriction map has been prepared. The 4.9-kb plasmid has a G + C content of 57%. It replicates in C. glutamicum ATCC13032 and is compatible with the three other plasmids, pCC1, pBL1 and pHM1519, commonly used for vector construction for amino acid-producing corynebacteria. Fusions of pGA1 with different Escherichia coli replicons (transferred from E. coli to Corynebacterium via transformation of spheroplasts or by filter mating experiments with intact cells) are shown to be suitable as shuttle plasmids; some of them are highly stable in C. glutamicum, even when propagated without any selection pressure.     

Bacterial Plasmids in Antarctic Natural Microbial Assemblages

Samples of psychrophilic and psychrotrophic bacteria were collected from sea ice, seawater, sediments, and benthic or ice-associated animals in McMurdo Sound, Antarctica. A total of 155 strains were isolated and tested for the presence of plasmids by DNA agarose gel electrophoresis. Thirty-one percent of the isolates carried at least one kind of plasmid. Bacterial isolates taken from sediments showed the highest plasmid incidence (42%), and isolates from seawater showed the lowest plasmid incidence (20%). Plasmids were significantly more frequent in the strains which had been first isolated from low-nutrient media (46%) than in the strains which had been isolated from high-nutrient media (25%). Multiple forms of plasmids were observed in two-thirds of the plasmid-carrying strains. A majority of the plasmids detected were estimated to have a mass of 10 megadaltons or less. Among 48 plasmid-carrying strains, 7 showed antibiotic resistance. It is concluded that bacterial plasmids are ubiquitous in natural microbial assemblages of the pristine marine ecosystem of Antarctica.     

Purification of supercoiled plasmids from crude cell lysates using high performance anion exchange chromatography

High performance liquid chromatography is of increasing importance in the purification of nucleic acids. Recently, a new anion exchange column called Gen-Pak FAX has been introduced for this purpose. Previously, it has been used in the purification of restriction fragments and oligonucleotides. In this paper we present the use of the Gen-Pak FAX column for the purification of plasmids from crude E. coli lysates. The different conformational forms of the plasmid can be well separated and collected with high recoveries of both mass and activity. Up to 50 micrograms of supercoiled plasmid can be purified in a single 30 min run with up to 98% purity.     

Detection and characterization of broad-host-range plasmids in environmental bacteria by PCR.

Primer systems for PCR amplification of different replicon-specific DNA regions were designed on the basis of published sequences for plasmids belonging to the incompatibility (Inc) groups IncP, IncN, IncW, and IncQ. The specificities of these primer systems for the respective Inc groups were tested with a collection of reference plasmids belonging to 21 different Inc groups. Almost all primer systems were found to be highly specific for the reference plasmid for which they were designed. In addition, the primers were tested with plasmids which had previously been grouped by traditional incompatibility testing to the IncN, IncW, IncP, or IncQ group. All IncQ plasmids gave PCR products with the IncQ primer systems tested. However, PCR products were obtained for only some of the IncN, IncP, and IncW group plasmids. Dot blot and Southern blot analyses of the plasmids revealed that PCR-negative plasmids also failed to hybridize with probes derived from the reference plasmids. The results indicated that plasmids assigned to the same Inc group by traditional methods might be partially or completely different from their respective reference plasmids at the DNA level. With a few exceptions, all plasmids related to the reference plasmid at the DNA level also reacted with the primer systems tested. PCR amplification of total DNA extracted directly from different soil and manure slurry samples revealed the prevalence of IncQ- and IncP-specific sequences in several of these samples. In contrast, IncN- and IncW-specific sequences were detected mainly in DNA obtained from manure slurries.