Plasmid RFLP profiling and DNA homology inThermus isolated from hot springs of different geographical areas

Thirty-four strains belonging to various species of the genus Thermus (T. aquaticus, "T. thermophilus," "T. brockianus," T. scotoductus, and genomic species 2) isolated from hot springs of different geographical areas were examined for plasmid content and restriction fragment length polymorphism (RFLP) of plasmid DNAs. The four strains of the numerical taxonomy cluster E of genomic species 2 did not harbor plasmid DNA. Overall examination of the HindIII-RFLP profiling of plasmid DNA showed considerable variability between and within genomic species, with the exception of presumed clonal isolates. In spite of this heterogeneity, HindIII plasmid digests within a numerical taxonomic cluster gave a subset of restriction fragments of similar or identical length. Strains belonging to genomic species 2 or unclassified isolates from S. Pedro do Sul that harbored plasmid DNA (7 of the 14 strains studied) exhibited strong DNA homology between plasmid regions. No homologous sequences to these plasmid regions were found in chromosomal DNA from strains isolated from S. Pedro do Sul in which no plasmids were detected. The strains belonging to T. scotoductus formed two plasmid DNA homology groups, as estimated by probing with a plasmid fragment that coincided with the two numerical taxonomy clusters proposed previously. Among the other species, homology of plasmid regions was also found between some strains. Strong homology was also found between plasmid regions from some strains of different taxonomic groups, isolated from the same and from different sources, suggesting that these sequences are highly conserved in plasmids present in Thermus. For plasmid-containing strains, results of plasmid RFLP profiling/DNA homology appear promising for the typing of Thermus at the level of biotypes or of individual strains, namely, for monitoring the diversity and frequency of isolates from a particular hot spring. Received: 24 October 1994 / Accepted: 6 March 1995     

Occurrence of megaplasmids in halobacteria

Sixty-five halobacteria, including culture collection and freshly isolated strains from widely differing geographical areas, were examined for the presence of high molecular weight plasmids by agarose gel electrophoresis. Seventy-five per cent of all the strains were shown to harbour at least one plasmid. In the majority of strains three or four megaplasmids were detected. Approximate molecular weights of the plasmids were in the range < 100 to 300 megadaltons (Mdal). In most culture collection strains, two or three plasmids were demonstrated, except in two in which no plasmid was detected, and in two Haloarcula strains which were found to contain five and eight plasmids; four and six of the latter were more than 100 Mdal. No relationship between the plasmid profile of each strain and its taxonomic assignation nor its isolation source was found. Evidence is presented for the first time on the occurrence of megaplasmids in halobacteria.     

Restriction Fragment Length Polymorphism Analyses of the Plasmid DNAs in Strains of Xanthomonas campestris pv. vesicatoria from Different Geographic Areas

Restriction fragment length polymorphisms (RFLPs) of plasmid DNAs in Xanthomonas campestris pv. vesicatoria were analysed using 77 strains from the United states, Argentina, Australia, Taiwan, and Korea. One or more plasmids were detected in all tested strains, irrespective of geographic origin, host plant from which isolated, or chemical resistance. All Korean strains contained a few plasmids of similar high molecular weight, whereas some small plasmids occured only in strains from the United States, Argentina, and Taiwan. After digesting total plasmid DNAs with each of four restriction endonucleases, 18 fragments with sizes from about 1 to 23 kb were visualized. Seventy-seven strains of diverse geographic origins, with different levels of resistance to streptomycin and copper, were classified into the 14 RFLP groups based on the restriction endonuclease digestion patterns of their plasmid DNAs. Strains belonging to each group shared DNA fragments of identical size, suggesting the possible presence of similar plasmids in these strains. A 5.8-kb EcoRI plasmid DNA probe prepared from the United States strain 81-23 hybridized to EcoRI plasmid digests from all tested strains. Other plasmid DNA fragments of the strain81-2,3 used as probes had no homology to plasmid DNA fragments from several strains around the world. The variation in hybridization profiles of plasmid DNA was very similar to the results obtained by RFLP analysis of plasmid DNA digested by four restriction enzymes. Most of the Korean strains tested were highly sensitive to streptomycin and copper, whereas most strains from other geographic areas showed a high level of resistance to one or two of the chemicals. Cluster analysis of genetic distance between the strains based on the data obtained generated the dendrograms that separated all Korean strains from the other strains, suggesting that plasmid DNA of the Korean strains may be genetically very different from those of the others.     

Evaluation of Plasmid Content and Tetracycline Resistance Conjugative Transfer in <Emphasis Type="Italic">Enterococcus italicus</Emphasis> Strains of Dairy Origin

Five Enterococcus italicus strains harbouring tet genes responsible for the tetracycline resistance were subjected to plasmid profile determination studies. For four strains tested the profiles showed between three and six plasmid bands, the size of which ranged between 1.6 and 18.5 kb. Southern hybridization experiments associated tetS and tetK genes with chromosomal DNA in all strains and tetM gene with plasmids of around the same size (18.5 kb) in two of the tested strains. The ability of the new species to transfer tetM gene was studied by transfer experiments with the tetracycline-susceptible recipient strains E. faecalis JH2-2 and OG1RF; mobilization experiments were performed with E. faecalis JH 2-2 harbouring the conjugative plasmid pIP501as helper plasmid. The results obtained show that the new enterococcal species was able to acquire antibiotic resistance by conjugation, but not to transfer its plasmids to other bacteria. Further PCR and hybridization experiments carried out to assess the presence of mobilization sequences also suggest that the tetM plasmid from E. italicus is a non-mobilizable plasmid.     

Transfer and stability of drug resistance plasmids inEscherichia coli K12

Mating experiments between pairs of strains ofEscherichia coli containing either the compatible plasmids TP120 (Inc N) and R1 (Inc FII) or the incompatible plasmids TP125 (Inc B) and TP113 (Inc B) were undertaken in mixed continuous-flow cultures and in dialysis sacs suspended in pond water. Plasmid transfer was readily demonstrated between strains carrying compatible plasmids TP120 and R1 in both continuous-flow culture and pond water. In mixed cultures of strains carrying plasmids TP125 and TP113, transfer was only observed in continuous-flow culture systems. Strains ofE. coli containing aggregates of plasmids TP120 and R1 were shown to be stable over 5 months continuous cultivation under carbon limited conditions at a growth rate of 0.1 hours^–1 in the presence of drugs which select for the maintenance of both plasmids. In the strains containing plasmid aggregates, a gene dosage effect was observed with respect to the levels of resistance to drugs whose resistance was encoded by both plasmids. Chemostat experiments showed that no cointegrate plasmids were found from the strains ofE. coli initially containing both plasmid TP120 and plasmid R1.     

An evolutionary comparison of homologous mitochondrial plasmid DNAs from three Neurospora species

Summary We have discovered a mitochondrial DNA plasmid in N. crassa 516 (Roanoke, LA) which is homologous to those previously described from N. intermedia 435 (Fiji) and N. tetrasperma 2510 (Hanalei, HA). Subsequent analysis by DNA-DNA hybridization showed that 6 of 14 other Louisiana N. crassa isolates possessed plasmids homologous to these three plasmids, but at lower copy number. Plasmids from the three named strains were studied to examine possible plasmid diversity within each isolate, the extent of the homology between the plasmids, and the possibility that these plasmids could be inherited separately from their host mitochondria. Comparison of cloned plasmids and covalently closed circular mitochondrial DNA showed that only one plasmid line was present in each of the three intensively studied isolates. DNA-DNA hybridization and restriction endonuclease site mapping showed that the mitochondrial plasmids from the three species were very similar; most of the variation was due to presumed nucleotide substitutions. Plasmids judged identical by our analysis were found in different species. The distribution of the homologous plasmids in nature and the presence of these identical plasmids in different species, suggested that these plasmids could be transmitted between isolates independently of their host mitochondria.     

Plasmid diversity within the genus Chlamydia

Examination of 12 Chlamydia psittaci strains recovered from nine different host species (three avian and six mammalian) revealed the presence of a 7.5 kb plasmid in all isolates except two ovine abortion strains, the human strain IOL207 and the Cal 10 strain. Restriction mapping analysis distinguished four different plasmids that were associated with avian, feline, equine and guinea-pig C. psittaci isolates, respectively. The restriction maps of these four C. psittaci plasmid types all differed from that of the plasmid recovered from C. trachomatis L2/434. Despite this plasmid diversity, which is likely to be of taxonomic importance, all four plasmids identified within the species C. psittaci were found to share some sequence homology, which was mapped to two separate regions in the plasmid molecules. One region, which showed a high degree of homology between C. psittaci plasmids and also detectable homology with the C. trachomatis plasmid, may represent a common replication control region for plasmids of this genus.     

Factors influencing the copy number of F-like plasmids in Escherichia coli and Salmonella typhimurium.

The copy numbers of Flac, four F-like plasmids and pLT2 were estimated in two strains of Salmonella typhimurium and (for all except pLT2) one strain of Escherichia coli. For organisms grown in casamino acids minimal medium, the plasmids spanned a 7--8 fold range of copy number with ColB-K98 having the highest copy number in each strain and R124 the lowest. The copy number of ColB-K98 was substantially greater than 1 in each of the strains tested. There was no clear relation between the plasmid size and copy number, although the plasmids studied spanned only a narrow size range. The copy number of individual plasmids was slightly reduced or not affected at all by the presence of a second plasmid in the same strain. Derivatives harbouring each of the plasmids were grown in three different media to ascertain how plasmid copy number responds to changes in growth rate. For each plasmid, the copy number increased with decreasing growth rate. Extracts from each of the three strains harbouring ColB-K98 contained two distinct plasmid species. One appeared to be about twice as large as the other and both were absent from Col- segregants.     

Genetic variation in IncI1-co1Ib plasmids

Nucleotide sequences of portions of three plasmid genes (cib, cir, and abi) present in IncI1-Co1Ib colicin plasmids obtained from strains of Salmonella typhimurium isolated in either 1974 (Barker strains) or between 1935 and 1941 (Murray strains) were examined along with sequences of the chromosomal gene for 6-phosphogluconate dehydrogenase (gnd). Our principal findings were: (1) The plasmid genes were virtually identical to those in IncI1-CoIIb plasmids from E. coli, suggesting that Salmonella and E. coli share overlapping pools of these plasmids. (2) The plasmid genes were much less polymorphic than gnd or any other known chromosomal gene from Salmonella, further suggesting horizontal transfer with rapid transmission and turnover. (3) No characteristic differences were found in either the plasmid genes or the chromosomal gene between the 1974 isolates and the Murray strains, indicating that these plasmids have been stable for at least several decades. (4) There was an excess of amino-acid replacement polymorphisms, relative to synonymous polymorphisms, in the plasmid genes, which is consistent with the hypothesis of diversifying selection among colicin-producing plasmid families. (5) The abi (abortive infection) gene present in each of the plasmids contained two single-nucleotide insertions relative to the published sequence. These result in a putative abi protein of 114 amino acids instead of 89.     

Plasmid profiles ofLegionella species

Forty-six strains ofLegionella species were assayed for plasmid DNA content using routine laboratory procedures. Large-molecular-weight cryptic plasmids were detected inLegionella pneumophila serogroups 2, 3, and 4,L. bozemanii, L. dumoffii, L. micdadei, L. gormanii, L. longbeachae, and as yet unclassifiedLegionella-like organisms. No plasmids were found in strains ofL. pneumophila serogroups 1, 5, and 6. No correlations could be made between the possession of a specific plasmid profile, or lack of one, and any phenotypic markers such as virulence or antibiotic resistance. Several parameters were identified in this study as critical to the isolation of plasmid DNA fromLegionella: (i) DNA preparations obtained from frozen egg or animal materials had a higher incidence of detectable plasmid DNA than subcultures on bacteriologic media. (ii) A newly formulated broth supported exponential growth in all of the 46 strains; one strain required the addition of CO₂. (iii) Considerable heterogeneity was seen in cell susceptibility to various detergents. Since no single lytic agent was suitable for all strains, both ionic and noninic lysis methods were used with each strain. Within the limitations of both crude lysate preparations and the agarose gel electrophoresis method, this study identified a large 60–80 megadalton plasmid species in over 50% of the plasmid-containing strains.     

Plasmid diversity in senescent and nonsenescent strains of Neurospora

Summary A sample of 171 natural isolates of Neurospora crassa and Neurospora intermedia was tested for senescence. Of these, 28 strains senesced within the duration of the experiment. These senescent strains, together with a selection of nonsenescent strains, were examined for the presence of mitochondrial plasmids. This was done by digesting mitochondrial DNA preparations with proteinase K, and running these samples on agarose gels. Most of the strains examined, both senescent and nonsenescent, contained plasmids, many of them new. Some new plasmids were linear, as inferred from their resistance to 5 exonuclease and sensitivity to 3 exonuclease. New circular plasmids were also found. Some strains carry several plasmids, and mixtures of circular and linear elements were common. A cross-homology study was performed on a sample of plasmid-bearing strains, and several cases of apparent relatedness were found, some between strains from distant geographical locations. Linear plasmids homologous to the maranhar linear senescence plasmid were quite common. A new member of the LaBelle circular plasmid homology group was found. In the sample tested for homology, no strains contained elements related to the kalilo linear senescence plasmid. The relationship of the new plasmids to senescence is not known. In addition to plasmid monomers, several different types of derivatives were found. The kalilo linear plasmid was found to occur in linear and circular forms of low mobility, presumed to be giant concatamers, and, in some strains, variant sibling structures and ladders of short derivatives were found. Circular plasmids also gave rise to extensive ladders on electrophoresis, probably representing different relaxation states and head-to-tail concatameric series. Some such forms migrated more slowly than mitochondrial DNA. One unique type of plasmid modification observed was a pair of linear elements that had apparently arisen de novo which showed homology to a circular plasmid.     

Plasmids in clinical isolates ofStreptococcus pneumoniae

Forty clinical isolates ofStreptococcus pneumoniae with various antibiotic resistance profiles were screened for the presence of plasmids. Plasmids were demonstrated in five isolates. Three procedures for plasmid isolation were evaluated. A 10.8-kb plasmid was demonstrated by all three methods, but a further four plasmids were detected with one method only. The sizes of these plasmids were 11.5 kb, 10.8 kb, and 3.0 kb (two strains). Curing experiments were performed, but no plasmid/antibiotic correlation was observed. Tetracycline, erythromycin, and clindamycin resistance was lost in one strain, although the plasmid was still present.     

Survey of plasmid profiles of <Emphasis Type="Italic">Shigella</Emphasis> species isolated in Malaysia during 1994–2000

Summary Plasmid profiling was used to characterize 219 strains of Shigellaspecies isolated from sporadic cases of shigellosis in Malaysia during the period 1994–2000. Heterogeneous plasmid patterns were observed in all Shigella spp. There was a correlation between plasmid patterns and serotypes of S. flexneri, S. dysenteriaeand S. sonnei. Five common small plasmids (>20.0 kb) were observed in S. flexneri1b and 2a, whereas six common small plasmids were found in serotype 3a. Some of these plasmids appeared to maintain their existence stably in each individual serotype. Plasmids of size 11.40 and 4.20 kb were present only in S. flexneri2a isolates, whereas the 4.40 kb plasmid was unique for serotype 3a. Large (>150 kb) or mid-range plasmid (20.0–150 kb) was not observed from any S. flexneri1b isolates. Eighty-nine percent of S. flexneriof various serotypes harboured the plasmid of 3.20 kb. All S. dysenteriaetype 2 isolates harboured the 9.00 kb plasmid, while four common small plasmids were found in S. sonneiisolates. The 2.10 kb plasmid was only seen in S. sonnei. Streptomycin resistance in S. dysenteriaetype 2 and multi-drug resistance in S. sonneimay be associated with the 9.00 and 14.8 kb plasmids, respectively. Plasmid profiling provided a further discrimination beyond serotyping and a useful alternative genotypic marker for differentiation of Shigellaspecies. To the best of our knowledge, this is the first report on the plasmid prevalence of the Malaysian Shigellaspecies.     

Distinct plasmid profiles of Pasteurella haemolytica serotypes and the characterization and amplification in Escherichia coli of ampicillin-resistance plasmids encoding ROB-1 beta-lactamase.

Thirty-five isolates of Pasteurella haemolytica from cattle or sheep were screened for the presence of plasmids and for resistance to a range of antibiotics. Eight strains (four of serotype A1, three of serotype A2 and one untypable) contained plasmid DNA and isolates of the same serotype had similar plasmid profiles, which were different from those of the other serotypes. All but one of the plasmid-bearing strains were isolated from pneumonic animals or from animals in contact with pneumonic cattle or sheep. In A2 and untypable strains, there was no obvious correlation between antibiotic resistance and the presence of a specific plasmid. In contrast, all plasmid-bearing A1 strains exhibited ampicillin resistance (ApR), which was shown by transfer studies to be plasmid-mediated. Plasmid DNA prepared from E. coli transformants was not routinely detected on ethidium-bromide-stained agarose gels, but could be amplified to detectable levels by treatment of cultures with chloramphenicol (Cm) or by modifying the growth conditions. The ApR plasmids from P. haemolytica were identical by restriction enzyme analysis. Restriction analysis and hybridization data indicated that these plasmids were closely related to the prototype ROB-1 beta-lactamase-encoding plasmid, originally isolated from Haemophilus influenzae. From substrate profiles and isoelectric focusing data, the beta-lactamases encoded by the P. haemolytica plasmids were indistinguishable from the ROB-1 beta-lactamase.     

Concatemer formation of ColE1-type plasmids in mutants of Escherichia coli lacking RNase H activity

rnh mutants harboring pBR322 were found to contain several slowly migrating DNA species when examined by agarose gel electrophoresis. The plasmid DNA from rnh mutants included large molecules, i.e. plasmids two, three or four times the size of a single plasmid unit. That this DNA contained concatemeric plasmid joined in a head-to-tail fashion was determined by digestion with restriction endonucleases that cleaved the monomeric plasmid DNA at a unique site. This treatment resulted in migration of the plasmid DNA at a mobility identical to that of linearized monomeric plasmid by agarose gel electrophoresis. This was confirmed by electron microscopy. Plasmid concatemer formation was detected with several high-copy-number (relaxed type) plasmids but not with low-copy-number (stringent) plasmids. Concatemer formation was dependent on RecA+ and RecF+ functions since several recA and recF mutations abolished concatemer formation. ColE1-type plasmids were previously shown to replicate in rnh mutants in the absence of DNA polymerase I (PolI) activity. This DNA PolI-independent plasmid replication was also examined for its dependence on the recF and recA gene products. rnh- polA(Ts) recF- strains were efficiently transformed with these plasmids at 30 degrees C and 42 degrees C, indicating the presence of DNA PolI-independent replication under recF- conditions. The presence or absence of plasmid replication in rnh- polA- recA(Ts) strains was also examined by measuring the increase in total amounts of plasmid. The results indicated that DNA PolI-independent replication occurred in these triple mutants at 42 degrees C as well as at 30 degrees C. It was concluded that the recombination event giving rise to concatemer formation was not essential for DNA PolI-independent replication in rnh mutants.     

Diversity ofFrankia strains isolated from a single alder stand

One hundredFrankia strains isolated from variousAlnus species in a single alder stand were tested for plasmid presence. Plasmid DNA was observed in five of the frankiae strains and was analyzed. We found that plasmids with a similar molecular weight exhibited in fact minor divergences in restriction patterns. The genetic diversity among the five isolates which contained plasmids and seven isolates which contained no plasmid DNA were examined by using restriction endonucleas digestions, Southern hybridization ofnifHDK,nifAB genes, andFrankia cryptic DNA fragments determined at random. Results indicate that genomic DNA digestion patterns and Southern hybridizations to anifHDK probe were not able to discriminate between closely related frankiae. On the other hand, plasmid presence, Southern hybridization to anifAB proble or to a crypticFrankia probe allowed us to delineate groupings of these isolates.     

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.     

Involvement of a Polyethylene glycol (PEG)-oxidizing Enzyme in the Bacterial Metabolism of PEG

Eighty-seven strains of acetic acid bacteria were surveyed for plasmids by CsCl-ethidium bromide equilibrium centrifugation of cell lysates. Twenty-seven of the 33 strains of Acetobacter were found to harbor plasmid DNA and most strains contained multiple species of low-molecular-weight plasmids. On the other hand, plasmid DNAs were detected in 23 of the 36 strains of Gluconobacter and most of them had molecular weights of more than 5 megadaltons. Of the 18 strains newly isolated from a vinegar factory, some of which were examined taxonomically, 17 contained plasmid DNA. The molecular weights of plasmids detected in this study were in the range of about one to over 17 megadaltons. The plasmids with molecular weights of less than 5 megadaltons were characterized with restriction endonucleases. The physical maps of two plasmids designated as pMV1O1 and pMV102, which were found in the isolated strains, were constructed.     

Characterization of new plasmids from methylotrophic bacteria

Several tens of methanol-utilizing bacterial strains isolated from soil were screened for the presence of plasmids. From the obligate methylotrophMethylomonas sp. strain R103a plasmid pIH36 (36 kb) was isolated and its restriction map was constructed. In pink-pigmented facultative methylotrophs (PPFM), belonging to the genusMethylobacterium four plasmids were detected: plasmids pIB200 (200 kb) and pIB14 (14 kb) in the strain R15d and plasmids pWU14 (14 kb) and pWU7 (7.8 kb) in the strain M17. Because of the small size and the presence of several unique REN sites (HindIII, EcoRI, NcoI), plasmid pWU7 was chosen for the construction of a vector for cloning in methylotrophs. Cointegrates pKWU7A and pKWU7B were formed between pWU7 and theE. coli plasmid pK19 Km^r, which were checked for conjugative transfer fromE. coli into the methylotrophic host.     

Plasmids from Lactobacillus helveticus: distribution and diversity among natural isolates

AIMS: To investigate the distribution and the level of diversity of extrachromosomal molecules in Lactobacillus helveticus strains in relation to their different ecological niches. METHODS AND RESULTS: The plasmid profile of 22 Lact. helveticus strains, isolated from five different Italian cheeses, was determined. Among the tested strains, there was a variable presence of plasmids: eight plasmid-free strains and the remaining with several plasmids that could be differentiated on the basis of number and molecular weight. The profiles showed between one and five plasmid bands, which size ranged between 2.3 and 31 kb. Four of these plasmids were further analyzed by restriction digestion and compared with the plasmids from Lact. helveticus ATCC 15009(T). Analyses and comparison of their primary structures and hybridization experiments revealed the presence of different DNA homology groups. CONCLUSIONS: This study indicates that within Lact. helveticus species, there is a high degree of variability in relation to the presence of plasmid molecules. Moreover, the structural diversity found among some of these plasmids allows to hypothesize the presence of different evolutionary lineages. SIGNIFICANCE AND IMPACT OF THE STUDY: Studies on plasmid distribution and diversity should be considered as an essential component in a continuing effort to explore microbial diversity as well as to understand the real role of plasmids in the flow of genetic information in natural bacterial communities.