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PubMed1998

Characterization of multiple regions involved in replication and mobilization of plasmid pNZ4000 coding for exopolysaccharide production in Lactococcus lactis.

We characterized the regions involved in replication and mobilization of the 40-kb plasmid pNZ4000, encoding exopolysaccharide (EPS) production in Lactococcus lactis NIZO B40. The plasmid contains four highly conserved replication regions with homologous rep genes (repB1, repB2, repB3, and repB4) that belong to the lactococcal theta replicon family. Subcloning of each replicon individually showed that all are functional and compatible in L. lactis. Plasmid pNZ4000 and genetically labeled derivatives could be transferred to different L. lactis strains by conjugation, and pNZ4000 was shown to be a mobilization plasmid. Two regions involved in mobilization were identified near two of the replicons; both included an oriT sequence rich in inverted repeats. Conjugative mobilization of the nonmobilizable plasmid pNZ124 was promoted by either one of these oriT sequences, demonstrating their functionality. One oriT sequence was followed by a mobA gene, coding for a trans-acting protein, which increased the frequency of conjugative transfer 100-fold. The predicted MobA protein and the oriT sequences show protein and nucleotide similarity, respectively, with the relaxase and with the inverted repeat and nic site of the oriT from the Escherichia coli plasmid R64. The presence on pNZ4000 of four functional replicons, two oriT sequences, and several insertion sequence-like elements strongly suggests that this EPS plasmid is a naturally occurring cointegrate.

Amino Acid Sequence↗
PubMed2004

Characterization of pC7 from Lactobacillus paraplantarum C7 derived from Kimchi and development of lactic acid bacteria--Escherichia coli shuttle vector.

A cryptic plasmid pC7 was characterized, which was isolated from Lactobacillus paraplantarum C7 derived from Kimchi, a traditional korean vegetable food. The plasmid pC7 is a circular molecule of 2,134 base-pairs in length with a G + C content of 38.5%. The nucleotide sequence analysis revealed the presence of an open reading frame encoding a putative 317 amino acids protein homologous to replication proteins RepA. Furthermore, a putative double and a single-strand origin were identified. Together with features of nucleotide sequences, the detection of single-stranded intermediate DNA in Lb. paraplantarum demonstrated that pC7 replicates via a rolling circle mechanism. A cloning vector for lactic acid bacteria was developed on the basis of the pC7 replicon, into which were inserted an erythromycin resistance gene as a marker, multiple cloning sites, and Escherichia coli ColE1 replication origin. E. coli and several species of Lactobacillus and Leuconostoc can be transformed with the resultant vector. Therefore, pC7 derivatives may be useful LAB-E. coli shuttle vectors, which are essential in engineering important strains in food fermentation.

Amino Acid Sequence↗
PubMed1984

Transfer of yeast telomeres to linear plasmids by recombination.

We have characterized two types of recombination events between linear plasmids and yeast chromosomal telomere-adjacent sequences (Y' elements). In one type of event, a linear plasmid restriction-cut within a Y' element regains the missing Y' DNA, and may also acquire additional Y' elements. This process is similar to the healing of broken chromosomes by recombination. In a second type of event, terminally added C1-3A sequences on the linear plasmid interact with C1-3A sequences located just internal to the chromosomal Y' elements, resulting in the addition of one to four Y' elements to the plasmid. Similar recombination events occurring between different chromosome ends could lead to the dispersal and amplification of telomere-adjacent sequences.

Base Sequence↗
PubMed1975

Accumulation of replicating bacterial plasmid DNA during thymine limitation or hydroxyurea treatment.

The rate of DNA chain elongation of a thymine auxotroph of Proteus mirabilis harboring an R factor was reduced by either thymine limitation or treatment with hydroxyurea. Using CsCl density gradient centrifugation, sedimentation in sucrose gradients, and electron microscopy, it has been shown that the fraction of total R factor DNA which is in the process of replication is greatly increased when the rate of DNA chain elongation is reduced while the initiation of plasmid replication continues at the normal rate. This makes possible the isolation and characterization of replicating plasmid DNA. Electron microscopy revealed that replicating R factor DNA consists of double-branched (theta type) circular molecules.

Centrifugation, Density Gradient↗
PubMed1985

SAL-TOL in vivo recombinant plasmid pKF439.

SAL-TOL in vivo recombinant plasmid pKF439 was characterized in a strain from a mixed culture of bacteria harboring various degradative plasmids. Analysis of the gene organization of pKF439 revealed that the 57-kilobase TOL fragment, including the 40-kilobase TOL metabolic region, was inserted into the complete SAL replicon at the position of SmaI-C within XhoI-B of SAL. The molecular size of pKF439 was calculated to be 138 kilobases. pKF439 could be transferred to Pseudomonas putida and Pseudomonas aeruginosa at high frequency, and the transconjugants gained the ability to grow with m-xylene, m-toluate, and salicylate as the sole carbon source.

DNA Transposable Elements↗
PubMed1991

Plasmid transformation of Mycoplasma mycoides subspecies mycoides is promoted by high concentrations of polyethylene glycol.

The recent isolation and characterization of two plasmids from Mycoplasma mycoides subspecies mycoides has opened up new possibilities for studying mycoplasmal genetics. In order to facilitate the development of a genetic system in M. mycoides subsp. mycoides, parameters of polyethylene glycol (PEG)-mediated transformation were examined, as existing protocols prove very inefficient in this organism. The effects of PEG concentration, DNA concentration, presence of Ca2+ ions, and choice of buffers on the transformation of the Tn916-containing plasmid pAM120 into M. mycoides subsp. mycoides were examined. The stability of Tn916 in the M. mycoides subsp. mycoides chromosome was also evaluated. The optimal PEG concentration (53-62% (w/v)) in the transformation mixture was substantially higher than the PEG concentration reported to be optimal for transformation of other mycoplasmas (36% (w/v)). The PEG concentrations used here were also higher than the concentration used to promote transformation or fusion of gram-positive bacterial protoplasts. A necessity for the presence of Ca2+ ions for optimal transformation was shown, as was the possible involvement of cell culture growth stage. Our results demonstrate the need for expanding current transformation techniques for mycoplasmas. Studies also indicate that once Tn916 inserts into the M. mycoides subsp. mycoides chromosome, it can transpose to other sites at a relatively high frequency.

Blotting, Southern↗
PubMed2004

The replication protein of the Sulfolobus islandicus plasmid pRN1.

The thermoacidophile crenarchaeote Sulfolobus ssp. is one of the best-studied Archaea. Cryptic and conjugative plasmids as well as viruses have been described for this genus. For the majority of the genetic elements only the genome sequence and the basic characteristics were determined. In contrast the fusellovirus SSV1 and the cryptic plasmid pRN1, which is the smallest known genetic element of the crenarchaeota, have been studied in more detail. The three gene products of the plasmid pRN1 have been characterized biochemically. The replication protein of the plasmid, a multifunctional enzyme, has a novel domain, termed prim/pol domain. This domain constitutes the first member of the DNA polymerase family E. Based on the biochemical characterization of the gene products a model of how pRN1 is replicated in vivo is proposed.

Amino Acid Sequence↗
PubMed1985

Transformation of Saccharomyces cerevisiae and Schizosaccharomyces pombe with linear plasmids containing 2 micron sequences.

Linear plasmids were constructed by adding telomeres prepared from Tetrahymena pyriformis rDNA to a circular hybrid Escherichia coli-yeast vector and transforming Saccharomyces cerevisiae. The parental vector contained the entire 2 mu yeast circle and the LEU gene from S. cerevisiae. Three transformed clones were shown to contain linear plasmids which were characterized by restriction analysis and shown to be rearranged versions of the desired linear plasmids. The plasmids obtained were imperfect palindromes: part of the parental vector was present in duplicated form, part as unique sequences and part was absent. The sequences that had been lost included a large portion of the 2 mu circle. The telomeres were approximately 450 bp longer than those of T. pyriformis. DNA prepared from transformed S. cerevisiae clones was used to transform Schizosaccharomyces pombe. The transformed S. pombe clones contained linear plasmids identical in structure to their linear parents in S. cerevisiae. No structural re-arrangements or integration into S. pombe was observed. Little or no telomere growth had occurred after transfer from S. cerevisiae to S. pombe. A model is proposed to explain the genesis of the plasmids.

Ascomycota↗
PubMed1990

[High-level tetracycline-resistance of Neisseria gonorrhoeae].

High level tetracycline resistance of Neisseria gonorrhoeae is mediated by a 25.2-MD plasmid and is characterized by a minimal inhibitory concentration of 16 micrograms/ml or more. The 25.2-MD plasmid originated through the insertion of the streptococcal tetM determinant into the 24.5-MD transfer plasmid of N. gonorrhoeae. In the present study the prevalence of N. gonorrhoeae with high-level tetracycline resistance in the Heidelberg gonococcal population between 1981 and 1989 was determined. Among 1765 N. gonorrhoeae isolates, one strain with high-level tetracycline resistance was identified. The presence of the 25.2-MD plasmid in this strain was demonstrated by plasmid-agarose gel electrophoresis. The auxotype/serovar class proline-/IB-1 indicates the importation of this strain from the USA. The 25.2-MD recombinant plasmid, in contrast to the 24.5 MD transfer plasmid, has a rather wide host range, which is favourable to the occurrence both of high-level tetracycline resistance and of beta-lactamase plasmids in N. meningitidis.

Bacterial Proteins↗
PubMed1980

[Identification and characteristics of the conjugative R plasmids of conditionally pathogenic E. coli isolated from chronic pyelonephritis patients].

The study on 63 strains of conditionally pathogenic E. coli isolated from patients with chronic pyelonephritis showed that the bacteria of the 47 strains (74.6%) were resistant to one or several antibiotics. 7 strains of such bacteria carried R-plasmids capable of transferring into the cells of E. coli K-12 during conjugation. Most of the identified 9 R-plasmids belonged to the class of the F-like plasmids and were characterized by similar lots of antibiotic resistance markers. DNA of the detected R-plasmids were treated with endonuclease (restrictase) EcoRI. After that the fragments were analysed by means of electrophoresis in agarose gel. On the basis of the restrictogrames it was concluded that the plasmids studied were not identical. A possibility of detecting complexes consisting of different plasmids carrying similar lots of antibiotic resistance markers in the cells of the same strain was shown.

Chemical Phenomena↗
PubMed1983

Characterization and molecular cloning in Escherichia coli of a plasmid from the mollicute Spiroplasma citri.

Two plasmids, pMH1 with 7 kilobase pairs and pM41 with 8 kilobase pairs, were purified from the plant pathogen Spiroplasma citri and characterized by restriction mapping. Upon in vitro DNA recombination with plasmid pBR328 as a vector, we have cloned pMH1 in Escherichia coli. A radioactive probe obtained upon nick translation of the recombinant plasmid was used to further characterize and compare pMH1 and pM41.

Cloning, Molecular↗
PubMed2005

Comparison of multidrug resistance gene regions between two geographically unrelated Salmonella serotypes.

OBJECTIVES: The aim of this study was to identify chromosomally integrated genes conferring multidrug resistance to a Salmonella enterica (S.) serotype Typhimurium isolate, phage type DT193, isolated in Ireland and to compare them with resistance genes conferring plasmid-mediated multidrug resistance to a S. Enteritidis isolate from Italy. METHODS: A complete DNA sequence of the regions containing the resistance genes was obtained from the chromosome of the S. Typhimurium DT193 isolate and from the IncI plasmid of the S. Enteritidis isolate. The plasmid was also characterized by conjugation and incompatibility grouping. RESULTS: Two 10 kb multidrug resistance non-Salmonella Genomic Island 1 type clusters were independently identified in the S. Enteritidis plasmid and in the chromosome of the S. Typhimurium isolate. Detailed characterization identified an IP-type 2 integron containing a dfrA1-aadA1 gene cassette and other common resistance determinants derived from the RSF1010 plasmid. CONCLUSIONS: These multidrug resistance regions originate following chromosomal integration of key resistance markers encountered on plasmids circulating in other Salmonella serotypes. This mechanism of marker acquisition may have future implications for the evolution of similar structures in previously susceptible serotypes, leading to an increased public health risk.

Chromosome Mapping↗
PubMed1996

The isolation of recombinant plasmids expressing secreted antigens of Pasteurella haemolytica A1 and the characterization of an immunogenic 60 kDa antigen.

A collection of recombinant plasmids had been isolated which express secreted antigens of Pasteurella haemolytica A1 likely to be important in protection. The recombinant Escherichia coli clones were found to express the P. haemolytica A1 antigens by Western immunoblot analysis, using sera from calves which had been vaccinated with Presponse and were subsequently resistant to experimental challenge with P. haemolytica A1. Detailed analysis of three of the recombinant plasmids (pGS1-17, pGS3-19 and pSA1-50) showed that they all carry the same 4.2 kbp insert DNA. E. coli clones which carry the recombinant plasmids all express a strongly antigenic protein of approximately 60 kDa. Nucleotide sequence analysis of the cloned DNA showed that it codes for a polypeptide with an estimated M.W. of 60.8 kDa. A partial clone of this gene has been reported previously and an antibody response to the antigen was shown to be significantly correlated to resistance to disease. This gene was found to be present in only the A biotypes of P. haemolytica and not in the T biotypes, which have been reclassified Pasteurella trehalosi. This demonstrates that this recombinant plasmid collection codes for antigens of P. haemolytica A1 important to protection and warrants further characterization to identify additional recombinant antigens.

Amino Acid Sequence↗
PubMed1975

Five control systems preventing transfer of Escherichia coli K-12 sex factor F.

The transfer inhibition systems of 28 Fin+ plasmids have been characterized, using Flac mutants insensitive to inhibition by R100 or R62. All F-like plasmids (except R455) and one N group plasmid determined systems analogous to that of R100; this is designated the FinOP system. None of these plasmids could supply a FinP component of the transfer inhibitor able to replace that of F itself. In addition to the FinOP and R62 transfer inhibition systems described previously, new systems were encoded by the F-like plasmid R455, the I-like plasmid JR66a, and the group X plasmid R485. Besides inhibiting F transfer, JR66a also inhibited F pilus formation and surface exclusion, whereas R485 inhibited only pilus formation and R455 inhibited neither. All three R factors inhibited transfer of J-independent Flac elements, indicating that they act directly on one or more genes (or products) of the transfer operon, rather than directly via traJ. The tral products and transfer origin sequences of two Fin+ F-like plasmids, ColB2 and R124, appear to have similar specificities to those of F itself.

Coliphages↗
PubMed2000

Repetition, conservation, and variation: the multiple cp32 plasmids of Borrelia species.

Members of the spirochete genus Borrelia contain large numbers of extrachromosomal DNAs. Sequence analysis of the B. burgdorferi strain B31 genome indicated that its many plasmids contain large quantities of repeated sequences, the most obvious of which are the cp32 plasmid family. Individual spirochetes may carry nine or more different, but homologous, cp32 plasmids. Every other species of Borrelia examined thus far also contains multiple plasmids related to the B. burgdorferi cp32s. These plasmids are arguably the best characterized of all the borrelial plasmids, and epitomize the apparent redundancy evident in the many plasmids carried by these bacteria. Despite their extensive similarities, cp32 plasmids contain some open reading frames whose sequences often vary between plasmids, and which encode proteins synthesized by the bacteria during vertebrate infection. In this review, we analyze the hypervariable and conserved regions of the cp32 plasmid family, and discuss possible reasons why borreliae harbor multiple gene paralogs.

Amino Acid Sequence↗
PubMed1989

Characterization of a gram-positive broad-host-range plasmid isolated from Lactobacillus hilgardii.

Two plasmids, pLAB1000 and pLAB2000 (3.3 and 9.1 kb, respectively), have been isolated from a grass silage strain of Lactobacillus hilgardii. Both plasmids were cloned in Escherichia coli and characterized through restriction mapping. A 1.6-kb XbaI-SacI fragment of pLAB1000 appeared to be sufficient for autonomous replication in Lactobacillus plantarum and in Bacillus subtilis. Different shuttle vectors for E. coli and gram-positive bacteria were developed using the pLAB1000 plasmid. These could stably be maintained in Lactobacillus, Enterococcus, and Bacillus under selective conditions. Plasmids sharing DNA homologies with pLAB1000 have been observed in different strains of the related species L. plantarum.

DNA, Bacterial↗
PubMed1980

Tn2301, a transposon construct carrying the entire transfer region of the F plasmid.

The largest R . BamHI fragment of the plasmid F, which carries the entire F conjugation system, has been cloned into the single R . BamHI site of the ampicillin (Ap) resistance transposon TN1. pDS1106 (ColE1 mob::Tn1) was the vector plasmid, and the resultant conjugative plasmid, pED830, was characterized both genetically and by restriction enzyme analysis. The transposon construct, denoted Tn2301, was transposable at frequencies similar to Tn1 to small nonconjugative plasmids or to the Escherichia coli host chromosome. In the former case, Apr conjugative plasmids were obtained, whereas in the latter case, Hfr strains resulted. Representative Hfr strains were characterized by quantitative and interrupted mating experiments. Extension of this technique for Hfvr formation should aid chromosome mapping both in E. coli and in other bacterial genera.

Ampicillin↗