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PubMed1984

Characterization of a mini plasmid isolated from Shigella sonnei.

A multicopy plasmid, 2.1 kb in size, was isolated from Shigella sonnei and named pKYM. This plasmid is cryptic and isolated along with pKY-1, a ColE1-like plasmid. In this paper, we report the physical map of pKYM and some characters required for its multiplication. The replication of the plasmid DNA does not require DNA polymerase I but depends on protein(s) produced by itself. The plasmid is poorly mobilized by the F factor.

Cloning, Molecular↗

PubMed1997

Characterization of two plasmid-borne lps beta loci of Rhizobium etli required for lipopolysaccharide synthesis and for optimal interaction with plants.

In Rhizobium etli CFN42, both the symbiotic plasmid (pd) and plasmid b (pb) are required for effective bean nodulation. This is due to the presence on pb of a region (lps beta) involved in lipopolysaccharide (LPS) biosynthesis. We report here the genetic array and functional features of this plasmid-borne region. The sequence analysis of a 3,595-bp fragment revealed the presence of a transcriptional unit integrated by two open reading frames (lps beta 1 and lps beta 2) essential for LPS biosynthesis and symblosis. The lps beta 1 encodes a putative 193 amino acid polypeptide that shows strong homology with glucosyl-1P and galactosyl-1P transferases. The deduced amino acid sequence of the protein encoded by lps beta 2 was very similar to that of proteins involved in surface polysaccharide biosynthesis, such as Pseudomonas aeruginosa WpbM, Bordetella pertussis BpIL, and Yersinia enterocolitica TrsG. DNA sequences homologous to lps beta 1 and lps beta 2 of R. etli CFN42 were consistently found in functionally equivalent plasmids of R. etli, R. leguminosarum bv. viciae, and R. leguminosarum hv. trifolii strains, but not in R. meliloti, R. loti, R. tropici, R. fredii, Bradyrhizobium, Azorhizobium, and Agrobacterium tumefaciens. Even though Rhizobium and Agrobacterium do not share lps beta sequences, their presence is required for crown-gall tumor induction by R. etli transconjugants carrying the Ti plasmid.

Amino Acid Sequence↗

PubMed1980

Characterization of Bacillus stearothermophilus plasmid pab124 and construction of deletion variants.

A restriction endonuclease cleavage map of the tetracycline resistance plasmid pAB124, originally isolated from Bacillus stearothermophilus, was constructed using ten enzymes. Tetracycline resistance was associated with a 1 x 95 megadalton (Md) region of pAB124 lying between two EcoRI sites, and this region was circularized to produce a viable tetracycline resistance plasmid (pAB224), with two EcoRI fragments of pAB124 deleted amounting to 0 x 95 Md. A second plasmid (pAB524) with one EcoRI fragment (0 x 6 Md) of pAB124 deleted was also constructed. Restriction endonuclease cleavage maps of pAB224 and pAB524 were constructed.

Cloning, Molecular↗

PubMed1982

Isolation and characterization of a recombinant plasmid carrying a functional part of the Bacillus subtilis spoIIA locus.

Plasmid pHM2 consists of a 3.3 kb insert of Bacillus subtilis DNA in the chimeric plasmid pHV33, and can replicate in Escherichia coli and B. subtilis. In B. subtilis, pHM2 complements the defects resulting from mutations spo-42, spo-50, spo-69 and sas-1 in the spoIIA locus. This complementation can occur in recE4 strains where recombination of the plasmid with the chromosome is prevented, and the chromosome retains the mutant allele. Thus the plasmid carries a functional part of the spoIIA locus; it does not contain the complete locus as it cannot complement several other spoIIA mutations. It is likely that the locus is complex, containing at least two genes.

Bacillus subtilis↗

PubMed1983

Generalized transduction between Salmonella typhi and Salmonella typhimurium by phage j2 and characterization of the j2 plasmid in Escherichia coli.

Phage j2, a P1-like phage in Salmonella typhi, was heteroimmune to phage P1 and existed in the lysogenic state as a plasmid of molecular size 58.6 MDal. The phage j2 plasmid was incompatible with the P1 plasmid (IncY group). A j2-sensitive mutant of Salmonella typhimurium LT2 was isolated by transduction of j2Ap phage into LT2 followed by curing of the prophage. The mutant was used to demonstrate transduction between S. typhi and S. typhimurium by phage j2.

Coliphages↗

PubMed1987

Characterization and localization of plasmid functions involved in pock formation and pock resistance of plasmid pSK3 of Streptomyces kasugaensis MB273.

Deletion derivatives and recombinants of the plasmid pSK3, which is a cointegrate of pSK1 and pSK2 in Streptomyces kasugaensis, were constructed and analysed for their ability to transfer and 'pock' on strains carrying pSK1 or pSK2. Various deletions in the pSK1 and/or pSK2 regions of pSK3 were grouped into nine classes on the basis of their pock-forming ability and pock resistance. Analysis of these deletions and insertions provided tentative locations of DNA regions for two pock-resistance determinants (Por1 and Por2), two pock-forming determinants (Poc1 and Poc2) consisting of plasmid transfer and spread determinants (Tra/Spr), and two replication determinants (Rep1 and Rep2) corresponding to the pSK1 and pSK2 regions of pSK3. In particular, the Por2 function in the pSK2 region was determined to be located in a 1.35 kb segment.

DNA, Bacterial↗

PubMed1991

Cloning and characterization of the ColE7 plasmid.

The 6.2 kb ColE7-K317 plasmid was mapped and the DNA fragments of the colicin E7 operon subcloned into pUC18 and pUC19. The size of the functional colicin E7 operon deduced by subcloning was 2.3 kb. The colicin E7 gene product was purified by carboxymethylcellulose chromatography. Both colicin E7 and E9 were demonstrated to exhibit a non-specific DNAase-type activity by in vitro biological assay. The molecular mass of colicin E7 was 61 kDa, as determined by SDS-PAGE. From DNA sequence data, the estimated sizes of the E7 immunity protein and the E7 lysis protein were 9926 Da and 4847 Da, respectively. Comparison of restriction maps and DNA sequence data suggests that ColE7 and ColE2 are more closely related than other E colicin plasmids.

Amino Acid Sequence↗

PubMed1992

Characterization of a circular plasmid from the yeast Kluyveromyces waltii.

A new plasmid was found in the yeast Kluyveromyces waltii. This high-copy-number plasmid, named pKW1, is a double-stranded circular DNA plasmid of 5619 bp. It has several features characteristic of the 2 mu-type plasmids: presence of two inverted repeats and four open reading frames, as well as the interconversion of two isomeric forms. However, the nucleotide sequence shows little homology with known yeast plasmids. An ARS function was localized within a segment of 545 bp near one of the inverted repeats. Chimeric plasmids carrying this segment efficiently transformed K. waltii. A strain of K. waltii cured of the plasmid (cir degree) was also obtained. In the pKW1 sequence, a functionally neutral region was found at which foreign DNA can be inserted with little effect on plasmid stability. Such constructions carrying the full sequence of pKW1 replicated autonomously in a cir degree host and were particularly stable. pKW1-derived full-sequence plasmids also transformed K. thermotolerans, but not K. lactis.

Base Sequence↗

PubMed1992

Isolation and characterization of a conjugative plasmid from Legionella pneumophila.

The conjugative properties of an indigenous 85 MDa plasmid (designated pCH1) from Legionella pneumophila were studied. To determine if pCH1 was transmissible by conjugation, mating experiments were performed between legionellae that harboured pCH1 and several plasmid-less recipients. Plasmid transfer was monitored by colony hybridization, using a cloned 21.0 kb SalI restriction fragment from pCH1 as a probe. The results from these experiments showed that pCH1 could be conjugatively transferred into several strains of L. pneumophila serogroup 1 but not into strain Bloomington-2 (serogroup 3) or Escherichia coli. Southern hybridization experiments in which pCH1 DNA was used as a probe showed that pCH1 does not share homology with other indigenous L. pneumophila plasmids. There was no detectable DNA homology between pCH1 and L. pneumophila chromosomal DNA. Additional mating experiments revealed that pCH1 was unable to mobilize the L. pneumophila chromosome. The conjugative transfer of pCH1 into plasmid-less avirulent or virulent serogroup 1 strains did not alter the intracellular growth characteristics of these strains in U937 cells, a human-monocyte-like cell line, or in the amoeba Hartmannella vermiformis. These results suggest that pCH1 does not contribute to the ability of L. pneumophila to enter or grow within eukaryotic cells.

Cloning, Molecular↗

PubMed2000

Molecular characterization of the lactococcal plasmid pCIS3: natural stacking of specificity subunits of a type I restriction/modification system in a single lactococcal strain.

A 6.1 kb plasmid from the Lactococcus lactis subsp. cremoris strain UC509.9, named pCIS3, was found to mediate a restriction/modification (R/M) phenotype. Nucleotide sequence analysis of pCIS3 revealed the presence of an hsdS gene, typical of type I R/M systems. The presence of this plasmid resulted in a 10(4)-fold reduction in the efficiency of plating (e.o.p.) of unmodified phage. In addition to the hsdS gene of pCIS3, two more hsdS genes were identified in strain UC509.9, one located on the chromosome downstream of a gene highly homologous to hsdM genes and a third on the smallest (4 kb) plasmid, named pCIS1. The replication region of pCIS3 was highly similar to that of a large family of lactococcal theta replicons. In addition, pCIS3 was found to encode a member of the CorA family of magnesium transporters.

Amino Acid Sequence↗

PubMed1996

Identification and characterization of a mobilizing plasmid, pND300, in Lactococcus lactis M189 and its encoded nisin resistance determinant.

A 60 kb conjugative plasmid, pND300, which encodes nisin resistance, was identified in Lactococcus lactis ssp. lactis (L. lactis) M189. pND300 was found to mobilize the transfer of some other plasmids as indicated by the mobilization of plasmids encoding lactose utilization. The nisin resistance determinant from pND300 was initially subcloned on a 12 kb DNA fragment and subsequently reduced to 10.4 kb. Restriction analysis, PCR, Southern hybridization and sequencing illustrated that the nisin resistance of pND300 is very similar to that encoded by the transposon involved in nisin production. pND300 encodes nisR as well as nisK and the recently reported nisF, nisE and nisG, but does not encode nisI. The DNA fragment encoding the nis genes is flanked by IS946 with a copy at each end in reverse orientation. The expression of these nis genes is probably controlled by a putative promoter upstream of nisR, which is composed of the TTGCAA hexanucleotide on the insertion sequence IS946 and the TATAAT sequence 21 bp downstream.

Base Sequence↗

PubMed1993

Molecular cloning and characterization of a sym plasmid locus that regulates cultivar-specific nodulation of soybean by Rhizobium fredii USDA257.

Rhizobium fredii strain USDA257 produces nitrogen-fixing nodules on primitive soybean cultivars such as Peking but fails to nodulate agronomically improved cultivars such as McCall. Transposon-mutant 257DH4 has two new phenotypes: it nodulates McCall, and its ability to do so is sensitive to the presence of parental strain USDA257, i.e. it is subject to competitive nodulation blocking. We have isolated a cosmid containing DNA that corresponds to the site of transposon insertion in 257DH4 and have localized Tn5 on an 8.0 kb EcoRI fragment. The 5596 bp DNA sequence that surrounds the insertion site contains seven open reading frames. Five of these, designated nolBTU, ORF4, and nolV, are closely spaced and of the same polarity. nolW and nolX are of the opposite polarity. The initiation codon for nolW lies 155 bp upstream from that of nolB, and its is separated from nolX by 281 bp. The predicted NolT and NolW proteins have putative membrane-spanning regions. The N-terminus of the hypothetical NolW protein also has limited homology to NodH of Rhizobium meliloti, but none of the deduced protein sequences has significant homology to known nodulation gene products. Site-directed mutagenesis with mudII1734 confirms that inactivation of nolB, nolT, nolU, nolV, nolW, or nolX extends host range for nodulation to McCall soybean. This phenotype could not be genetically dissected from sensitivity to competitive nodulation blocking. Expression of nolBTU and nolX is induced as much as 30-fold by flavonoid signal molecules, even though these genes lack nod-box promoters. Histochemical staining of McCall roots inoculated with nolB-, nolU-, or nolX-lacZ fusions verifies that these genes are expressed continuously from preinfection to the stage of the functional nodule. Although a nolU-ORF4-nolV clone hybridizes to a single 8.0 kb EcoRI fragment from 10 strains of R. fredii and broad-host-range Rhizobium sp. NGR234, hybridizing sequences are not detectable in other rhizobia.

Amino Acid Sequence↗

PubMed1994

Construction, expression and characterization of a plasmid-encoded Na(+)-specific ATPase hybrid consisting of Propionigenium modestum F0-ATPase and Escherichia coli F1-ATPase.

The Escherichia coli strain DK8, a deletion mutant lacking the complete unc operon, was transformed with a plasmid containing the genes encoding the a, b, c, delta and part of the alpha subunit of the Na(+)-dependent ATPase of Propionigenium modestum and the genes encoding the alpha, gamma, beta and epsilon subunits of the H(+)-dependent E. coli ATPase. The transformants showed Na(+)-dependent growth on succinate as non-fermentable carbon source. The functionally expressed hybrid ATPase was activated 13-fold at pH 7.5 by the addition of Na+ and inhibited by 1,3-dicyclohexylcarbodiimide, azide and tributyltin chloride. At pH 7.5 and pH 9.0, the hybrid enzyme was protected from inhibition by 1,3-dicyclohexylcarbodiimide in the presence of 50 mM NaCl and 5 mM NaCl, respectively. The hybrid ATPase was reconstituted into proteoliposomes and catalyzed the transport of Na+ upon ATP addition. ATP-dependent fluorescence quenching of 9-amino-6-chloro-2-methoxyacridine proved that the ATPase hybrid was able to pump protons in the absence of Na+. Furthermore, ATP synthesis could be measured under conditions where a valinomycin-mediated K+ diffusion potential (delta psi) and a Na+ concentration gradient (delta p Na+) were imposed.

Adenosine Triphosphatases↗

PubMed1991

Isolation and characterization of a cryptic plasmid from mesophilic aeromonads: potential as a cloning vector.

A 2.6 kb covalently closed circular plasmid has been isolated from clinical and environmental isolates of Aeromonas sobria and A. hydrophila. The possibility that the plasmid carries genetic determinants that mediate resistance to a variety of anti-microbial agents has been eliminated. The plasmid is stable at approximately 20-25 copies per chromosome equivalent which, together with its relatively small size and the presence of unique restriction sites, makes it a good candidate for development as a cloning vector.

Aeromonas↗

PubMed2002

Characterization of a novel plasmid pXZ608 from Corynebacterium glutamicum.

The complete nucleotide sequence of a novel cryptic plasmid pXZ608 from Corynebacterium glutamicum 227 was determined. pXZ608 was 5949 bp with six open reading frames (ORF1-6). The predicted ORF1 gene product was homologous to replication proteins of rolling circle replication plasmids. The conserved single- and double-stranded origins of rolling circle replication were found, and interestingly, the two origins were both located on ORF1, which indicated that the Rep protein encoded by ORF1 could bind to its own gene region. Deletion analysis revealed that the minimal replicon was located on the 2.14-kb SacI-BstEII fragment.

Amino Acid Sequence↗

PubMed1987

Molecular characterization and comparison of plasmid content in seven different strains of Neisseria gonorrhoeae.

The plasmid content of one penicillin sensitive and six penicillin resistant strains of Neisseria gonorrhoeae has been examined. All strains harbour a small, phenotypically cryptic plasmid of 4.1 kilo base pairs (kb). Four of the penicillin resistant strains carry a beta-lactamase-producing plasmid of 7.3 kb. One of these also carries a large plasmid of about 40 kb. The two remaining penicillin resistant strains harbour a smaller beta-lactamase-producing plasmid of 5.5 kb. The plasmids have been subjected to digestion with a number of restriction endonucleases, and their restriction maps have been compared. Judging by the maps, the cryptic (C-) plasmids show great similarities. Except that two of them have 54 additional base-pairs (bp), and two have a HpaII site instead of a DdeI site, no differences were found. The larger beta-lactamase-producing (B-) plasmids have identical maps. The smaller seem to be homologous with the larger, except for a deletion of 1.8 kb. There is no correlation between the variant of C-plasmid and type of B-plasmid harboured in the penicillin resistant strains. The evolutionary implications suggested by this finding are discussed.

DNA Restriction Enzymes↗

PubMed1978

Characterization of a small plasmid determining resistance to erythromycin, lincomycin, and vernamycin Balpha in a strain of Streptococcus sanguis isolated from dental plaque.

A plasmid determining resistance to erythromycin (inducible), lincomycin, and vernamycin B(alpha) was isolated from a strain of Streptococcus sanguis isolated from dental plaque of a patient who had been on erythromycin therapy for an extended period of time. The plasmid, designated pAM77, had a molecular weight of 4.5 x 10(6) and was present to the extent of one to two copies per bacterial chromosome. The plasmid could transform S. sanguis strain Challis.

Anti-Bacterial Agents↗

PubMed1978

Partial characterization of R-plasmids from Pasteurella multocida isolated from turkeys.

Pasteurella multocida, isolated from turkeys during an outbreak of septicemic disease ("fowl cholera"), was found to be resistant to tetracycline, streptomycin, and sulfonamides. Agarose gel electrophoretic analysis of DNA from these isolates indicated the presence of extrachromosomal elements. Plasmid DNA was isolated by cesium chloride-ethidium bromide density centrifugation. Escherichia coli was transformed to antimicrobic resistance with this DNA. Two plasmids were isolated. One of these plasmids had a buoyant density of 1.7158 g/cm(3) (56.9 mol% guanine plus cytosine) and a molecular weight of 4.4 x 10(6) and conferred resistance to tetracycline, streptomycin, and sulfonamides. The other, having a buoyant density of 1.7198 g/cm(3) (61 mol% guanine plus cytosine) and a molecular weight of 3.44 x 10(6), conferred resistance to streptomycin and sulfonamides. Streptomycin resistance was mediated by streptomycin phosphotransferase. Compatibility group testing indicated that neither plasmid belonged to any of 13 compatibility groups (of conjugal plasmids). Both plasmids were also found to be compatible with three small, nonconjugative resistance plasmids.

Animals↗