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PubMed2006

Isolation and characterization of pHW15, a small cryptic plasmid from Rahnella genomospecies 2.

A small cryptic plasmid designated pHW15 was isolated from Rahnella genomospecies 2 WMR15 and its complete nucleotide sequence was determined. The plasmid contained 3002 bp with a G+C content of 47.4%. The origin of replication was identified by deletion analysis as a region of about 600 bp. This region had an identity of 70% to the replication origin of the ColE1 plasmid at the nucleotide level. Sequence analysis revealed the typical elements: RNA I, RNA II and their corresponding promoters, a sequence allowing hybridisation of RNA II to the DNA and favouring processing by RNaseH, a single-strand initiation determinant (ssi) that allows initiation of lagging-strand synthesis, and a terH sequence required for termination of lagging-strand synthesis. The plasmid contained three expressed open reading frames, one of which showed homology to a ColE1 plasmid-encoded protein. Furthermore, a multimer resolution site was identified by sequence analysis. Its deletion resulted in formation of plasmid multimers during growth leading to an increased plasmid loss rate.

Base Composition↗

PubMed2006

Identification and characterization of novel ColE1-type, high-copy number plasmid mutants in Legionella pneumophila.

ColE1-type plasmids are commonly used in bacterial genetics research, and replication of these plasmids is regulated by interaction of RNA I and RNA II. Although these plasmids are narrow-host-range, they can be maintained in Legionella pneumophila under antibiotic selection, with low-copy number and instability. Here, we have described the isolation of two novel spontaneous mutants of pBC(gfp)Pmip, pBG307 and pBG309, which are able to mark the L. pneumophila with strong green fluorescence when exposed to visible light. One of the mutants, pBG307, has a single CG-->TA mutation in RNA II promoter located 2-bases upstream the - 10 region. Another one, pBG309, has the same mutation, as well as an additional CG-->AT mutation in the 76th nucleotide of RNA I, or in the 6th nucleotide of RNA II. A plasmid with the single mutation in RNA I, pBG308, was also constructed. Characterization of these plasmids carrying the enhanced green fluorescent protein (gfpmut2) gene revealed that the green fluorescence intensities of these plasmids were 2- to 30-fold higher than that of the wild type and both of the mutations contribute to increase the plasmid copy number and/or plasmid stability. The mutation located in RNA II promoter played a more dominant role in elevating the copy number, compared to the mutation in RNA I. We also tested the mutant plasmids for replication in Escherichia coli, and found that their copy number and stability were dramatically decreased, except pBG307. Our data suggest that these plasmids might be useful and convenient in genetic studies in L. pneumophila.

Base Pairing↗

PubMed2002

Characterization of pKU701, a 2.5-kb plasmid, in a Japanese Helicobacter pylori isolate.

A cryptic plasmid of Helicobacter pylori, pKU701 (accession number AB078638), was isolated and the complete nucleotide sequence was determined. No drug resistance properties were mediated by pKU701. The 2454b pKU701 sequence, which had a 38% content of G-C residues, generated one polypeptide from a single open reading frame (ORF1). Extensive sequence homology was evident between pKU701 and ORF1 of H. pylori plasmid pHPO100 (repA, 88.8% identity) as well as ORF3 of plasmid pHPS1 (repB, 80.2% identity), but pKU701 showed only 46.3% homology with ORF1 of plasmid pHPK255 (repA). Tandem direct repeats of a 33-bp segment were found in pKU701 outside ORF1, but there were no inverted repeat ends such as those found in typical insertion sequences. The ability of drug resistance plasmids to replicate in H. pylori is probably limited, so chromosomal mutation may be a more likely cause of resistance.

Amino Acid Sequence↗

PubMed1997

Genetic characterization of insertion sequence ISJP4 on plasmid pJP4 from Ralstonia eutropha JMP134.

Directly adjacent to the (tfdT-) tfdCDEF gene cluster for chlorocatechol breakdown on plasmid pJP4 of Ralstonia eutropha (formerly Alcaligenes eutrophus) JMP134, we identified a 0.9-kb DNA element, designated ISJP4, with the typical features of a bacterial insertion sequence. ISJP4 occurs as a single complete copy on plasmid pJP4. About 9 kb away from this copy, in the tfdA-tfdS intergenic region, we found a 71-bp duplication of the ISJP4 right-hand extremity. In addition, we discovered a complete copy of ISJP4 on the chromosome of the R. eutropha JMP134 strain that we use routinely in our laboratory. We suppose that this copy resulted from a recent transposition of the plasmid-borne ISJP4, since it was shown to be lacking from the chromosomes of R. eutropha JMP222 and JMP289, two previously pJP4-cured derivatives of JMP134. By comparing both complete copies and their flanking regions, we could establish that element ISJP4 has a size of 915 bp and is bordered by 18-bp inverted repeats with one mismatch. Based on sequence similarity of its coding regions, ISJP4 could be classified into the IS5 group of the IS4 family of bacterial insertion sequences, where it is mostly related to IS402 of Burkholderia cepacia. A TAA direct repeat, presumably resulting from a duplication of the target site, flanked the chromosomal copy of ISJP4. We could demonstrate that a piece of DNA that is flanked by two complete copies of ISJP4 can be transposed. Even more so, one complete ISJP4 plus its tfdA-tfdS intergenic remnant were sufficient to mediate transposition of intervening DNA. A possible role of ISJP4 in the formation of the tfd pathway genes will be discussed.

Alcaligenes↗

PubMed1994

Use of plasmid profile typing for surveillance of Salmonella enteritidis phage type 4 from humans, poultry and eggs.

Plasmids were found in 1022 of 1089 (94%) of drug-sensitive strains of Salmonella enteritidis phage type 4 from humans (sporadic and outbreak cases), poultry (chickens) and eggs in England and Wales in the 5-year period 1988-92 and 25 plasmid profile patterns were identified. Strains characterized by a single plasmid of 38 MDa predominated (= plasmid profile type SE 38), comprising over 90% of isolates from humans, 70% from poultry and 92% from eggs. Eleven profile types were identified in strains from humans, 21 in strains from poultry and 3 in strains from eggs. Eight of the 11 patterns identified in human isolates were found in strains from poultry and 2 in strains from eggs. In contrast 15 patterns seen in poultry were not found in strains from humans. Four percent of strains from humans and 13% from poultry did not carry the 38 MDa plasmid but all strains from eggs were found to carry this plasmid. The second most common profile type in strains isolated between 1981 and 1988 was not identified in strains isolated from 1988-92. It is concluded that plasmid profile typing is a useful method for rapid differentiation within phage type 4 of S. enteritidis but that methods which can discriminate within the predominant profile type, SE 38, are now required.

Animals↗

PubMed1975

Cloning, isolation, and characterization of replication regions of complex plasmid genomes.

EcoRI endonuclease-generated DNA fragments carrying replication regions of the F'lac and R6-5 plasmids have been cloned and isolated, using as a selection vehicle a nonreplicating ampicillin-resistance DNA fragment derived from a Staphylococcus aureus plasmid. Heteroduplex analysis of the constructed plasmid chimeras and the parent replicons has localized the cloned R6-5 replication region to a DNA segment between kilobase pair coordinates 1.0 and 88.0 on the R6-5 map. Physical proximity between the plasmid replication functions and the locus governing plasmid incompatibility has been shown for both parent replicons. The cloning method reported appears to be generally applicable for the identification and isolation of replication regions of a variety of complex genomes.

Centrifugation, Density Gradient↗

PubMed1982

Characterization of a ColE1-like plasmid isolated from Shigella sonnei.

A multicopy plasmid, 4.7 kb in size, was isolated from Shigella sonnei and named pKY1. This plasmid produces a colicin E1-like bacteriocin (colicin E1*) in E. coli cells. The cells harboring pKY1 are immune not only to this bacteriocin but also to colicin E1, and the cells harboring Co1E1 show immunity to colicin E1* as well. Although these two plasmid DNAs have different cleavage maps and are compatible with each other, pKY1 shows partial DNA homology with ColE1 DNA. In this paper, we report the isolation and properties of several Tn3 inserted pKY1 mutants, and propose a preliminary genetic map of pKY1. It was also found that this plasmid is not capable of self-transmission and is poorly mobilized by the F factor.

Chromosome Mapping↗

PubMedDate not supplied

Characterization of two Azospirillum brasilense Sp7 plasmid genes homologous to Rhizobium meliloti nodPQ.

Bacteria belonging to the Azospirillum genus are nitrogen fixers that colonize the roots of grasses, but do not cause the formation of differentiated structures. Sequences from total DNA of several Azospirillum strains are homologous to restriction fragments containing Rhizobium meliloti nodulation genes. A 10-kilobase (kb) EcoRI fragment from A. brasilense Sp7, sharing homology with a 6.8-kb EcoRI fragment carrying nodGEFH and part of nodP of R. meliloti 41, was cloned in pUC18 to yield pAB502. The nucleotide sequence of a 3.5-kb EcoRI-SmaI fragment of the pAB502 insert revealed 60% homology with R. meliloti nodP and nodQ genes. The nodP gene product shares no homology to any known protein sequence. The Azospirillum nodQ gene product shares homology with a family of initiation and elongation factors as does the R. meliloti nodQ gene product. Since the nodQ gene overlaps the nodP gene, the two genes might be cotranscribed. Azospirillum contains large plasmids, and the nodPQ genes were found on the 90-MDa plasmid (p90). A translational nodP-lacZ fusion was constructed in the broad host range plasmid pGD926. No beta-galactosidase activity was detected in Escherichia coli, but the fusion was functional in Azospirillum and constitutively expressed. Deletions and mutations of nodPQ did not modify growth, nitrogen fixation, or interaction with wheat seedlings.

Amino Acid Sequence↗

PubMedDate not supplied

Construction and characterization of chimeric beta-lactamase plasmids of Neisseria gonorrhoeae with altered ability to be mobilized during conjugation.

A series of chimeric plasmids derived from the transfer factor, beta-lactamase-encoding R factor, and cryptic plasmid of Neisseria gonorrhoeae was constructed. Two of these plasmids, each lacking a 1.9-kilobase-pair (kb) HinfI fragment, could not be mobilized by the 40-kb gonococcal transfer factor to recipient strains of N. gonorrhoeae or Escherichia coli during conjugation. The proteins encoded by both the naturally occurring and chimeric plasmids were examined with an E. coli cell-free transcription-translation system and in E. coli maxicells. Six plasmid-specific proteins were identified when DNA from a naturally occurring 7.1-kb R factor was used as template. A small protein (16,000 daltons), which is apparently not encoded by the two plasmids lacking the 1.9-kb HinfI fragment, appears to be necessary for plasmid transfer during conjugation.

Bacterial Proteins↗

PubMed1979

Molecular characterization of a small Haemophilus influenzae plasmid specifying beta-lactamase and its relationship to R factors from Neisseria gonorrhoeae.

The ampicillin-resistant Haemophilus influenzae strain Ve445 which caused purulent meningitis and septicaemia in a newborn child in Germany contained a 4.4 megadalton (Mdal) plasmid (pVe445) and produced a TEM type beta-lactamase. The transformation to ampicillin resistance of a sensitive Escherichia coli strain with isolated pVe445 DNA proved that the structural gene for the beta-lactamase resided on this plasmid genome. Molecular DNA-DNA hybridization studies and electron microscope DNA heteroduplex analysis indicated that pVe445 probably contained 38 to 41% of the ampicillin translocation DNA segment (TnA) found on R factors of enteric origin. The TnA fragment present in pVe445 most likely does not contain both of the inverted repeat sequences of TnA. DNA-DNA polynucleotide sequence studies indicated that the 4.4 Mdal plasmid pVe445 was unrelated to the 30 to 38 Mdal H. influenzae R plasmids but was closely related to the 4.1 Mdal ampicillin resistance specifying H. influenzae plasmid RSF0885 isolated in the U.S.A. The H. influenzae plasmid pVe445 shared 91% of its base sequences with the beta-lactamase specifying Neisseria gonorrhoeae plasmid pMR0360 (4.4 Mdal) and had 85% of its base sequences in common with the beta-lactamase specifying N. gonorrhoeae plasmid pMR0200 (3.2 Mdal). All of the four 3.2 to 4.4 Mdal beta-lactamase specifying R plasmids of H. influenzae and N. gonorrhoeae investigated probably have a common evolutionary origin.

Ampicillin↗

PubMed1986

Characterization of the ColE9-J plasmid and analysis of its genetic organization.

We have determined the restriction and functional map of the ColE9-J plasmid. By sub-cloning and transposon mutagenesis we have shown that the ColE9imm gene and the ColE5imm gene present on the ColE9-J plasmid are located on separate EcoRI fragments. Using an expression vector we have demonstrated the presence of two lys genes on the ColE9-J plasmid, both of which are dependent upon the colicin E9 structural gene promoter. Promoter mapping studies imply that the colicin E9 structural gene and the ColE5imm gene are transcribed in the same direction, but that the ColE9imm gene is transcribed in the opposite orientation.

Bacteriocin Plasmids↗

PubMed1987

Characterization of three group A klebicin plasmids: localization of their E colicin immunity genes.

We have investigated the immunity to E colicins conferred by three group A klebicin plasmids. pP5a, which encodes klebicin A1-P5, like pClo-DF13, confers immunity to colicin E6 on Escherichia coli K12, whilst pP5b and pP3, which encode klebicins A2-P5 and A3-P3 respectively, both confer immunity to colicin E3. We have determined the restriction endonuclease and functional maps of the three group A klebicin plasmids. By sub-cloning and transposon mutagenesis we have investigated the relationship between the klebicin immunity and the E colicin immunity conferred by these plasmids. The colicin E6 and the klebicin A1 immunity are encoded by a single gene present on pP5a. The colicin E3 and the klebicin A2 immunity are encoded by a single gene present on pP5b. The colicin E3 and the klebicin A3 immunity are encoded by separate genes present on pP3. Recombinant pML8412, which is derived from the ColE6-CT14 plasmid and encodes colicin E6 immunity, confers klebicin A1-P5 immunity upon Klebsiella pneumoniae UNF5023. Recombinant pKC23, which is derived from the ColE3-CA38 plasmid and confers colicin E3 immunity, confers immunity to klebicin A2-P5, but not to klebicin A3-P3.

Bacteriocins↗

PubMed2006

Characterization of the theta-type plasmid pCD3.4 from Carnobacterium divergens, and modulation of its host range by RepA mutation.

The complete nucleotide sequence of the 3475 bp plasmid pCD3.4 from Carnobacterium divergens LV13, which encodes the bacteriocin divergicin A, was determined. Nucleotide sequence, deletion and complementation analyses revealed the presence of a trans-acting replication protein, RepA, and DNA sequences involved in plasmid replication and copy-number control. The DNA region preceding the repA gene probably contains the origin of replication. This sequence includes four and a half direct repeats (iterons) of 22 bp, to which RepA is thought to bind, and an AT-rich region containing a 12 bp repeat, at which initiation of DNA might occur. Further upstream of this sequence resides a fifth iteron required for optimal plasmid replication. The RepA protein shows homology to replication proteins of the pUCL287 subfamily of theta-type replicons. Two ORFs were found downstream of the repA gene that could be deleted without affecting replication and stability of the plasmid. pCD3.4 has a narrow host range, and could only be maintained in Carnobacterium spp.; however, a mutant of the plasmid was obtained that enabled the pCD3.4 replicon to replicate in Enterococcus faecium, but not in Carnobacterium spp. The mutation was located in the C-terminal region of the RepA protein, changing a proline into a serine. This is believed to be the first example of such plasmid-host-range modulation in Gram-positive bacteria.

Bacterial Proteins↗

PubMed2002

Characterization of the Streptomyces violaceoruber SANK95570 plasmids pSV1 and pSV2.

We have analyzed the structure of two extrachromosomal elements of the methylenomycin producing actinomycete Streptomyces violaceoruber SANK95570. The presence of the circular plasmid pSV1 which was supposed to contain the genes for methylenomycin biosynthesis could be verified. Physical mapping of pSV1 revealed a size of 175.35 kb for this plasmid. In addition we generated a restriction map for the 100-kb linear plasmid pSV2. Cloning and sequencing of the terminal ends of pSV2 indicated the presence of 426-bp terminal inverted repeats. Both pSV2 termini show significant homology to the chromosome ends of Streptomyces coelicolor A3(2) which is a closely related strain to S. violaceoruber SANK95570.

Amino Acid Sequence↗

PubMed1995

Characterization of an erythromycin resistance (erm) plasmid in Streptococcus pyogenes.

Three erythromyxin-resistant Swedish isolates of Streptococcus pyogenes, representing different T-types, were studied. Two of the strains showed constitutive high-level (MIC > 200 micrograms/ml) and one showed moderate (MIC 6.4 micrograms/ml) resistance; the latter strain was sensitive to lincosamide and clindamycin, and resistance was not induced by erythromycin. In each of the strains, a plasmid with an estimated Mw of 17.6 +/- 0.9 x 10(6) was isolated in addition to smaller cryptic plasmids. The three plasmids pSE701, pSE702, and pSE703 had very similar restriction enzyme cleavage patterns. Novobiocin curing of the high-level resistance strain ER559 showed the resistance to be linked to its 17.6 x 10(6) plasmid, pSE703. Furthermore, by electroporation this rather large plasmid was reintroduced into an erythromycin-sensitive cured derivative, acquiring resistance, and the plasmid was again recovered from the transconjugant. One of the plasmids, pSE702, was shown by filter mating to be conjugative within S. pyogenes. DNA-DNA hybridization showed that the resistance determinant of the present three isolates was related to the erm gene on plasmid pAM beta 1 of Enterococcus faecalis but not to that of plasmid pE194 of Staphylococcus aureus. The copy numbers of pSE702 and pSE703, derived from the two high-level resistant strains, were 11 +/- 3 and 17 +/- 5 compared to 2 +/- 1 for pSE701, derived from the moderately resistant strain, possibly accounting for the phenotypic variation observed. The plasmids pSE702 and pAM beta 1 showed about 80% homology in DNA-DNA hybridization tests and high similarity in their restriction maps.

Drug Resistance, Microbial↗

PubMed1989

Characterization of a multiple antibiotic resistance plasmid from Haemophilus ducreyi.

Plasmid pLS88 from a clinical isolate of Haemophilus ducreyi encoded resistance determinants for sulfonamides and streptomycin related to those of RSF1010 and for kanamycin related to Tn903 but lacked the inverted repeats of the transposon. Its host range included Haemophilus influenzae, Actinobacillus pleuropneumoniae, and Escherichia coli; and it was compatible with pDM2 and RSF1010.

Autoradiography↗

PubMed1999

Characterization of SFO-1, a plasmid-mediated inducible class A beta-lactamase from Enterobacter cloacae.

Enterobacter cloacae 8009 produced an inducible class A beta-lactamase which hydrolyzed cefotaxime efficiently. It also hydrolyzed other beta-lactams except cephamycins and carbapenems. The activity was inhibited by clavulanic acid and imipenem. The bla gene was transferable to Escherichia coli by electroporation of plasmid DNA. The molecular mass of the beta-lactamase was 29 kDa and its pI was 7.3. All of these phenotypic characteristics of the enzyme except for inducible production resemble those of some extended-spectrum class A beta-lactamases like FEC-1. The gene encoding this beta-lactamase was cloned and sequenced. The deduced amino acid sequence of the beta-lactamase was homologous to the AmpA sequences of the Serratia fonticola chromosomal enzyme (96%), MEN-1 (78%), Klebsiella oxytoca chromosomal enzymes (77%), TOHO-1 (75%), and FEC-1 (72%). The conserved sequences of class A beta-lactamases, including the S-X(T)-X(S)-K motif, in the active site were all conserved in this enzyme. On the basis of the high degree of homology to the beta-lactamase of S. fonticola, the enzyme was named SFO-1. The ampR gene was located upstream of the ampA gene, and the AmpR sequence of SFO-1 had homology with the AmpR sequences of the chromosomal beta-lactamases from Citrobacter diversus (80%), Proteus vulgaris (68%), and Pseudomonas aeruginosa (60%). SFO-1 was also inducible in E. coli. However, a transformant harboring plasmid without intact ampR produced a small amount of beta-lactamase constitutively, suggesting that AmpR works as an activator of ampA of SFO-1. This is the first report from Japan describing an inducible plasmid-mediated class A beta-lactamase in gram-negative bacteria.

Amino Acid Sequence↗

PubMed2002

Characterization of a self-transferable plasmid from Salmonella enterica serotype typhimurium clinical isolates carrying two integron-borne gene cassettes together with virulence and drug resistance genes.

An unusual self-transferable virulence-resistance plasmid (pUO-StVR2) was found in nine multidrug-resistant (ACSSuT phenotype) Salmonella enterica serotype Typhimurium clinical isolates that were assigned to four different phage types and a single and distinctive XbaI pulsed-field gel electrophoresis profile. pUO-StVR2 is an IncFII plasmid of about 140 kb in length carrying the spvA, spvB, and spvC (Salmonella plasmid virulence) and rck (resistance to complement killing) genes. It also carries the oxa1/aadA1a (ampicillin resistance and streptomycin-spectinomycin resistance) gene cassette configuration located within a class 1 integron with qacEDelta1/sul1 (ammonium antiseptics resistance and sulfadiazine resistance); the transposon genes merA, tnpA, and tnpR (mercury resistance, transposase, and resolvase of Tn21, respectively); and the catA1 (chloramphenicol resistance) and tet(B) (tetracycline resistance) genes. The insertion of resistance genes into a Salmonella virulence plasmid constitutes a new and interesting example of plasmid evolution and presents a serious public health problem.

Bacteriophage Typing↗