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PubMed1987

Isolation and structure of the replicon of the promiscuous plasmid pCU1.

Evidence is presented to indicate that a PvuII fragment of approx. 2 kb isolated from the 39-kb IncN-group plasmid pCU-1 contains all plasmid-borne determinants for stable maintenance as an extrachromosomal element in Escherichia coli K-12. The fragment was sequenced. The features of this sequence include a group of 13 direct tandem repeats of 37 bp and a second group of two other direct repeats of 30 bp flanking a third partial member of this group. In addition, for a 19-bp sequence that overlaps a member of this second group, there are inverted repeats that straddle the members of the first group. There are three open reading frames within the fragment. We compare features of this sequence with that of other plasmid replicons and draw attention to similar and to dissimilar features.

Base Composition↗
PubMed2003

Molecular structure and evolution of the conjugative multiresistance plasmid pRE25 of Enterococcus faecalis isolated from a raw-fermented sausage.

Plasmid pRE25 from Enterococcus faecalis transfers resistances against kanamycin, neomycin, streptomycin, clindamycin, lincomycin, azithromycin, clarithromycin, erythromycin, roxithromycin, tylosin, chloramphenicol, and nourseothricin sulfate by conjugation in vitro to E. faecalis JH2-2, Lactococcus lactis Bu2, and Listeria innocua L19. Its nucleotide sequence of 50237 base pairs represents the largest, fully sequenced conjugative multiresistance plasmid of enterococci (Plasmid 46 (2001) 170). The gene for chloramphenicol resistance (cat) was identified as an acetyltransferase identical to the one of plasmid pIP501 of Streptococcus agalactiae. Erythromycin resistance is due to a 23S ribosomal RNA methyl transferase, again as found in pIP501 (ermB). The aminoglycoside resistance genes are packed in tandem as in transposon Tn5405 of Staphylococcus aureus: an aminoglycoside 6-adenyltransferase, a streptothricin acetyl transferase, and an aminoglycoside phosphotransferase.). Identical resistance genes are known from pathogens like Streptococcus pyogenes, S. agalactiae, S. aureus, Campylobacter coli, Clostridium perfringens, and Clostridium difficile. pRE25 is composed of a 30.5-kbp segment almost identical to pIP501. Of the 15 genes involved in conjugative transfer, 10 codes for putative transmembrane proteins (e.g. trsB, traC, trsF, trsJ, and trsL). The enterococcal part is joined into the pIP501 part by insertion elements IS1216V of E. faecium Tn1545 (three copies), and homologs of IS1062 (E. faecalis) and IS1485 (E. faecium). pRE25 demonstrates that enterococci from fermented food do participate in the molecular communication between Gram-positive and Gram-negative bacteria of the human and animal microflora.

Animals↗
PubMed1983

Primary structure of the essential replicon of the plasmid pSC101.

The replicon of the low copy number plasmid pSC101 has an obligatory requirement for the dnaA initiator protein of Escherichia coli as well as a plasmid-encoded initiator protein. We have identified the cistron of the plasmid-encoded initiator by DNA sequence analysis. Fusion of the initiator cistron with the lacZ gene of E. coli yielded a fusion protein of approximately equal to 150 kilodaltons, thus confirming that the open reading frame detected by DNA sequence analysis actually encoded a 37.5-kilodalton protein. Deletion of 26 amino acid residues from the COOH terminus of the plasmid initiator abolished autonomous replication from pSC101 origin. By in vitro deletion analysis we have shown that, although sequences downstream from the initiator cistron are dispensable, a maximum of 400 base pairs immediately upstream from the NH2-terminal region of the initiator is necessary for plasmid replication. These upstream sequences contain an A + T-rich region and three tandem repeats of a 21-base pair sequence; these features are characteristics of other replication origins.

Bacterial Proteins↗
PubMed1988

Z DNA and loop structures by immunoelectronmicroscopy of supercoiled pRW751, a plasmid containing left-handed helices.

Single and multiple loops were seen when the plasmid pRW751 was allowed to react with anti-Z-DNA or with a Z-specific cross-linking agent. Loop formation was dependent upon negative supercoiling and the presence of Z-specific antibody or cross-linking agent. Restriction enzyme mapping located 18 sites at the bottoms of loops, in addition to the two (dG-dC)n inserts of pRW751. No more than 5 loops were seen in any of the measured molecules; thus, not all potential Z-sites assume the Z conformation at any particular time. Stretches of alternating purine-pyrimidine sequences occur at all 20 sites. Almost all of the Z sites could be mapped to regions located at the beginnings or ends of reading frames or at various regulatory sites. Our findings support the concept that supercoiling brings distant sequences to within 5A of one another, allowing joint participation in regulatory processes controlled by DNA-binding proteins.

DNA Restriction Enzymes↗
PubMed1999

Structural organization and regulation of the plasmid-borne type II restriction-modification system Kpn2I from Klebsiella pneumoniae RFL2.

Kpn 2I enzymes of a type II restriction-modification (R-M) system from the bacterium Klebsiella pneumoniae strain RFL2 recognize the sequence 5'-TCCGGA-3'. The Kpn 2I R-M genes have been cloned and expressed in Escherichia coli. DNA sequence analysis revealed the presence of two convergently transcribed open reading frames (ORFs) coding for a restriction endonuclease (Enase) of 301 amino acids (34. 8 kDa) and methyltransferase (Mtase) of 375 amino acids (42.1 kDa). The 3'-terminal ends of these genes ( kpn2IR and kpn2IM, respectively) overlap by 11 bp. In addition, a small ORF (gene kpn2IC ) capable of coding for a protein of 96 amino acids in length (10.6 kDa) was found upstream of kpn2IM. The direction of kpn2IC transcription is opposite to that of kpn2IM. The predicted amino acid sequence of this ORF includes a probable helix-turn-helix motif. We show that the product of kpn2IC represses expression of the Kpn 2I Mtase but has no influence on expression of the Enase gene. Such a mode of regulation is unique among R-M systems analyzed so far. The Kpn 2I R-M is located on the K.pneumoniae RFL2 plasmid pKp4.3, which is able to replicate in E.coli cells.

Amino Acid Motifs↗
PubMed1997

Structural analysis of the 6 kb cryptic plasmid pFAJ2600 from Rhodococcus erythropolis NI86/21 and construction of Escherichia coli-Rhodococcus shuttle vectors.

The complete nucleotide sequence of the 5936 bp cryptic plasmid pFAJ2600 from Rhodococcus erythropolis NI86/21 was determined. Based on the characteristics of its putative replication genes, repA and repB, pFAJ2600 was assigned to the family of pAL5000-related small replicons identified in Mycobacterium (pAL5000), Corynebacterium (pXZ10142), Brevibacterium (pRBL1), Bifidobacterium (pMB1) and Neisseria (pJD1). The replication systems of these plasmids show striking similarities to the ones used by the ColE2 family of plasmids from Enterobacteria with respect to both trans-acting factors and ori sequences. Two possible plasmid stabilization systems are encoded on pFAJ2600: a site-specific recombinase (PmrA) related to the Escherichia coli Xer proteins for plasmid multimer resolution and an ATPase (ParA) related to the A-type of proteins in sop/par partitioning systems. The proposed replication termination region of pFAJ2600 has features in common with the Ter loci of Bacillus subtilis. Chimeras composed of a pUC18-Cmr derivative inserted in the parA-repA intergenic region of vector pFAJ2600 produced vectors that could be shuttled between Escherichia coli and several Rhodococcus species (R. erythropolis, R. fascians, R. rhodochrous, R. ruber). The pFAJ2600-based shuttle vector pFAJ2574 was stably maintained in R. erythropolis and R. fascians growing under non-selective conditions.

Amino Acid Sequence↗
PubMed1993

Contributions of promoter context and structure to regulated expression of the F plasmid traY promoter in Escherichia coli K-12.

Expression of the F plasmid traY promoter in vivo requires both host (E. coli) and plasmid encoded proteins. As judged by transcript size and primer extension analyses, the F plasmid traY promoter was utilized in vitro by purified E. coli sigma 70 RNA polymerase in the absence of other proteins. However, in vitro transcription required supercoiled templates. Endonuclease protection experiments showed that RNA polymerase is unable to form a stable complex at the traY promoter in linear or relaxed circular templates. In vitro transcription with linear templates could be elicited by altering the traY -10 and -35 hexamers to the consensus sequences. Alterations that reduced the effect of template supercoiling on apparent promoter strength in vitro also reduced the effect of the F plasmid TraJ protein on traY expression in vivo. Apparent traY promoter strength in vitro, estimated in template competition experiments, was unaltered by deletion of tra DNA normally upstream of the promoter, a change in promoter context that elicited high levels of promoter activity in TraJ- cells. These data suggest a model for regulated traY promoter activity in which a nucleoprotein complex involving tra DNA immediately upstream locally relaxes traY promoter DNA. TraJ and perhaps other activators could disrupt the complex, allowing promoter DNA to equilibrate at the prevailing negative superhelical density and thereby eliciting transcription initiation.

Bacterial Outer Membrane Proteins↗
PubMed2004

Structure and function of the shufflon in plasmid r64.

Conservative site-specific recombination plays key roles in creating biological diversity in prokaryotes. Most site-specific inversion systems consist of two recombination sites and a recombinase gene. In contrast, the shufflon multiple inversion system of plasmid R64 consists of seven sfx recombination sites, which separate four invertible DNA segments, and the rci gene encoding a site-specific recombinase of the integrase family. The rci product mediates recombination between any two inverted sfx sites, resulting in the inversion of four DNA segments independently or in groups. Random shufflon inversions construct seven pilV genes encoding constant N-terminal segment with different C-terminal segments. The pilV products are tip-located adhesins of the type IV pilus, called the thin pilus, of R64 and recognize lipopolysaccharides of recipient bacterial cells during R64 liquid matings. Thus, the shufflon determines the recipient specificity of liquid matings. Rci protein of R64 was overexpressed, purified, and used for in vitro recombination reactions. The cleavage and rejoining of DNA strands in shufflon recombinations were found to take place in the form of a 5' protruding 7-hp staggered cut within sfx sequences. Thus, the sfx sequence is asymmetric: only the 7-bp spacer sequence and the right arm sequence are conserved among various R64 sfxs, whereas the sfx left arm sequences are not conserved. Rci protein was shown to bind to entire sfx sequences, suggesting that it binds to the right arms of the sfx sequences in a sequence-specific manner and to their left arms in a non-sequence-specific manner. The sfx left arm sequences greatly affected the shufflon inversion frequency. The artificial symmetric sfx sequence, in which the sfx left arm was changed to the inverted repeat sequence of the right arm, exhibited the highest inversion frequency. Rci-dependent deletion of a DNA segment flanked by two symmetric sfx sequences in direct orientation was observed, suggesting that the asymmetry of sfx sequences may prevent recombination between sfx sequences in direct orientation in the R64 shufflon. The Rci C-terminal domain was not required for recombination using the symmetric sfx sequence. A model, where the C-terminal domain of Rci protein plays a key role in the sequence-specific and non-specific binding of Rci to asymmetric sfx sites, was proposed. Site-specific recombination in the temperate phage Mx8 of M. xanthus was also described. The Mx8 attP site is located within the coding sequence of the Mx8 intP gene. Therefore, the integration of Mx8 into the M. xanthus chromosome results in the conversion of the intP gene into a new gene, intP. As a result of this conversion, the 112-amino-acid C-terminal sequence of the intP product is replaced with a 13-amino acid sequence of the intR product. The C-terminal domain of Mx8 IntP recombinase is only required for integration and not for excision.

Journal Article↗
PubMed2005

[Study on the genetic structure and transmission mechanism of a plasmid-mediated AmpC beta-lactamase].

OBJECTIVE: To clone the gene of plasmid-mediated AmpC beta-lactamase from the plasmid of multiple-drug resistance Klebsiella pneumoniae producing plasmid-mediated AmpC beta-lactamase and demonstrate its mechanism of transmission. METHODS: Plasmids of the transconjugant were extracted and digested with restriction endonuclease HindIII. Taq DNA polymerase was applied to fill the recessed 3' termini, and a single deoxyadenosine was added to the 3' termimi of fragments. Then these fragments were ligated with pGEM-T Easy vector. E. coli DH5alpha containing recombinant plasmid was selected on MacConkey agar plates containing ampicillin and cefoxitin. Insert fragments were sequenced by primer walking. MIC determinations and isoelectric focusing electrophoresis (IFE) were utilized to analyze recombinant. RESULTS: The recombinant plasmid pT948 containing a 5.2-kb insert was obtained. The inserted fragment contained a bla(DHA-1) and a regulatory gene ampR. The insertion sequence (IS26), qacEDelta1 and sulI genes of the I type integron were obtained near the bla(DHA-1) gene. Recombinant expressed a beta-lactamase with pI of 7.7. MIC determinations showed that recombinant was resistant to cefoxitin and the resistance to ceftazidime could be induced by the cefoxitin. CONCLUSION: The plasmid-mediated ampC gene cloned was identified as bla(DHA-1). IS26 observed on the flanks of the bla(DHA-1) maybe relate to the translocation of bla(DHA-1) gene region from the chromosome to plasmid.

Anti-Bacterial Agents↗
PubMed1985

Rapid large-scale purification of plasmid DNA by medium or low pressure gel filtration. Application: construction of thermoamplifiable expression vectors.

This paper describes a new method of plasmid DNA purification which is fast and reliable enough for most purposes in recombinant DNA technology. The present method does not require the use of toxic chemicals such as phenol or ethidium bromide, costly ultracentrifugation procedures or other processes which can modify the supercoiled structure of the plasmids, such as adsorption on glass fiber. This method is based on the principle of gel filtration chromatography, at low pressure (1 bar) or medium pressure (between 5 and 10 bars), using Sephacryl S1000 or Superose 6B. It permits recovery of plasmids: in preparative quantities (from 300 micrograms to 4 mg), exempt from RNA, DNA and protein contamination, and suitable for various common genetic engineering procedures immediately after purification. To test the reliability of the technique as well as the degree of purification, the plasmids were used to construct thermoamplifiable vectors, carrying the lacUV5 promoter and the 5' end of the beta-galactosidase gene with a single EcoR1 site in each of the three possible translational phases. This set of vectors is designed for the expression of foreign genes as hybrid proteins in Escherichia coli.

Chromatography, Gel↗
PubMed1988

[Recombination of plasmids with inverted repeats in Bacillus subtilis cells].

The plasmid pNB48 carring a pair of genetically distinct inverted repeats is constructed on the basis of plasmid pSM19035 replicon. The single Campbell-type recombination between inverted repeats of two circular monomers leads to the formation of the so called "inverted" dimers in Bacillus subtilis cells. The gene conversion is observed in the course of this recombination. The mechanism of structural rearrangements of plasmids with inverted repeats that was postulated earlier is confirmed now. Some replicon features may influence the recombination of plasmids in Bacillus subtilis cells. The plasmid pNB48 may be a suitable model for studying the genetic recombination in Bacillus subtilis.

Bacillus subtilis↗
PubMed1982

Hepatitis B virus core antigen: synthesis in Escherichia coli and application in diagnosis.

Fragments of hepatitis B virus DNA cloned in plasmid pBR322 carrying the gene for the viral core antigen have been placed under the control of the lac promoter of Escherichia coli. Several of the new recombinants direct higher levels of synthesis of the antigen, but the degree of enhancement varies with the different structures of the plasmids and hence the mRNAs produced. The antigen in crude bacterial lysates is a satisfactory diagnostic reagent for antibodies to the core antigen in serum samples.

Antibodies, Viral↗
PubMed2004

[Construction of DNA vaccine of Trichinella spiralis muscle larvae and its expression in mice].

OBJECTIVE: To construct and express recombinant plasmid containing the structural gene encoding Mr 31 000 antigen of Trichinella spiralis muscle larvae. METHODS: The target gene TspE1 was amplified by RT-PCR, cloned into pUC18 vector, and sub-cloned into the eukaryotic expression vector pcDNA3. BALB/c mice were immunized with the purified recombinant plasmid pcDNA3-TspE1 by gene-gun bombardment. The expression of recombinant plasmid in the skin tissue was observed by HE staining and immunohistochemical staining. RESULTS AND CONCLUSION: The recombinant plasmid pcDNA3-TspE1 was successfully constructed and expressed in the BALB/c mice.

Animals↗
PubMed1992

A general system to integrate lacZ fusions into the chromosomes of gram-negative eubacteria: regulation of the Pm promoter of the TOL plasmid studied with all controlling elements in monocopy.

A new procedure is described to recombine plasmid-borne lacZ fusions into the chromosome of gram-negative eubacteria in order to study promoter activity in monocopy. The procedure is based upon the insertion into the chromosome of a target bacterium of a recombinant transposon that carries DNA sequence homology to the regions flanking lacZ fusions present in multicopy promotor-probe vectors, which can be mobilized via RP4-mediated transfer but are unable to replicate in non-enteric bacteria. Double recombination between the promoter-probe vectors and the chromosomal homology region of the transposon is genetically selected by reconstruction and expression of wild-type sequences from truncated lacZ and aadA (streptomycin/spectinomycin) resistance genes in the homology fragment and from an amber mutation carrying lacZ and aadA genes present in the plasmid vectors. The structure of desired clones is confirmed by screening for loss of the transposon-encoded kanamycin resistance marker. We have used this procedure to assemble in monocopy in Pseudomonas putida the regulatory elements controlling expression of the XylS-activated Pm promoter of the TOL catabolic plasmid pWWO. We show here that the Pm promoter undergoes a XylS-independent, strictly growth-phase-controlled activation by benzoate but not meta-toluate. In the presence of XylS, however, activation by both effectors involves a combination of growth phase-dependent and -independent controls.

Blotting, Southern↗
PubMed1990

Cloning and sequence analysis of linear plasmid telomeres of the bacterium Borrelia burgdorferi.

Borrelia burgdorferi, the Lyme disease agent, has double-stranded linear plasmids with covalently closed ends. DNA at the ends, or telomeres, of two linear plasmids of B. burgdorferi strain B31 was examined. Telomeric sequences from both ends of a 16 kb linear plasmid and from one end of a 49 kb linear plasmid were cloned and sequenced. An 18 bp AT-rich inverted repeat was found at each end of the 16 kb linear plasmid. The sequences of the two ends of this plasmid were different beyond these short inverted terminal repeats. The cloned end of the 49 kb linear plasmid had sequence identity with one end of the 16 kb linear plasmid. The end sequence common to both plasmids contained a series of phased, short direct repeats and a 52 bp palindrome adjacent to a highly AT-rich region. These findings indicate that Borrelia linear plasmid telomeres have structural features different from those of other known replicons.

Base Sequence↗
PubMed2005

Molecular basis of resistance to trimethoprim, chloramphenicol and sulphonamides in Bordetella bronchiseptica.

OBJECTIVES: To date, little is known about the molecular basis of antimicrobial resistance in Bordetella bronchiseptica, an important respiratory tract pathogen in pigs, dogs and cats. The aim of this study was to identify genes coding for trimethoprim resistance present in porcine B. bronchiseptica and to determine their localization, transferability and association with other resistance genes. METHODS: Six B. bronchiseptica isolates with elevated MICs of trimethoprim were investigated by PCR for the presence of trimethoprim resistance genes and their association with class 1 integrons. The amplicons obtained were cloned and sequenced. Plasmid localization of these integrons was confirmed by transformation and conjugation. Isolates carrying the same integron were compared for their genetic relatedness by XbaI and SpeI pulsed-field gel electrophoresis (PFGE). RESULTS: Five B. bronchiseptica isolates carried a class 1 integron with two gene cassettes, one carrying the trimethoprim resistance gene dfrA1 and the other the chloramphenicol resistance gene catB3. This integron was present on a common conjugative plasmid in four of the five isolates and on the chromosome in the remaining isolate. All five B. bronchiseptica isolates proved to be related on the basis of their PFGE patterns. Another isolate had a class 1 integron with a dfrB1 and a catB2 cassette on a structurally different conjugative plasmid. The sulphonamide resistance gene sul1 was detected in the 3'-conserved segment of both types of integrons. CONCLUSIONS: This is the first report of trimethoprim, chloramphenicol and sulphonamide resistance genes and class 1 integrons in B. bronchiseptica isolates.

Animals↗