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PubMed2001

DnaA boxes in the P1 plasmid origin: the effect of their position on the directionality of replication and plasmid copy number.

The DnaA protein is essential for initiation of DNA replication in a wide variety of bacterial and plasmid replicons. The replication origin in these replicons invariably contains specific binding sites for the protein, called DnaA boxes. Plasmid P1 contains a set of DnaA boxes at each end of its origin but can function with either one of the sets. Here we report that the location of origin-opening, initiation site of replication forks and directionality of replication do not change whether the boxes are present at both or at one of the ends of the origin. Replication was bidirectional in all cases. These results imply that DnaA functions similarly from the two ends of the origin. However, origins with DnaA boxes proximal to the origin-opening location opened more efficiently and maintained plasmids at higher copy numbers. Origins with the distal set were inactive unless the adjacent P1 DNA sequences beyond the boxes were included. At either end, phasing of the boxes with respect to the remainder of the origin influenced the copy number. Thus, although the boxes can be at either end, their precise context is critical for efficient origin function.

Bacterial Proteins↗
PubMed1994

The large nonsymbiotic plasmid pRmeGR4a of Rhizobium meliloti GR4 encodes a protein involved in replication that has homology with the RepC protein of Agrobacterium plasmids.

The large plasmid, pRmeGR4a, of Rhizobium meliloti GR4 is a nonsymbiotic, self-transmissible replicon that shows a high degree of stability. Plasmid replication and stabilization functions are located on a 4.8-kb PstI fragment. Analysis of the nucleotide sequence of this DNA region revealed the presence of six open reading frames (ORFs) with coding capabilities; all are transcribed in the same direction. Only one of the ORFs (ORF3) seems to be essential for replication. Its predicted protein sequence shows homology with RepC, a protein that has been suggested to be involved in the replication of Agrobacterium Ri and Ti plasmids.

Amino Acid Sequence↗
PubMed2000

Transcription reporters that shuttle cloned DNA between high-copy Escherichia coli plasmids and low-copy broad-host-range plasmids.

We describe and apply lacZ transcription reporter plasmids designed for both biochemical analyses requiring high DNA yield and physiological studies requiring low gene dosage. Standard DNA ligations are performed at seven unique restriction sites 5' to the lacZ gene on high-copy ColE1 plasmids suitable for double- or single-strand DNA sequencing. A divergent gusA transcription reporter is included and serves as an internal control. Rec(+) Escherichia coli cells readily shuttle DNA placed between gusA and lacZ by allelic exchange with pRK290-based plasmids that subsequently conjugate and replicate in most gram-negative bacteria. We applied this system to study Caulobacter crescentus cell cycle promoters directed by the CtrA response-regulator protein. Synthetic oligonucleotides were ligated to create altered CtrA binding sites and corresponding promoters with varied transcription strength. We also document the phenomenon of long-range promoter interference. A strong promoter can repress up to twofold the transcription from a divergent promoter located 100 bp away. However, the cell cycle timing of both promoters is not changed. Additional applications of our system and theoretical aspects of promoter organization are discussed.

Bacterial Proteins↗
PubMed2001

Sequence and gene expression analyses of plasmid pHPM8 from Helicobacter pylori reveal the presence of two operons with putative roles in plasmid replication and antibiotic activity.

The DNA sequence of a 7.8-kb Helicobacter pylori plasmid, pHPM8, was determined. Six open reading frames (ORFs) were present. Ribonuclease protection studies showed that ORF1/ORF2 and ORF3/ORF4 genes are organized in operons possibly involved in plasmid replication and in production of a peptide with antibiotic activity, respectively. Finding areas of pHPM8 with a high level of identity to H. pylori chromosomal DNA supported the hypothesis that recombination occurs between plasmids and the chromosome of H. pylori.

Amino Acid Sequence↗
PubMed1989

Nucleotide sequence of the leading region adjacent to the origin of transfer on plasmid F and its conservation among conjugative plasmids.

The leading region of the Escherichia coli K12 F plasmid is the first segment of DNA to be transferred into the recipient cell during conjugal transfer. We report the nucleotide sequence of the 64.20-66.77F portion of the leading region immediately adjacent to the origin of transfer, oriT. The 2582 bp region encodes three open reading frames, ORF95, ORF169 and ORF273; the product of ORF273, is equivalent in size and map location to the 35 kDa protein, 6d, previously described (Cram et al. 1984). S1 nuclease analyses of mRNA transcripts have identified a potential promoter for ORF95 and ORF273 and indicated that these ORFs are transcribed as a single transcript; in contrast, ORF169 appears to be transcribed from two overlapping promoters on the complementary DNA strand. The products of ORF95 and ORF273 are mainly hydrophilic and are probably located in the cytoplasm. ORF273 shares some homology with DNA-binding proteins. There is a signal peptide sequence at the NH2-terminus of ORF169 and the mature form of ORF169 probably resides in the periplasm due to its hydrophilic nature. Both ORF273 and ORF169 are well conserved among conjugative F-like and a few non-F-like plasmids. On the other hand, ORF95 sequences are only present on some of these plasmids. Several primosome and integration host factor recognition sites are present implicating this region in DNA metabolism and/or replication functions.

Amino Acid Sequence↗
PubMed1979

Construction in vitro of "phage-plasmid" chimerae: a new tool to analyse the mechanism of plasmid maintenance.

In this paper, we report the construction in vitro of chimerae between lambdoid replacement vectors (Murray et al., 1977) and the miniF Apr plasmid: pSC138 (Timmis et al., 1975). lambdaF recombinants were shown to be chimerae between the lambda and the F replicons. By genetical tests, we have demonstrated that both lambda and F replication mechanisms are functional: the lambdaF recombinant behaves as a non defective plaque forming phage on lambda sensitive bacteria and establishes itself as a stable plasmid on recA F- homoimmune bacteria. In the extra-chromosomal state, the lambdaF recombinant apparently retains the controlled autonomous replication and the FI incompatibility characteristics of the F plasmid. The potential experimental uses of these phages are discussed.

Coliphages↗
PubMed1979

Construction of an HpaI and HindII plasmid vector allowing direct selection of transformants harboring recombinant plasmids.

The construction of the vector plasmid PKN80 is described, which can be used as HpaI or HindII cloning vehicle with direct selection on transformants harboring hybrid plasmids. pKN80 carries the EcoRI.C fragment of phage Mu DNA coding for a killing function which is efficiently expressed upon transformation of pKN80 into Mu-sensitive bacteria. Cloning of DNA fragments at the single HpaI site of pKN80 results in insertional inactivation of the killing function. Whereas religated pKN80 molecules yielded only a few transformants, the transformation efficiency had been increased by a factor of at least ten when HpaI fagments of lambda DNA were added to the linearized vector prior to ligation. More than 90% of the transformants tested containted hybrid plasmids.

DNA Restriction Enzymes↗
PubMed1994

Control of ColE2 plasmid replication: regulation of Rep expression by a plasmid-coded antisense RNA.

We isolated and characterized mutants of ColE2 with increased copy number (cop) and those with reduced sensitivity to the wild-type incA gene (inc). Both types of mutations were single-base substitutions in the incA region and simultaneously increased the plasmid copy number and reduced the inhibitory activity of the incA gene on ColE2 DNA replication. Most of the cop mutations also reduced sensitivity to the wild-type incA gene. These mutations were located in the region specifying the large stem-and-loop structures of RNA I and the 5' portion of the Rep mRNA. All these results indicate that RNA I interacts with the Rep mRNA and thereby inhibits expression of the Rep protein at a post-transcriptional step and that this is probably the only mechanism that controls the ColE2 Rep protein expression. It is suggested that only portions of the nucleotides in the loop region are involved in initial (kissing) interaction of these RNAs. The total level of rep gene expression in the host cells appears to be kept constant (at a level characteristic for each cop allele) irrespective of the actual plasmid copy number above a certain level, when rep gene expression is regulated by the incA gene on the same plasmid. These seem to be the basic mechanisms for the replication control of ColE2.

Bacterial Proteins↗
PubMed1993

Interaction between mitochondrial DNA and mitochondrial plasmids in Claviceps purpurea: analysis of plasmid-homologous sequences upstream of the lrRNA-gene.

Homology of two linear, mitochondrial (mt) Claviceps purpurea plasmids, pClK1 and pClT5, to the upstream region of the large ribosomal RNA gene in the mtDNA of three strains (W3, T5 and K) has been investigated in detail to explore the widespread phenomenon of homology between mt plasmids and mtDNA in C. purpurea. Sequence comparison indicates that recombination between free plasmids and mtDNA is the cause of the observed homology. The process is similar to the integration of the structurally related adenoviruses into the mammalian genome. As in other fungi, palindromic sequences seem to be involved in this mitochondrial recombination process.

Base Sequence↗
PubMed1987

Sequence of psi, a gene on the symbiotic plasmid of Rhizobium phaseoli which inhibits exopolysaccharide synthesis and nodulation and demonstration that its transcription is inhibited by psr, another gene on the symbiotic plasmid.

A gene termed psi (polysaccharide inhibition), located close to the nodulation genes of the Rhizobium phaseoli symbiotic plasmid pRP2JI inhibited exopolysaccharide synthesis (EPS) and nodulation ability (Nod) in Rhizobium when it was cloned in a multicopy plasmid. The sequence of psi showed that it specified a polypeptide of mol. wt. 10,000 that may be associated with the membrane of Rhizobium. A second gene, psr (polysaccharide restoration), was located on pRP2JI. When cloned in multicopy plasmids, psr overcame the EPS and Nod defects in strains carrying multicopy psi. Strains with multicopy psr induced non-fixing nodules on Phaseolus beans. Using gene fusions between psi and lacZ, it was found that psr [corrected] inhibited transcription of psi [corrected].

Gene Expression↗
PubMed1983

Occurrence of deletion plasmids at high rates after conjugative transfer of the plasmids RP4 and RK2 from Escherichia coli to Alcaligenes eutrophus H16.

The broad host-range IncP-1 plasmids RP4 and RK2 were transferred by conjugation from Escherichia coli to Alcaligenes eutrophus H16. Among the transconjugants selected on media containing tetracycline, a considerable number did not express kanamycin resistance. By comparing restriction patterns of plasmids isolated from a large number of transconjugants a variety of different deletion derivatives were found. All of these possess more or less extended deletions always including parts of the tra 1-region. The plasmids RP4 and RK2, once established in A. eutrophus H16 showed a high stability and it can be concluded that deletion formation is connected with the conjugation process. Evidence is given that degradation of DNA entering an A. eutrophus recipient cell during the conjugative transfer process may be involved in deletion formation. Furthermore, the finding of a small deletion derivative of RP4 lacking the transacting replication function trfB and the entire kil-kor-system may allow the assumption that these gene functions are not essential for replication and maintenance of RP4 in A. eutrophus hosts.

Alcaligenes↗
PubMed1990

Construction of plasmid-free derivatives of Salmonella typhimurium LT2 using temperature-sensitive mutants of pKZ1 for displacement of the resident plasmid, pSLT.

Replication (or partitioning) temperature-sensitive mutants of pKZ1 were isolated and shown to exhibit incompatibility with the resident plasmid (pSLT) of Salmonella typhimurium LT2. Following displacement of pSLT, the mutant plasmids were effectively eliminated from the cell population by passage at 42 degrees C, yielding plasmid-free isolates.

Blotting, Southern↗
PubMed1993

Polypyrimidine/polypurine sequence in plasmid DNA enhances formation of dimer molecules in Escherichia coli. Dimerization of plasmid DNA in Escherichia coli.

Formation of dimer molecules of a recombinant plasmid, pTIR10, which carries a pyrimidine/purine-biased stretch occurs about 6-fold more efficiently than for the control plasmid pUC19 in Escherichia coli strain JM107. Since pyrimidine/purine-biased sequences have a potential to form unusual DNA structures, this observation suggests that the inserted sequence affects the replication process of plasmid DNA, probably by forming a triple helix under physiological conditions.

Animals↗
PubMed1996

Comparative analysis of 18 sex pheromone plasmids from Enterococcus faecalis: detection of a new insertion element on pPD1 and implications for the evolution of this plasmid family.

A new IS element, IS1062, related to the enterococcal IS elements IS6770 and IS1252, was detected in the 3'-terminus of the surface exclusion gene, sep1, of sex pheromone plasmid pPD1 in Enterococcus faecalis. pPD1-bearing cells lack the surface exclusion function, probably as a consequence of this insertion. Analysis of pAD1 and pPD1 sequences (7.5 kb and 2.7 kb, respectively) downstream of their aggregation substance genes revealed no similarity in these DNA regions. Detailed DNA/DNA hybridization studies using DNA probes specific for various pAD1-encoded genes needed for plasmid transfer indicated that the sex pheromone plasmids have evolved by repeated recombination and insertion of diverse transposable elements which presumably account for recent acquisition of antibiotic resistances.

Amino Acid Sequence↗
PubMed1998

Electroporation of, plasmid isolation from and plasmid conservation in Clostridium acetobutylicum DSM 792.

Procedures have been developed allowing recombinant DNA work with Clostridium acetobutylicum DSM 792. Electroporation was used to introduce plasmid DNA into exponentially growing clostridial cells and 6 x 10(2) transformants/microgram DNA could be obtained at a time constant of 5.5 ms, 1.8 kV, 50 microF, and 600 omega. The method also allowed the taxonomic group IV strain NI-4082 to be transformed (10(1) transformants/microgram DNA). Plasmid preparation from recombinant clostridia was optimal when a modification of the alkaline lysis method was employed. It was also important to use cells from the mid-logarithmic growth phase. Recombinant strains could be easily preserved as spore suspensions; under all conditions tested plasmids were maintained.

Anaerobiosis↗
PubMed2002

F-like plasmid sequences in enteric bacteria of diverse origin, with implication of horizontal transfer and plasmid host range.

Seventy-eight bacterial isolates from human, animal, and plant hosts, representing eight species of the family Enterobacteriaceae, were screened for F-like plasmid sequences. Of the examined human Escherichia coli strains, 28% harbored one or two of the three F-like, RepFI replication regions, while 35% of the examined animal and all phytopathogenic strains harbored RepFIA-specific sequences. Comparative analysis of Salmonella, Shigella, Erwinia, and E. coli plasmid RepFI sequences showed 100% or very high homology, indicating frequent and recent interspecies gene transfer. The high incidence of RepFIA sequences in enteric bacterial species, including Klebsiella and Erwinia, showed that F-like plasmids are successful in avoiding natural barriers to establishment of horizontally transferred DNA and that in the natural environment conjugal transfer is efficient in diverse ecological niches.

Amino Acid Sequence↗
PubMed1993

Purification of the beta product encoded by the Streptococcus pyogenes plasmid pSM19035. A putative DNA recombinase required to resolve plasmid oligomers.

Genetic evidence suggests that the gene beta product of Streptococcus pyogenes plasmid pSM19035 is required for converting plasmid multimers into monomers. The beta protein was purified from cells overexpressing the cloned gene. N-terminal protein sequence analysis demonstrated that the purified protein had the predicted sequence, except that the N-terminal initiator methionine was not present. Native beta protein consists of a dimer of two identical subunits with a molecular mass of 23.8 kDa (25 kDa in SDS-PAGE). The beta protein (isoelectric point of 9.7) binds specifically to a DNA fragment (312 bp in length) which contains the promoter region of the orf alpha-gene beta operon and two regions (sites I and II) that show dyad axes of symmetry. It is proposed that protein beta binds to sites I and II to mediate resolution of plasmid oligomers.

Bacterial Proteins↗
PubMed1976

Recombination between bacterial plasmids leading to the formation of plasmid multimers.

We report here the formation of plasmid multimers in E. coli. Multimers are covalently closed, circular molecules composed of tandem repeats of the monomer plasmid. Multimer formation occurs at high frequency in rec+, recB-C-, and recF- hosts. Multimer formation is not detected in recA- hosts and occurs at reduced frequency in recB-C-F- hosts. We conclude that multimer formation is mediated by a single reciprocal recombination event occurring at homologous regions on the two plasmids.

Coliphages↗