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PubMed1983

Analysis of IS21-mediated mobilization of plasmid pACYC184 by R68.45 in Escherichia coli.

Escherichia coli plasmids like pACYC184 or pBR325 can be mobilized by the P-type plasmid R68.45, which carries a tandem duplication of insertion element IS21, at a frequency of 10(-3)-10(-5) per donor cell. Analysis of exconjugant cells revealed that plasmid mobilization occurs via cointegrate formation involving transposition of IS21. No resolution of cointegrates of pACYC184 and the P-type plasmid could be found in recA recipient cells. In the cointegrate, the E. coli plasmid is flanked by single copies of IS21 in direct orientation. After resolution of the cointegrate in recA+ recipients, the mobilizing plasmid R68.45 lost one copy of IS21 becoming indistinguishable from plasmid R68. It was shown that during mobilization, insertion element IS21 transposes to the mobilized plasmid. Insertion sites and orientations of IS21 in 33 pACYC184::IS21 insertion mutants have been determined: IS21 was found to be integrated in plasmid pACYC184 in different regions but only in one orientation. The IS21 tandem structure of plasmid R68.45 and its role in the mobilization process is discussed.

Chromosome Mapping↗

PubMed2005

The interaction between resistance modifiers such as pyrido[3,2-g]quinoline, aza-oxafluorene and pregnane derivatives with DNA, plasmid DNA and tRNA.

Various resistance mechanisms such as complex formation with DNA, tRNA and MDR1 p-glycoprotein were modified in bacteria and cancer cells in presence of pregnane, pyridoquinoline, and aza-oxafluorene derivatives. Interaction between the compounds, plasmid DNA and tRNA was shown and compared to the interaction with calf thymus DNA. Complex formation with MDR1 p-glycoprotein and drug accumulation increased in cancer cells. Both plasmid DNA and p-gp complex formation were related to the chemical structures of the resistance modifiers.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2000

Replication of staphylococcal multiresistance plasmids.

Based on structural and functional properties, three groups of large staphylococcal multiresistance plasmids have been recognized, viz., the pSK1 family, pSK41-like conjugative plasmids, and beta-lactamase-heavy-metal resistance plasmids. Here we describe an analysis of the replication functions of a representative of each of these plasmid groups. The replication initiation genes from the Staphylococcus aureus plasmids pSK1, pSK41, and pI9789::Tn552 were found to be related to each other and to the Staphylococcus xylosus plasmid pSX267 and are also related to rep genes of several plasmids from other gram-positive genera. Nucleotide sequence similarity between pSK1 and pI9789::Tn552 extended beyond their rep genes, encompassing upstream divergently transcribed genes, orf245 and orf256, respectively. Our analyses revealed that genes encoding proteins related to the deduced orf245 product are variously represented, in several types of organization, on plasmids possessing six seemingly evolutionarily distinct types of replication initiation genes and including both theta-mode and rolling-circle replicons. Construction of minireplicons and subsequent functional analysis demonstrated that orf245 is required for the segregational stability of the pSK1 replicon. In contrast, no gene equivalent to orf245 is evident on the conjugative plasmid pSK41, and a minireplicon encoding only the pSK41 rep gene was found to exhibit a segregational stability approaching that of the parent plasmid. Significantly, the results described establish that many of the large multiresistance plasmids that have been identified in clinical staphylococci, which were formerly presumed to be unrelated, actually utilize an evolutionarily related theta-mode replication system.

Amino Acid Sequence↗

PubMedDate not supplied

Primary structure of a chloramphenicol acetyltransferase specified by R plasmids.

Naturally occurring isolates of chloramphenicol-resistant bacteria commonly synthesise chloramphenicol acetyltransferase (EC 2.3.28; CAT) in amounts which are sufficient to account for the resistance phenotype and often harbour plasmids which carry the structural gene for CAT. The findings of CAT in such diverse prokaryotes as Proteus mirabilis, Agrobacterium tumefaciens, Streptomyces sp., and a soil Flavobacterium has led to speculation concerning the origin and evolution of the more commonly observed CAT variants specified by plasmids in clinically important bacteria. To provide a more solid basis for studying the evolution and spread of CAT within prokaryotes we chose to determine the complete amino acid sequence of a type I variant of CAT, the variant known to be associated with most F-like plasmids conferring chloramphenicol resistance. The sequence has been determined by combining the results obtained from manual and automated sequential degradation with those obtained by mass spectrometry of peptides generated by enzymatic digestion. The directly determined primary structure is identical with that predicted by the DNA sequence analysis of the chloramphenicol resistance transponson Tn9 known to specify a type I variant of chloramphenicol acetyltransferase.

Acetyltransferases↗

PubMed2007

Mosaic structure of p1658/97, a 125-kilobase plasmid harboring an active amplicon with the extended-spectrum beta-lactamase gene blaSHV-5.

Escherichia coli isolates recovered from patients during a clonal outbreak in a Warsaw, Poland, hospital in 1997 produced different levels of an extended-spectrum beta-lactamase (ESBL) of the SHV type. The beta-lactamase hyperproduction correlated with the multiplication of ESBL gene copies within a plasmid. Here, we present the complete nucleotide sequence of plasmid p1658/97 carried by the isolates recovered during the outbreak. The plasmid is 125,491 bp and shows a mosaic structure in which all modules constituting the plasmid core are homologous to those found in plasmids F and R100 and are separated by segments of homology to other known regions (plasmid R64, Providencia rettgeri genomic island R391, Vibrio cholerae STX transposon, Klebsiella pneumoniae or E. coli chromosomes). Plasmid p1658/97 bears two replication systems, IncFII and IncFIB; we demonstrated that both are active in E. coli. The presence of an active partition system (sopABC locus) and two postsegregational killing systems (pemIK and hok/sok) indicates that the plasmid should be stably maintained in E. coli populations. The conjugative transfer is ensured by the operons of the tra and trb genes. We also demonstrate that the plasmidic segment undergoing amplification contains the blaSHV-5 gene and is homologous to a 7.9-kb fragment of the K. pneumoniae chromosome. The amplicon displays the structure of a composite transposon of type I.

DNA, Bacterial↗

PubMed1988

Genetics of naphthalene catabolism in pseudomonads.

In pseudomonads, naphthalene is catabolized in a series of reactions to salicylic acid, which is further degraded via the catechol meta-cleavage, ortho-cleavage, or gentisic acid pathway to Krebs cycle intermediates. The naphthalene catabolic genes have been located on self-transmissible plasmids, in most cases, and implicated to have chromosomal locations in other cases. The best-studied naphthalene catabolic plasmid is NAH7. It carries two operons, one of which enables the host to utilize naphthalene and the other to utilize salicylate as a carbon and energy source. The product of another NAH7 gene, nahR, is required to turn on both operons in the presence of the inducer, salicylate. Several different naphthalene and salicylate catabolic plasmids have been shown to share sequence homology with NAH7. These plasmids can undergo structural alterations involving insertions and deletions during conjugations and changes in nutritional conditions. Available evidence suggests that salicylate catabolic plasmids can form from the naphthalene catabolic plasmids by structural alterations of the plasmid DNA. The gene organization and regulation, as well as the genetic instability of the naphthalene catabolic plasmids, are reminiscent of the TOL plasmids and suggest that the naphthalene catabolic plasmids and other catabolic plasmids may have evolved in a short period of time by acquiring and modifying preevolved gene clusters from host chromosomes or other plasmids.

Naphthalenes↗

PubMed1991

Plasmid deletion formation between short direct repeats in Bacillus subtilis is stimulated by single-stranded rolling-circle replication intermediates.

The effects of the rolling-circle mode of replication and the generation of single-stranded DNA (ss DNA) on plasmid deletion formation between short direct repeats in Bacillus subtilis were studied. Deletion units consisting of direct repeats (9, 18, or 27 bp) that do or do not flank inverted repeats (300 bp) were introduced into various plasmid replicons that generate different amounts of ss DNA (from 0% to 40% of the total plasmid DNA). With ss DNA-generating rolling-circle-type plasmids, deletion frequencies between the direct repeats were 3- to 13-fold higher than in plasmids not generating ss DNA. When the direct repeats flanked inverted repeats the deletion frequencies in ss DNA-generating plasmids were increased by as much as 20- to 140-fold. These results support models for deletion formation based on template-switching errors during complementary strand synthesis of rolling-circle-type plasmids. The structural instability (deletion formation between short direct repeats) of the ss DNA-generating plasmid pTA1060 in B. subtilis was very low in the presence of a functional initiation site for complementary strand synthesis (minus origin). This observation suggests that it will be possible to develop stable host-vector cloning systems for B. subtilis.

Bacillus subtilis↗

PubMed1991

Molecular mechanisms of deletion formation in Escherichia coli plasmids. I. Deletion formation mediated by long direct repeats.

Derivatives of plasmid pBR327 with the tet gene interrupted by 165 pb or 401 bp direct repeats were constructed. In cells harboring these plasmids, deletions which restored the wild-type tet gene gave rise to tetracycline-resistant colonies, thereby allowing a simple phenotypic test for deletion formation. The frequencies of deletions in these plasmids were measured in Escherichia coli strains proficient or deficient in general recombination. The structure of plasmid DNA isolated from tetracycline-resistant transformants was analyzed by agarose gel electrophoresis, restriction mapping and sequencing. The data presented here demonstrate that deletion formation is always associated with dimerization of plasmid DNA. Dimeric plasmids were of two types. Those which carried both a deletion and a compensating duplication were the major type in a Rec+ background and were rare in recA, recF, recJ and recO backgrounds. Dimers of the second type contained deletions, but no compensating duplications, and their formation was RecA-independent. The data presented demonstrate that deletion formation mediated by long direct repeats is mainly the result of unequal crossing-over between two plasmid molecules.

Chromosome Mapping↗

PubMedDate not supplied

Structural effects of carbohydrate-containing polycations on gene delivery. 2. Charge center type.

Recent polycation structure-gene delivery studies reveal that subtle changes in the molecular structure of polycations have substantial influences on DNA-binding and condensation and on in vitro toxicity and gene delivery efficiency. In Part 1 of this structure-property study using carbohydrate-containing polycations (1), it is demonstrated that as the amidine charge center is removed further from the carbohydrate unit within the polycation structure, the toxicity increases. Inclusion of larger carbohydrate species within the polycation backbone also reduces the toxicity. Here, the effect that polycation charge center type has on toxicity and gene delivery efficiency is investigated. A series of quaternary ammonium polycations containing N,N,N',N'-tetramethyl-1,6-hexanediamine, d-trehalose, and beta-cyclodextrin are synthesized in order to elucidate the effects of charge center type (by comparison to the data given in Part 1) on gene delivery. In all cases, it is found that the quaternary ammonium analogues exhibit lower gene expression values and similar toxicities to their amidine analogues. Additionally, transfection experiments conducted in the presence of chloroquine reveal increased gene expression from quaternary ammonium containing polycations and not from their amidine analogues.

Animals↗

PubMed1997

Role of enzymes of homologous recombination in illegitimate plasmid recombination in Bacillus subtilis.

The structural stability of plasmid pGP1, which encodes a fusion between the penicillinase gene (penP) of Bacillus licheniformis and the Escherichia coli lacZ gene, was investigated in Bacillus subtilis strains expressing mutated subunits of the ATP-dependent nuclease, AddAB, and strains lacking the major recombination enzyme, RecA. Strains carrying a mutation in the ATP-binding site of the AddB subunit exhibited high levels of plasmid instability, whereas a comparable mutation in the A subunit did not affect plasmid stability. Using an alternative plasmid system, pGP100, we were able to demonstrate that the differences in stability reflected differences in initial recombination frequencies. Based on a comparison of endpoint sequences observed in the various hosts, we speculate that at least two different mechanisms underlie the deletion events involved, the first (type I) occurring between nonrepeated sequences, and the second (type II) occurring between short direct repeats (DRs). The latter event was independent of single-strand replication intermediates and the mode of replication and possibly requires the introduction of double-strand breaks (DSBs) between the repeats. In the absence of functional AddAB complex, or the AddB subunit, DSBs are likely to be processed via a recA-independent mechanism, resulting in intramolecular recombination between the DRs. In wild-type cells, such DSBs are supposed to be either repaired by a mechanism involving AddAB-dependent recombination or degraded by the AddAB-associated exonuclease activity. Plasmid stability assays in a recA mutant showed that (i) the level of deletion formation was considerably higher in this host and (ii) that deletions between short DRs occurred at higher frequencies than those described previously for the parental strain. We propose that in wild-type cells, the recA gene product is involved in recombinational repair of DSBs.

Adenosine Triphosphate↗

PubMed1985

[Replication of Streptomyces plasmids: the DNA nucleotide sequence of plasmid pSB 24.2].

The nucleotide sequence of DNA in plasmid pSB 24.2, a natural deletion derivative of plasmid pSB 24.1 isolated from S. cyanogenus was studied. The plasmid amounted by its size to 3706 nucleotide pairs. The G-C composition was equal to 73 per cent. The analysis of the DNA structure in plasmid pSB 24.2 revealed the protein-encoding sequence of DNA, the continuity of which was significant for replication of the plasmid containing more than 1300 nucleotide pairs. The analysis also revealed two A-T-rich areas of DNA, the G-C composition of which was less than 55 per cent and a DNA area with a branched pin structure. The results may be of value in investigation of plasmid replication in actinomycetes and experimental cloning of DNA with this plasmid as a vector.

Base Composition↗

PubMed2002

Characterization of the tetracycline resistance plasmid pMD5057 from Lactobacillus plantarum 5057 reveals a composite structure.

The 10,877bp tetracycline resistance plasmid pMD5057 from Lactobacillus plantarum 5057 was completely sequenced. The sequence revealed a composite structure containing DNA from up to four different sources. The replication region had homology to other plasmids of lactic acid bacteria while the tetracycline resistance region, containing a tet(M) gene, had high homology to sequences from Clostridium perfringens and Staphylococcus aureus. Within the tetracycline resistance region a Lactobacillus IS-element was found. The remaining part of the plasmid contained three open reading frames with unknown functions. The composite structure with several truncated genes suggests a recent assembly of the plasmid. This is the first sequence of an antibiotic resistance plasmid isolated from L. plantarum.

Base Sequence↗

PubMed1987

Mitochondrial plasmid DNAs of broad bean: nucleotide sequences, complex secondary structures, and transcription.

Three circular plasmid DNA molecules of 1704, 1695 and 1476 nucleotide pairs from broad bean mitochondria (mt-plasmids 1-3) have been sequenced. Within a highly homologous segment of mt-plasmid 1 and 2 are found a series of six directly repeated, inverted repeat sequences, separated by unique sequences. Mt-plasmid 3 contains a series of four inverted repeat sequences, unrelated to the inverted repeat sequences of mt-plasmids 1 and 2. Two RNA molecules of about 440 and 320 nucleotides that are complementary to mt-plasmid 2 were detected. Mapping of 5' and 3' termini of these complementary RNA molecules indicated that all transcription from mt-plasmid 2 occurs within a 441 nucleotide region of the molecule. Evidence for transcription of mt-plasmids 1 and 3 was not found.

Base Sequence↗

PubMed1983

Transfer of plasmids from Escherichia coli to Pseudomonas aeruginosa: characterization of a Pseudomonas aeruginosa mutant with enhanced recipient ability for enterobacterial plasmids.

Pseudomonas aeruginosa strain GT424, derived by the transfer of plasmid R45 to strain GT1, was shown to have an Era+ phenotype (enhanced recipient ability) for enterobacterial plasmids from incompatibility (Inc) groups C, FI, FII, J, N, W, and X. Increase in transfer frequency ranged from 10 to 10(6)-fold. Plasmids of IncFII, IncJ, and IncX were found to be transmissible only to the Era+ strain. Plasmids of Inc groups A-C, M, and T transferred at low frequency (less than 10(-7)) to both GT1 and GT424 and did not respond to the Era+ character of the latter. Also not responsive to the Era character were plasmids R144drd3 (IncI alpha) and RP1 (IncP), which transferred to both GT424 and GT1 at intermediate (10(-4)) and high frequencies (10(-1)), respectively. All plasmid-mediated antibiotic resistances that could be measured as well as UV resistance and susceptibility were found to be expressed in P. aeruginosa. Plasmid-coded phage susceptibilities, however, generally were not phenotypically manifested in the P. aeruginosa strains. The physical structure of plasmids in P. aeruginosa could be classified into four major types. Some of the plasmids underwent additions or deletions and were unstable; the majority, however, appeared to integrate into the chromosome. Some members of IncN, IncC, IncJ, and IncP were found to be transmissible from P. aeruginosa back to Escherichia coli. This is the first report of the successful transfer to P. aeruginosa of plasmids from many of the Inc groups that have been defined in Enterobacteriaceae, namely, FI, I alpha, J, M, T, and X.

Conjugation, Genetic↗

PubMed1983

The role of lipopolysaccharide structure in the recipient cell during plasmid-mediated bacterial conjugation.

The role of the lipopolysaccharide (LPS) structure in the recipient cell during bacterial conjugation was studied using a series of well-defined LPS mutations in Salmonella minnesota. The plasmids Flac (IncFI) and R1drd19 (IncFII) transferred at a high frequency to the smooth S218 parent strain and the rough LPS mutants. However, R64drd1 1 (IncI alpha) transferred poorly to the LPS mutants compared with transfer to the smooth LPS parent strain. The decrease in R64drd1 1 transfer frequency correlated with the extent of the defect in LPS structure, suggesting that intact LPS on the recipient is a major requirement for R64drd1 1 mating. Transfer of the P-group plasmid, RK2, was not affected by changes in LPS structure. These results show that plasmids use different cell surface structures during conjugation, and that LPS is particularly important for R64drd1 1 transfer.

Cell Membrane↗

PubMed1992

A deletion derivative of the kalilo senescence plasmid forms hairpin and duplex DNA structures in the mitochondria of Neurospora.

A novel deletion derivative, delta kal, of the kalilo senescence plasmid from Neurospora intermedia, was recovered from a culture treated with chloramphenicol. The deletion derivative exists in mitochondria as two different, equally abundant forms: a 2.8 kb duplex DNA molecule (delta kal-2.8) and a 1.4 kb hairpin form (delta kal-1.4). The delta kal-2.8 plasmid contains the 1366 bp terminal inverted repeats and a partially duplicated 102 bp segment of the unique sequence of the 8.6 kb kalilo plasmid. In contrast, the delta kal-1.4 hairpin plasmid appears to result from the folding of single strands that are generated during the replication of delta kal-2.8. Both forms of delta kal have covalently linked terminal proteins. Sequence analysis suggests that delta kal was generated either by slippage of the tip of a growing strand during the replication of kalilo, or by illegitimate recombination between two copies of the plasmid at non-homologous palindromic sequences that might form cruciform structures. In either case, the deletion process was mediated at least in part by an inverted repeat of 5 bp in the unique region of kalilo. Since the terminal segments of kalilo DNA that are implicated in plasmid integration might also form cruciform structures, it is possible, but improbable, that the process that generated the first delta kal molecule is related to that which mediates integration of the plasmid into mitochondrial DNA.

Base Sequence↗

PubMed1987

Plasmidal maintenance of composite DNA derived from polyoma related plasmid, L factor.

Recently, we reported a multicopy mammalian plasmid with a structure related to polyoma. The plasmid, named L factor, was found at a high copy number (5,000 or more per cell) in a subclone derived from mouse L cells. We attempted to utilize L factor as a plasmid vector for mammalian cells. A series of composite DNA consisting of L factor and a foreign (herpes simplex virus tk) were constructed. These DNA could be established as plasmids after transfection to several mouse cell lines, although the copy number of the re-established plasmids was considerably less than that observed for the original subclone. The composite DNA maintained the structure of the original DNA after prolonged culture and the copy number remained constant even with no selective pressure. A composite DNA, with no DNA sequence corresponding to polyoma T antigen, could also be established as a plasmid in a mouse L cell line in which polyoma T antigen is expressed. The potential use of the plasmid is discussed.

Animals↗

PubMed2004

Drosophila melanogaster topoisomerase IIIalpha preferentially relaxes a positively or negatively supercoiled bubble substrate and is essential during development.

Eukaryotic type IA topoisomerases are important for the normal function of the cell, and in some cases essential for the organism, although their role in DNA metabolism remains to be elucidated. In this study, we cloned Drosophila melanogaster topoisomerase (topo) IIIalpha from an embryonic cDNA library and expressed and purified the protein to >95% homogeneity. This enzyme partially relaxes a hypernegatively supercoiled plasmid substrate consistent with other purified topo IIIs. A novel, covalently closed bubble substrate was prepared for this study, which topo IIIalpha fully relaxed, regardless of the handedness of the supercoils. Experiments with the bubble substrate demonstrate that topo IIIalpha has much different reaction preferences from those obtained by plasmid substrate-based assays. This is presumably due to the fact that solution conditions can affect the structure of plasmid based substrates and therefore their suitability as a substrate. A mutant allele of the Top3alpha gene, Top3alpha191, was isolated through imprecise excision mutagenesis of an existing P-element inserted in the first intron of the gene. Top3alpha191 is recessive lethal, with most of the homozygous individuals surviving to pupation but never emerging to adulthood. Whereas this mutation can be rescued by a Top3alpha transgene, ubiquitous overexpression of D. melanogaster topo IIIbeta cannot rescue this allele.

Animals↗