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PubMed1990

Structural and functional stability of IncP plasmids during stepwise transmission by trans-kingdom mating: promiscuous conjugation of Escherichia coli and Saccharomyces cerevisiae.

In order to establish a gene transfer system for yeast by promiscuous conjugation, we constructed plasmid pAY101 which contained an oriT sequence derived from RK2 (IncP) and the yeast TRP1 and ARS1 genes. A conjugation mixture consisted of yeast Saccharomyces cerevisiae, E. coli harboring pAY101, and E. coli carrying a helper plasmid with mob and tra. In the conjugation mixture a tryptophan-requiring yeast mutant (trp1) was converted to be prototrophic for tryptophan at a frequency of about 10(-5) to 10(-3) per recipient cell. This E. coli-yeast conjugation system required the mob, tra, oriT, TRP1 and ARS1 genes. The mob and tra genes were trans-acting elements as in an E. coli conjugation system. The mobilization was inhibited by nalidixic acid as in a typical bacterial conjugation. DNA analysis indicated that the plasmid pAY101 was transferred from E. coli to S. cerevisiae, and retained its original structure and function in yeast host cells.

Blotting, Southern↗

PubMed2002

Structural and functional analysis of the kid toxin protein from E. coli plasmid R1.

We have determined the structure of Kid toxin protein from E. coli plasmid R1 involved in stable plasmid inheritance by postsegregational killing of plasmid-less daughter cells. Kid forms a two-component system with its antagonist, Kis antitoxin. Our 1.4 A crystal structure of Kid reveals a 2-fold symmetric dimer that closely resembles the DNA gyrase-inhibitory toxin protein CcdB from E. coli F plasmid despite the lack of any notable sequence similarity. Analysis of nontoxic mutants of Kid suggests a target interaction interface associated with toxicity that is in marked contrast to that proposed for CcdB. A possible region for interaction of Kid with the antitoxin is proposed that overlaps with the target binding site and may explain the mode of antitoxin action.

Amino Acid Sequence↗

PubMed2001

Viewing of complex molecules of ethidium bromide and plasmid DNA in solution by atomic force microscopy.

Tertiary structure changes in plasmid DNA, induced by ethidium bromide intercalation, have been observed in aqueous solutions by the use of an atomic force microscope. A relaxed closed circular pBR322 molecule became a positively supercoiled complex on the drug binding. The supercoiling always resulted in an interwound (or a plectonemic) form, but never a solenoidal (or a toroidal) form. A quantitative analysis of the compactness of such supercoiled complexes has been carried out.

DNA↗

PubMed1975

Detection of nonintegrated plasmid deoxyribonucleic acid in the folded chromosome of Escherichia coli: physiochemical approach to studying the unit of segregation.

Physiocochemical evidence presented indicates plasmid deoxyribonucleic acid (DNA) can associate with host chromosome without linear insertion of the former into the latter. This conclusion is based on the observation that covalently closed circular (CCC) plasmid DNA can cosediment with undegraded host chromosome in a neutral sucrose gradient. When F plus bacteria are lysed under conditions that preserve chromosome, approximately 90% of CCC F sex factor plasmid (about 1% of the total DNA) is found in folded chromosomes sedimenting at rates between 1,500 and 4,000s. The remaining 10% of the CCC F DNA sediments at the rate (80S) indicative of the free CCC plasmid form. Reconstruction experiments in which 80S, CCC F DNA is added to F plus or F minus bacteria before cell lysis show that exogenous F DNA does not associate with folded chromosomes. In F plus bacteria, F plasmid is harbored at a level of one or two copies per chromosomal equivalent. In bacteria producing colicin E1, the genetic determinant of this colicin, the Col E1 plasmid, is harbored at levels of 10 to 13 copies per chromosomal equivalent; yet, greater than 90% of these plasmids do not cosediment with the 1,800S species of folded chromosome. However, preliminary evidence suggests one or two Col E1 plasmids may associate with the 1,800S folded chromosome. Based on evidence presented in this and other papers, we postulate F plasmid can link to folded chromosome because the physicochemical structure of the plasmid resembles a supercoiled region of the chromosome and, therefore, is able to interact with the ribonucleic acid that stabilizes the folded chromosome structure. Implications of this model for F plasmid replication and segregation are discussed.

Carbon Radioisotopes↗

PubMed2002

Structural heterogeneity of pyrimidine/purine-biased DNA sequence analyzed by atomic force microscopy.

We report here the direct evidence for the formation of alternative DNA structures in a plasmid DNA, termed pTIR10, containing a 0.23-kb pyrimidine/purine-biased (Pyr/Pur) stretch isolated from the rat genome. Long Pyr/Pur sequences are abundant in eukaryotic genomes, and they may modulate the biological activity of genes and genomes via formation of various types of triplex-related structures. The plasmid DNA in sodium acetate buffer (pH 4.35) was deposited on APS-modified mica, and after drying it was imaged with an atomic force microscope in air. Various types of thick protrusions have been observed on pTIR10 DNA. Structural parameters (width and height) of DNA molecules suggest that the alternative structures observed here are variations on the theme of an intramolecular triplex. The biological relevance of the structural features within Pyr/Pur stretches is discussed.

DNA↗

PubMed1989

Nucleotide sequences from the colicin E5, E6 and E9 operons: presence of a degenerate transposon-like structure in the ColE9-J plasmid.

The nucleotide sequences of 1288 bp of plasmid ColE5-099, 1609 bp of ColE6-CT14 and 2099 bp of ColE9-J were determined. These sequences encompass the structural genes for the C-terminal receptor-binding and nuclease domains of colicins E5, E6 and E9, their cis- or trans-acting immunity proteins and four lysis proteins including an atypical one of non-lipoprotein nature (Lys) present in the ColE9-J plasmid. The ColE6 gene organisation, in the order col-imm-E8imm-lys, is identical to that found in the previously described double-immunity gene system of ColE3-CA38 (an RNase producer). The corresponding genes in the two plasmids are 87%-94% homologous. In ColE9-J, the genes are organised as col-imm-lys-E5imm-lys. The E9 col-imm gene pair is homologous to the colicin E2-P9 type (a DNase producer). Downstream from E9imm is an E5imm (designated E5imm[E9]) which is trans-acting. Neither the predicted structures of E5Imm[E9] nor the cis-acting Imm resident in the ColE5-099 plasmid which differs by a single amino acid shows any resemblance to other immunity structures which have been sequenced. Furthermore, the E5col sequences differ from those predicted previously for other colicins except for the conserved btuB-specified receptor-binding domain. A novel 205 nucleotide long insertion sequence is found in the ColE9-J plasmid. This insertion sequence, which we named ISE9, has features reminiscent of the degenerate transposon IS101 previously found in plasmid pSC101. One effect of ISE9 is the presence of the atypical lysis gene, lys.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1988

Extranuclear gene expression in yeast: evidence for a plasmid-encoded RNA polymerase of unique structure.

Strains of the yeast Kluyveromyces lactis that produce killer-toxin have been found to contain two linear dsDNA plasmids, k1 (8.9 Kb) and k2 (13.4 Kb). The four transcribed open reading frames of plasmid k1 contain no recognisable yeast nuclear expression signals. Moreover, a toxin subunit gene fused with the lacZ gene of Escherichia coli is not detectably expressed when introduced to K.lactis or Saccharomyces cerevisiae on a nuclear vector, even when native k1 and k2 are present in the cell. This and other evidence is consistent with the hypothesis that k1 and k2 reside in an extranuclear location, and do not utilise the nuclear RNA polymerases I, II or III for transcription of their genes. Sequencing of plasmid k2, which is thought to encode factors necessary for the maintenance or expression of k1, reveals an open reading frame predicted to encode a 974 amino acid polypeptide with homology to several DNA-directed RNA polymerases. We suggest that this is a component of a novel plasmid-specific extranuclear gene expression system.

Amino Acid Sequence↗

PubMed2002

Characterization of pECL18 and pKPN2: a proposed pathway for the evolution of two plasmids that carry identical genes for a Type II restriction-modification system.

The primary structures of the plasmids pECL18 (5571 bp) and pKPN2 (4196 bp) from Escherichia coli and Klebsiella pneumoniae, respectively, which carry genes for a Type II restriction-modification system (RMS2) with the specificity 5'-CCNGG-3', were determined in order to elucidate the structural relationship between them. The data suggest a possible role for recombination events at bom (basis of mobility) regions and the sites of resolution of multimer plasmid forms (so-called cer sequences) in the structural evolution of multicopy plasmids. Analysis of the sequences of pECL18 and pKPN2 showed that the genes for RM* Ecl18kI and RM* Kpn2kI, and the sequences of the rep (replication) regions in the two plasmids, are almost identical. In both plasmids, these regions are localized between the bom regions and the cer sites. The rest of the pECL18 sequence is almost identical to that of the mob (mobilization) region of ColE1, and the corresponding segment of pKPN2 is almost identical to part of pHS-2 from Shigella flexneri. The difference in primary structures results in different mobilization properties of pECL18 and pKPN2. The complete sequences of pECL18, pKPN2 and the pairwise comparison of the sequences of pECL18, pKPN2, ColE1 and pHS-2 suggest that plasmids may exchange DNA units via site-specific recombination events at bom and cer sites. In the course of BLASTN database searches using the cer sites of pECL18 and pKPN2 as queries, we found twenty cer sites of natural plasmids. Alignment of these sequences reveals that they fall into two classes. The plasmids in each group possess related segments between their cer and bom sites.

Base Sequence↗

PubMed1997

Plasmid DNA-based immunization for hepatitis C virus structural proteins: immune responses in mice.

BACKGROUND & AIMS: Plasmid DNA-based immunization has been shown to be an effective means of vaccination in animal models. In this study, the immune responses to various hepatitis C virus structural protein antigens were evaluated using this technique. METHODS: Six recombinant plasmids were constructed. These include, individually, the coding regions for the core protein (pC); E1 (pE1) and E2 (pE2); as well as core, E1, and E2 together (pCE1E2); E1 and E2 together (pE1E2); and finally an E2 construct from which the N-terminal hypervariable region had been deleted (pE2 deltaHVR). These plasmids were transfected into mammalian cells to test their protein expression and were injected into the quadriceps muscles of BALB/c mice to measure specific antibodies and cytotoxic T-lymphocyte responses. RESULTS: All the recombinant plasmids were shown to express specific antigens transiently in cells and elicited specific antibody responses to core, E1, and E2 in mice. Specific cytotoxic T lymphocyte responses were detected only in mice injected with plasmid constructs encoding the core. CONCLUSIONS: Genetic immunization can aid the development of hepatitis C virus vaccines by allowing for the rapid construction and evaluation of different expression plasmids as potential immunogens.

Animals↗

PubMed1990

Processing and intracellular transport of rubella virus structural proteins in COS cells.

Plasmids encoding rubella virus (RV) structural proteins C-E2-E1, E2-E1, E2, and E1 have been constructed in the eukaryotic expression vector pCMV5. The processing and intracellular transport of these proteins have been examined by transient expression of the cDNAs in COS cells. Compared to alphaviruses, processing of RV glycoprotein moieties occurred relatively slowly and the transport of glycoproteins E2 and E1 to the plasma membrane was inefficient. Indirect immunofluorescence revealed that the majority of RV antigen in transfected and infected COS cells was localized to the Golgi region, including the capsid protein. Accumulation of capsid protein in the juxtanuclear region was determined to be RV glycoprotein dependent. Unlike alphaviruses, RV E1 did not require E2 for targeting to the Golgi where it was retained. E2 was however necessary for cell surface expression of E1. This study revealed that the processing and transport of RV structural proteins is quite different from alphaviruses and that the accumulation of antigens in the Golgi region may be significant in light of previous reports which suggest that RV buds from the internal membranes in some cell types.

Acylation↗

PubMed1982

Physical map of pColA-CA31, an amplifiable plasmid, and location of colicin A structural gene.

Evidence showing that the plasmic ColA, derived from strain CA31[pColA] can be amplified in the presence of chloramphenicol is presented. This plasmid has been purified and its Mr-value has been found to be 4.6 X 10(6) or 7 kb. Twelve cleavage sites have been mapped in pColA by using single and double restriction endonuclease digestions. These sites were ordered in relation to the single HindIII site. The other restriction endonucleases used were, respectively, SmaI, AvaI, PstI and HincII. Establishment of the map was helped by hybridization of pColA endonuclease digest products with 32P-labeled colicin A-mRNA. The structural gene for colicin A was contained in a 2.17-kb HincII fragment.

Chloramphenicol↗

PubMed1994

Nucleotide sequence and structural organization of the small, broad-host-range plasmid pCI411 from Leuconostoc lactis 533.

The nucleotide sequence of the Leuconostoc lactis 533 cryptic plasmid pCI411 (2926 bp) was determined. Analysis revealed the presence of three open reading frames (ORFs). ORF 1 was capable of encoding a 24.9 kDa peptide which shared homology with the replication initiation protein (RepB) from a number of Gram-positive rolling circle plasmids. ORF 2 could encode a peptide of 6.6 kDa which was homologous to the RepC protein of the lactococcal plasmid pWV01. A function could not be assigned to ORF 3, which was capable of encoding a 12.1 kDa peptide. Transcription-translation analysis indicated the presence of three peptides of the predicted molecular masses. A putative double strand origin of replication (DSO) was identified which showed strong similarity with the DSO of a number of Gram-positive plasmids including pE194 from Staphylococcus. Structural analysis identified a number of direct and indirect repeats in addition to putative recombination-specific sites (RSA and RSB) in the non-coding region of pCI411. The observed characteristics suggest that this plasmid replicates using the rolling circle mechanism. pCI411, which could be introduced into Leuconostoc, Lactococcus, Streptococcus, Lactobacillus and Bacillus is the first plasmid from the genus Leuconostoc to be characterized in such detail.

Amino Acid Sequence↗

PubMedDate not supplied

Inhibition of tumor induction in tobacco by Agrobacterium tumefaciens and nodulation induced by Rhizobium meliloti in the presence of phenothiazines and structurally related compounds.

Plasmids of Agrobacterium tumefaciens and Rhizobium meliloti carrying Kanamycin resistance genes were eliminated from 1.4 to 0.2% of the growing bacterial cultures by promethazine and imipramine. As a result of plasmid elimination, the A. tumefaciens plasmidless isolate was not able to induce crown gall tumor on tobacco plants. The plasmidless R. meliloti strain failed to induce nodule formation on alfalfa plants. The efficiency of nodulation was decreased when the bacteria were grown in the presence of the drugs. The antiplasmid effects of the drugs were not prevented by opines, (nopaline and octopine) in Escherichia coli F'lac cells.

Agrobacterium tumefaciens↗

PubMed1998

Structural basis for binding of the plasmid ColIb-P9 antisense Inc RNA to its target RNA with the 5'-rUUGGCG-3' motif in the loop sequence.

The sequence 5'-rUUGGCG-3' is conserved within the loop regions of antisense RNAs or their targets involved in replication of various prokaryotic plasmids. In IncIalpha plasmid ColIb-P9, the partially base paired 21-nucleotide loop of a stem-loop called structure I within RepZ mRNA contains this hexanucleotide sequence, and comprises the target site for the antisense Inc RNA. In this report, we find that the base pairing interaction at the 5'-rGGC-3' sequence in the hexanucleotide motif is important for interaction between Inc RNA and structure I. In addition, the 21-base loop domain of structure I is folded tighter than predicted, with the hexanucleotide sequence at the top. The second U residue in the sequence is favored for Inc RNA binding in a base-specific manner. On the other hand, the upper domain of the Inc RNA stem-loop is loosely structured, and maintaining the loop sequence single-stranded is important for the intermolecular interaction. Based on these results, we propose that a structural feature in the loop I domain, conferred probably by the conserved 5'-rUUGGCG-3' sequence, favors binding to a complementary, single-stranded RNA. This model also explains how the RepZ mRNA pseudoknot, described in the accompanying paper (Asano, K., and Mizobuchi, K. (1998) J. Biol. Chem. 273, 11815-11825) is formed specifically with structure I. A possible conformation adopted by the 5'-rUUGGCG-3' loop sequence is discussed.

Bacterial Proteins↗

PubMed1981

Large plasmids of fast-growing rhizobia: homology studies and location of structural nitrogen fixation (nif) genes.

A single large plasmid was isolated from multiplasmid-harboring strains Rhizobium leguminosarum 1001 and R. trifolii 5. These single plasmids, as well as the largest plasmid detectable in R. phaseoli 3622, hybridized with part of the nif structural genes of Klebsiella pneumoniae. In contrast, the plasmids of R. meliloti strains V7 and L5-30 did not show hybridization with the nif genes of K. pneumoniae, indicating that these genes might be located either on the chromosome or on a much larger plasmid which as yet has not been isolated. Studies of the homology between plasmids of fast-growing Rhizobium species showed that a specific deoxyribonucleic acid sequence, which carries the structural genes for nitrogenase, is highly conserved on a plasmid in R. leguminosarum, R. trifolii, and R. phaseoli. Furthermore, it was found that this type of plasmid in the different species shares extensive deoxyribonucleic acid homology, suggesting that strains in the R. leguminosarum cluster have preserved a nif plasmid.

Base Sequence↗

PubMed1988

Packaging and transduction of non-T3 DNA by bacteriophage T3.

A defined in vitro system for packaging T3 DNA also packaged other linear DNAs, including T4, lambda, and plasmid DNAs. The packaging capacity was determined to be 40 kb (kilobase pairs) by measuring the packaged length of T4 DNA. Packaged lambda and plasmid DNAs were injected into host cells to form plaques and transductants, respectively. The yield of transducers increased by using artificially ligated plasmid oligomers. The T3 mutant in gene 3 endonuclease (T3 3-) packaged plasmid DNA during abortive infection and transduced it into the recipient. Transduction of recombinant plasmids was not affected by the presence of the terminally redundant sequence (TR sequence) but increased by 4 orders of magnitudes when the genetic right-end 2.7-kb sequences, containing gene 19 (E1) but lacking TR, were present and by 7 orders when both E1 and TR sequences were present. However, these sequences did not increase transduction of these plasmids by T7 3-. Analysis of the structure of transduced plasmid DNAs indicates that transducing particles carry head-to-tail oligomers of plasmid DNA with the same termini as those of T3 genomic DNA. The mechanism of formation of transducing particles is discussed.

Bacteriophage lambda↗

PubMed1992

Plasmid instabilities of single and three-plasmid systems in Escherichia coli during continuous cultivation.

Plasmid instabilities in E. coli JM103 carrying three plasmids (pRK248cI, pMTC48, pEcoR4) and a single plasmid system (pTG206) for the production of fusion EcoRI (SPA::EcoRI) and catechol 2,3-dioxygenase, respectively, were investigated in continuous cultures under selective and non-selective conditions. In a three-plasmid system, pRK248cI was lost gradually together with pMTC48 from the host under non-selective conditions. The selective pressure against pRK248cI stabilized the pMTC48. This indicates that the loss of pMTC48 under non-selective conditions was caused by the loss of cI857 gene (coded by pRK248cI) which resulted in the overproduction of the toxic gene product (coded by pMTC48). In the case of single plasmid (pTG206) system, the plasmid lost from the host under non-selective conditions. This plasmid was stabilized in the host growing under selective conditions. During this period we obtained some ampicillin resistant colonies which gave low levels of enzyme activities compared to the normal plasmid bearing cells. Plasmid analysis from the above cells showed that the plasmid has undergone structural instability. Further, restriction analysis of this plasmid exhibited an additional PvuII site in a 0.9 kbp fragment that was integrated near the tet promoter which controls the expression of the xyl E gene, thereby resulting low levels of enzyme activities. Our results indicate that some of the IS elements which are present in the host chromosome were responsible for such instabilities to turn off the synthesis by inserting into the tet promoter region to lower the protein formation during the bioprocess.

Base Sequence↗

PubMed1982

Monitoring of plasmid-encoded, trimethoprim-resistant dihydrofolate reductase genes: detection of a new resistant enzyme.

Using-gene-specific radiolabeled probe DNAs, we analyzed 42 clinical bacterial isolates with high-level trimethoprim (Tp) resistance for the presence of a type I or a type II plasmid-specified dihydrofolate reductase (DHFR) gene. Plasmid DNA from 17 strains harbored a type I DHFR, whereas 11 isolates contained plasmids that harbored a type II DHFR structural gene. The plasmid DNAs from five strains appeared to hybridize with both type I and type II DHFR probe DNAs. In addition, eight isolates had type I resistance determinants integrated into the chromosomes, presumably on transposon 7 (Tn7). Among the strains analyzed in this survey, none of the chromosomally located, Tp-insensitive reductases were of the type II class. Both the plasmid and chromosomal DNAs of one isolate showed no homology with either the type I or type II DHFR probe DNA. The plasmid harbored by this strain encoded a "new" Tp-resistant enzyme that differed significantly, both in molecular weight and with respect to trimethoprim and methotrexate inhibition kinetics, from the previously characterized plasmid-associated dihydrofolate reductases.

Bacteria↗