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PubMed1982

Cloning and functional characterization of the plasmid-encoded hemolysin determinant of Escherichia coli.

We cloned the DNA containing the Escherichia coli hemolysin determinant on a small, high-copy plasmid. We generated plasmids containing fragments of this DNA and used them either alone or in two-plasmid complementation systems to define the limits of the structural genes. This system also allowed us to partially characterize the function of each of the gene products in the production and transport of hemolysin. Taken with previously published data, the present experiments indicate the following. (i) At least three cistrons, hlyC, hlyA, and hlyB (these were previously designated cisC, etc. [Noegel et al., Mol. Gen. Genet. 175:343-350, 1979]), contain the specific genetic information for the hemolytic phenotype, (ii) hlyA encodes a 107,000-kilodalton protein, which seems to be an inactive precursor of hemolysin. (iii) Normal amounts of hemolysin activity inactive precursor of hemolysin. (iii) Normal amounts of hemolysin activity require only the products of hlyA and hlyC. This activity was found in the periplasm; very little hemolysin activity was found in the cytoplasm, suggesting that the hlyC product is required for transport or activation of the hlyA product or both. (iv) Active hemolysin remains in the periplasm in the absence of hlyB function, hence the hlyB product seems to be necessary for the transport of hemolysin to the exterior of the cell. We further show that overproduction of the hlyA product is lethal, probably causing lysis of the cell.

Biological Transport↗
PubMed1994

Identification and characterization of a new plasmid carrying genes for degradation of 2,4-dichlorophenoxyacetate from Pseudomonas cepacia CSV90.

Pseudomonas cepacia CSV90 is able to utilize 2,4-dichlorophenoxyacetate (2,4-D) and 2-methyl-4-chlorophenoxyacetate as sole sources of carbon and energy. Mutants of the strain CSV90 which had lost this ability appeared spontaneously on a nonselective medium. The wild-type strain harbored a 90-kb plasmid, pMAB1, whereas 2,4-D-negative mutants either lost the plasmid or had a 70-kb plasmid, pMAB2. The plasmid pMAB2 was found to have undergone a deletion of a 20-kb fragment of pMAB1. The plasmid-free mutants regained the ability to degrade 2,4-D after introduction of purified pMAB1 by electroporation. Cloning in Escherichia coli of a 10-kb BamHI fragment from pMAB1, the region absent in pMAB2, resulted in the expression of the gene tfdC encoding 3,5-dichlorocatechol 1,2-dioxygenase. After subcloning, the tfdC gene was located in a 1.6-kb HindIII fragment. The nucleotide sequence of the tfdC gene and the restriction map of its contiguous region are identical to those of the well-characterized 2,4-D-degradative plasmid pJP4 of Alcaligenes eutrophus, whereas the overall restriction maps of the two plasmids are different. The N-terminal 44-amino-acid sequence of the enzyme purified from the strain CSV90 confirmed the reading frame in the DNA sequence for tfdC and indicated that the initiation codon GUG is read as methionine instead of valine.

2,4-Dichlorophenoxyacetic Acid↗
PubMed1978

Characterization of a bacteriocinogenic plasmid in Clostridium perfringens CW55.

A bacteriocinogenic strain of Clostridium perfringens was exposed to various curing agents known to accelerate the elimination of extrachromosomal DNA, and 20 independently derived mutants that had lost both the ability to produce bacteriocin and their immunity to it were isolated and characterized. All of the mutants were missing at least two specific plasmid bands seen in the agarose gel plasmid profile of the parent strain. Evidence that the two missing bands represented the open circular and closed circular forms of the same plasmid was obtained by X-ray nicking and restriction endonuclease digestion. The data indicated that bacteriocin production and immunity are controlled by a single plasmid, pCW4, with a molecular weight of 5.6 x 10(6) in this strain. Attempts to transfer the bacteriocinogenic plasmid were unsuccessful.

Bacteriocins↗
PubMed1993

Physical and functional characterization of the ColS8 plasmid.

The restriction map of the 5.1-kb colicinogenic plasmid ColS8 is reported. Transposon-insertion mutagenesis has been carried out to investigate the location of the various functional regions of this plasmid. Twenty-six independent ColS8::Tn1 insertions and six different deletion mutant plasmids were isolated, and the locations of the insertions and deletions were determined. The mapping of the transposon-insertion sites, together with characterization of the phenotypes of these mutants, permitted the localization of the regions of DNA involved in colicin production, colicin S8 immunity and mobilization by other plasmids. There is a polar effect in some mutants in which single insertions result in the non-expression of all three functions. In minicell preparations, the ColS8 plasmid directed the synthesis of colicin S8 (60 kDa) and a 14-kDa immunity protein. One deletion mutant with a colicin-less phenotype can synthesize colicin S8 in minicells, which placed the DNA region involved in colicin release between the colicin production and immunity regions. Both the 60-kDa and 14-kDa proteins are expressed from pColS8 in maxicell preparations.

Colicins↗
PubMed1994

Isolation and characterization of novel plasmid-encoded umuC mutants.

Most inducible mutagenesis in Escherichia coli is dependent upon the activity of the UmuDC proteins. The role of UmuC in this process is poorly understood, possibly because of the limited number of genetically characterized umuC mutants. To better understand the function of the UmuC protein in mutagenic DNA repair, we have isolated several novel plasmid-encoded umuC mutants. A multicopy plasmid that expressed UmuC at physiological levels was constructed and randomly mutagenized in vitro by exposure to hydroxylamine. Mutated plasmids were introduced into the umu tester strain RW126, and 16 plasmids that were unable to promote umuC-dependent spontaneous mutator activity were identified by a colorimetric papillation assay. Interestingly, these plasmid mutants fell into two classes: (i) 5 were expression mutants that produced either too little or too much wild-type UmuC protein, and (ii) 11 were plasmids with structural changes in the UmuC protein. Although hydroxylamine mutagenesis was random, most of the structural mutants identified in the screen were localized to two regions of the UmuC protein; four mutations were found in a stretch of 30 amino acids (residues 133 to 162) in the middle of the protein, while four other mutations (three of which resulted in a truncated UmuC protein) were localized in the last 50 carboxyl-terminal amino acid residues. These new plasmid umuC mutants, together with the previously identified chromosomal umuC25, umuC36, and umuC104 mutations that we have also cloned, should prove extremely useful in dissecting the genetic and biochemical activities of UmuC in mutagenic DNA repair.

Bacterial Proteins↗
PubMed1995

Replication of the broad-host-range plasmid RK2: isolation and characterization of a spontaneous deletion mutant that can replicate in Agrobacterium tumefaciens but not in Escherichia coli.

Two spontaneous deletions of a derivative of the broad-host-range plasmid RK2 were isolated from Agrobacterium tumefaciens. The two deletions have lost 56 and 505 bp, respectively, near the origin of replication (oriV). Of the eight 17-bp repeats present in the RK2 oriV, the smaller deletion has lost the first two while the larger one has lost the first three. The deletions led to a significant increase (3- to 7-fold) in plasmid copy number in A. tumefaciens, indicating their importance in copy number control. While the smaller deletion could replicate in Escherichia coli, the larger one could not. The role of the oriV sequences in the replication of pRK2 in A. tumefaciens and in E. coli is discussed.

Agrobacterium tumefaciens↗
PubMed1974

Studies on plasmid replication. III. Isolation and characterization of replication-defective mutants.

Some 85 Staphylococcus aureus mutants phenotypically thermosensitive for penicillinase plasmid segregation (Seg-) have been isolated and characterized. Some of the mutations were plasmid-linked and those studied in detail were found to be defective in plasmid replication, most problbly at the initiation stage. Analysis of the segregation behavior of these mutants suggested a figure of 2.7 for the average number of plasmid copies per cell in a random culture. Other mutations were host chromosome-linked and these could be divided into at least three classes on the basis of their ability to maintain plasmids of the two different incompatibility sets: some were defective for type I plasmids, some for type II, and some for both types. One host mutant, defective in segregation of type I but not type II plasmids, was defective in polymerization of both.

Chromosomes↗
PubMed1982

Isolation and characterization of Streptomyces fradiae plasmids which are prophage of the actinophage phiSF1.

Two plasmids, designated pUC1 and pUC13, have identified as different prophage states of the Streptomyces fradiae actinophage phiSF1. Plasmids pUC1 and pUC13, 51.1 Md, are identical as determined by restriction endonuclease analysis and other criteria. They differ in that while pUC1 specifies "type A" plaques, characterized by a narrow zone of growth inhibition around a transformant colony, pUC13 specifies "type B" plaques, characterized by concentric rings of growth and growth inhibition around a transformant colony. Additionally cultures carrying pUC13 release antinophage phiSF1 at a frequency about 10 3-10 4 times that pUC1 containing cultures. Restriction enzyme digestion, transfection, and transformation analyses show that phiSF1 DNA, 55.7 Md, is about 8-10% larger than pUC13 and appears to be circularly permuted and terminally redundant. The pUC1 and pUC13 prophage are interconvertible.

Bacteriophages↗
PubMed1997

Sequence analysis and characterization of pOM1, a small cryptic plasmid from Butyrivibrio fibrisolvens, and its use in construction of a new family of cloning vectors for Butyrivibrios.

As a preliminary step in the development of vector systems, we have isolated and begun to characterize small, cryptic plasmids from several strains of the rumen bacterium Butyrivibrio fibrisolvens. We present here the complete nucleotide sequence of Butyrivibrio plasmid pOM1, which was isolated from B. fibrisolvens Bu49. While it is very similar in size to the previously characterized Butyrivibrio plasmids pRJF1 and pRJF2, pOM1 exhibits a restriction pattern which is quite distinct. Analysis of sequence data reveals that pOM1 contains only two open reading frames of significant length (ORF1 and ORF2), both of which are required for self-replication and maintenance. The protein encoded in ORF1 shows homologies with Pre (plasmid recombination enzyme) proteins encoded in plasmids from gram-positive organisms such as Staphylococcus aureus, Streptococcus agalactiae, Lactobacillus plantarum, and Bacillus thuringiensis. The putative translation product of ORF2, on the other hand, resembles Rep (replication) proteins of a different group of gram-positive plasmids, for which the Staphylococcus plasmid pSN2 is a prototype. Unlike the other characterized-Butyrivibrio plasmids, pOM1 appears to replicate via a rolling-circle mechanism. Experimental evidence showing the presence of a single-stranded replication intermediate consistent with this mechanism is presented. pOM1 has been used in the construction of a new Escherichia coli-B. fibrisolvens shuttle vector, pSMerm1, which has been successfully used to introduce a cloned gene into B. fibrisolvens harboring the pRJF1 plasmid.

Amino Acid Sequence↗
PubMed1993

Strain characterization, genome size and plasmid content in the Lactobacillus acidophilus group (Hansen and Mocquot).

Chromosomal DNA of nine strains of the Lactobacillus acidophilus (Hansen and Mocquot) group in which heterogeneity had previously been revealed by biochemical characteristics and DNA-DNA hybridization studies were further investigated by restriction analysis, Southern hybridization, conventional and pulsed-field gel electrophoresis (PFGE) analyses. Industrial strains were characterized and identified as Lact. acidophilus. For this species, the number of rRNA gene clusters was estimated to be at least four from analyses of rRNA gene restriction patterns. The chromosome size of type-strains of Lact. acidophilus and Lact. gasseri was estimated from PFGE analysis to be 1.85 and 2.02 Mb respectively and Lact. gasseri strains were found to contain large-sized linear plasmids.

Bacterial Typing Techniques↗
PubMed1997

Molecular characterization of two large DNA plasmids in the red alga Porphyra pulchra.

Five plasmids occur in the red alga Porphyra pulchra. The two larger ones (6859 and 6427 bp) differ in their sequences. The three smaller (1896, 2100, and 2102 bp) have sequences that are similar to one another. These plasmids are circular, double-stranded DNA, present in high copy number, and maintained in this organism through successive generations of laboratory culture. Sequence analysis of the two larger plasmids reveals few prominent structural features, but several potential open reading frames (ORFs) occur, some of which are transcriptionally active. Sequence database comparisons find significant sequence similarity between ORF3 or PP6427 and a 411 amino-acid polypeptide previously characterized in plasmid GC2 from the red alga Gracilaria chilensis. These data support the presence of at least one conserved plasmid coding region in distant orders of red algae. Southern blots of total genomic DNA from other red algae probed with plasmids from P. pulchra demonstrate no hybridization to previously studied Gracilariales species but notable hybridization to several species within the genus Porphyra, although the sizes of detected bands vary.

Amino Acid Sequence↗
PubMed1984

Plasmids in methanotrophic bacteria: isolation, characterization and DNA hybridization analysis.

Ten strains of obligate methanotrophs were screened for the presence of plasmid DNA using a variety of methods. Plasmids were detected in all strains except Methylococcus capsulatus Bath. No significant similarity between plasmids was observed with respect to size or restriction digest patterns except for three strains of Methylosinus trichosporium, which appeared to contain the same three plasmids. Nitrocellulose filter hybridization revealed that the plasmid DNA from the M. trichosporium strains shared a small region of homology with the plasmid DNA from Methylosinus sporium 5. All of the plasmids remain cryptic. As the first step in characterization, a restriction digest map of the 55 kb plasmid found in Methylomonas albus BG8 was constructed.

Collodion↗
PubMed2000

Phenotypic and molecular characterization of conjugative antibiotic resistance plasmids isolated from bacterial communities of activated sludge.

In order to isolate antibiotic resistance plasmids from bacterial communities found in activated sludge, derivatives of the 3-chlorobenzoate-degrading strain Pseudomonas sp. B13, tagged with the green fluorescent protein as an identification marker, were used as recipients in filter crosses. Transconjugants were selected on agar plates containing 3-chlorobenzoate as the sole carbon source and the antibiotic tetracycline, streptomycin or spectinomycin, and were recovered at frequencies in the range of 10(-5) to 10(-8) per recipient. A total of 12 distinct plasmids, designated pB1-pB12, was identified. Their sizes ranged between 41 to 69 kb and they conferred various patterns of antibiotic resistance on their hosts. Two of the plasmids, pB10 and pB11, also mediated resistance to inorganic mercury. Seven of the 12 plasmids were identified as broad-host-range plasmids, displaying extremely high transfer frequencies in filter crosses, ranging from 10(-1) to 10(-2) per recipient cell. Ten of the 12 plasmids belonged to the IncP incompatibility group, based on replicon typing using IncP group-specific PCR primers. DNA sequencing of PCR amplification products further revealed that eight of the 12 plasmids belonged to the IncPbeta subgroup, whereas two plasmids were identified as IncPalpha plasmids. Analysis of the IncP-specific PCR products revealed considerable differences among the IncPbeta plasmids at the DNA sequence level. In order to characterize the gene "load" of the IncP plasmids, restriction fragments were cloned and their DNA sequences established. A remarkable diversity of putative proteins encoded by these fragments was identified. Besides transposases and proteins involved in antibiotic resistance, two putative DNA invertases belonging to the Din family, a methyltransferase of a type I restriction/modification system, a superoxide dismutase, parts of a putative efflux system belonging to the RND family, and proteins of unknown function were identified.

DNA Restriction Enzymes↗
PubMedDate not supplied

Characterization of two minicircular plasmid-like DNAs isolated from date-palm mitochondria.

We report here the identification and characterization of two minicircular plasmid-like DNAs isolated from mitochondria of a moroccan date-palm variety. Both molecules were cloned and used as probes in Southern analyses of mitochondrial and total-cellular DNA. Evidence was obtained that these plasmid-like DNAs cross-hybridized but did not show any homology to nuclear, chloroplastic, or main mitochondrial genomes. Sequence analysis revealed that both minicircles, 1,346- and 1,160-bp long, share several stretches of homology, the most important consisting of three identical clusters of lengths 42, 47 and 38 bp. In contrast, no major homology was observed with the other higher-plant plasmid-like DNAs reported so far. Sequence analysis also revealed the presence, in the same strand of one of the minicircles, of two open reading frames potentially encoding proteins 89 and 86 amino acids in length. Interestingly, Northern analyses, using single strands of each minicircle as probes, showed the presence of two transcripts hybridizing only with the strand bearing these two open reading frames. However, computer-assisted comparison of the predicted polypeptide sequences with a protein-sequence library failed to detect any significant homology to known sequences.

Base Sequence↗
PubMed1994

Transfer in Marine Sediments of the Naturally Occurring Plasmid pRAS1 Encoding Multiple Antibiotic Resistance.

The results of microcosm experiments performed with the fish-pathogenic bacterium Aeromonas salmonicida acting as a donor showed that promiscuous plasmid pRAS1, which encodes tetracycline resistance, is transferred at a high frequency in marine sediments even in the absence of a selective factor. The presence of oxytetracycline resulted in an increase in the transfer frequency compared with that of a microcosm to which no selective factor was added. Transfer frequencies of 3.4 x 10 transconjugant per recipient and 3.6 transconjugants per donor cell were obtained in a microcosm to which oxytetracycline had been added. Hybridization with a DNA probe specific for plasmid pRAS1 revealed that 45.8% of the oxytetracycline-resistant isolates obtained from a microcosm with no selective pressure carried the plasmid, while 86.8% of the isolates obtained from a microcosm to which oxytetracycline had been added carried the plasmid. Phenotypic characterization of the transconjugants revealed that the plasmid had been transferred to a variety of different biotypes in both microcosms. The diversity among the transconjugants isolated from the microcosm to which oxytetracycline had been added was substantially lower than the diversity among the transconjugants isolated from the microcosm to which no selective agent had been added.

Journal Article↗
PubMed2004

Novel antigen identification method for discovery of protective malaria antigens by rapid testing of DNA vaccines encoding exons from the parasite genome.

We describe a novel approach for identifying target antigens for preerythrocytic malaria vaccines. Our strategy is to rapidly test hundreds of DNA vaccines encoding exons from the Plasmodium yoelii yoelii genomic sequence. In this antigen identification method, we measure reduction in parasite burden in the liver after sporozoite challenge in mice. Orthologs of protective P. y. yoelii genes can then be identified in the genomic databases of Plasmodium falciparum and Plasmodium vivax and investigated as candidate antigens for a human vaccine. A pilot study to develop the antigen identification method approach used 192 P. y. yoelii exons from genes expressed during the sporozoite stage of the life cycle. A total of 182 (94%) exons were successfully cloned into a DNA immunization vector with the Gateway cloning technology. To assess immunization strategies, mice were vaccinated with 19 of the new DNA plasmids in addition to the well-characterized protective plasmid encoding P. y. yoelii circumsporozoite protein. Single plasmid immunization by gene gun identified a novel vaccine target antigen which decreased liver parasite burden by 95% and which has orthologs in P. vivax and P. knowlesi but not P. falciparum. Intramuscular injection of DNA plasmids produced a different pattern of protective responses from those seen with gene gun immunization. Intramuscular immunization with plasmid pools could reduce liver parasite burden in mice despite the fact that none of the plasmids was protective when given individually. We conclude that high-throughput cloning of exons into DNA vaccines and their screening is feasible and can rapidly identify new malaria vaccine candidate antigens.

Animals↗
PubMed1984

Transformation of Neurospora crassa with recombinant plasmids containing the cloned glutamate dehydrogenase (am) gene: evidence for autonomous replication of the transforming plasmid.

We have characterized Neurospora crassa transformants obtained with plasmid pJR2, which consists of the Neurospora glutamate dehydrogenase (am) gene cloned in pUC8 and an am132 host strain which contains a deletion encompassing the cloned fragment. Every one of 33 transformants tested showed extreme meiotic instability: less than 1 or 2% am+ progeny were obtained in initial or successive backcrosses between am+ transformants and am132 or in intercrosses between am+ progeny. Furthermore, am+ progeny from backcrosses gave a high proportion of auxotrophic (am) mitotic segregants during vegetative growth. These results indicate that the am+ character is not stably integrated into chromosomal DNA in any of the transformants tested. Nuclear DNAs from six transformants were analyzed by Southern hybridization. All six transformants contained sequences homologous to pJR2. Four showed restriction fragments expected for unmodified pJR2, but most showed additional bands. Southern blots of undigested DNAs showed that the plasmid sequences are present predominantly in high-molecular-weight form (larger than 20 kilobases). Southern blots showed that auxotrophic (am) progeny from a backcross to am132 had lost restriction bands corresponding to free plasmid but retained additional bands, apparently integrated into chromosomal DNA in a nonfunctional manner. Considered together, these results are most reasonably interpreted as follows: recombinant plasmids containing the am+ gene can replicate autonomously in N. crassa, the free plasmids are present in oligomeric or modified form or both, and plasmid sequences also integrate at multiple sites in the deletion host but in a nonfunctional manner. An alternate interpretation--that tandem repeats of the plasmid are integrated into chromosomal DNA but eliminated during meiosis--cannot be completely excluded. However, stable integration of the am gene can be obtained under a variety of other conditions, viz., using the am gene cloned in a phage lambda vector (J. A. Kinsey and J. A. Rambosek, Mol. Cell. Biol. 4:117-122, 1984), using derivatives of pJR2, or using pJR2 to transform a frameshift mutant.

DNA Replication↗