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At least 631 records · Page 35Linked to original sources
PubMed1991

Characterization of trbC, a new F plasmid tra operon gene that is essential to conjugative transfer.

We have characterized a previously unidentified gene, trbC, which is contained in the transfer region of the Escherichia coli K-12 fertility factor, F. Our data show that the trbC gene is located between the F plasmid genes traU and traN. The product of trbC was identified as a polypeptide with an apparent molecular weight (Ma) of 23,500 that is processed to an Ma-21,500 mature protein. When ethanol was present, the Ma-23,500 polypeptide accumulated; the removal of ethanol resulted in the appearance of the processed mature protein. Subcellular fractionation experiments demonstrated that the processed, Ma-21,500 mature protein was located in the periplasm. DNA sequence analysis showed that trbC encodes a 212-amino-acid Mr-23,432 polypeptide that could be processed to a 191-amino-acid Mr-21,225 mature protein through the removal of a typical amino-terminal signal sequence. We also constructed two different Kmr gene insertion mutations in trbC and crossed these onto the transmissible F plasmid derivative pOX38. We found that cells carrying pOX38 trbC mutant plasmids were transfer deficient and resistant to infection by F-pilus-specific phages. Transfer proficiency and bacteriophage sensitivity were restored by complementation when a trbC+ plasmid clone was introduced into these cells. These results showed that trbC function is essential to the F plasmid conjugative transfer system and suggested that the TrbC protein participates in F-pilus assembly.

Amino Acid Sequence↗
PubMed2006

Molecular characterization of Bifidobacterium longum biovar longum NAL8 plasmids and construction of a novel replicon screening system.

In this study, we performed molecular characterization and sequence analysis of three plasmids from the human intestinal isolate Bifidobacterium longum biovar longum NAL8 and developed a novel vector screening system. Plasmids pNAL8H (10 kb) and pNAL8M (4.9 kb) show close sequence similarity to and the same gene organization as the already characterized B. longum plasmids. The B. longum plasmid pNAC1 was identified as being most closely related to pNAL8L (3.5 kb). However, DNA sequence analysis suggested that direct repeat-rich sites could have promoted several recombination events to diversify the two plasmid molecules. We verified the likely rolling circle replication of plasmid pNAL8L and studied the phylogenetic relationship in all the Bifidobacterium plasmids fully sequenced to date based on in silico comparative sequence analysis of their replication proteins and iteron regions. Our transformation experiments confirmed that the ColE1 replication origin from high-copy-number pUC vectors could interfere with the replication apparatus of Bifidobacterium plasmids and give rise to false positive clones. As a result, we developed a system suitable for avoiding possible interference by other functional replication modules on the vector and for screening functional replicons from wild-type plasmids.

Bifidobacterium↗
PubMed1997

Characterization of a Pasteurella multocida plasmid and its use to express recombinant proteins in P. multocida.

The complete nucleotide sequence of a naturally occurring 5.36-kb streptomycin and sulphonamide resistance plasmid, designated pIG1, isolated from type D Pasteurella multocida was determined. A 1.6-kb noncoding region and a 1.4-kb region encoding three putative proteins were shown by sequence homologies and functional characterizations to be involved in the replication and mobilization of pIG1, respectively. The remaining sequence carried an unusual arrangement of streptomycin- and sulphonamide-resistant genes when compared to various other plasmids. It appears that the antibiotic resistance region of pIG1 may have evolved by recombination between three different short direct repeat DNA sequences. A 4.5-kb recombinant plasmid was constructed by replacing the antibiotic resistance genes of pIG1 with a kanamycin resistance gene and seven unique restriction sites. The resulting plasmid, designated pIG112, stably replicates in P. multocida, Pasteurella haemolytica, Actinobacillus pleuropneumoniae, and Escherichia coli and can be introduced into these organisms by either transformation or conjugation. This vector exists at approximately 70 copies per cell in P. multocida and approximately 20 copies per cell in E. coli. To demonstrate plasmid-borne gene expression in P. multocida, the P. multocida dermonecrotic toxin gene, toxA, and a genetically modified form of this gene were cloned into pIG112 and expressed in high amounts in a nontoxigenic P. multocida strain. Cell culture assays demonstrated that nontoxigenic P. multocida expressing toxA was cytopathic, whereas a strain expressing the modified toxA derivative was not.

Animals↗
PubMed1995

Identification of virulence-associated antigens and plasmids in Rhodococcus equi from patients with AIDS.

Rhodococcus equi is an emerging opportunistic pathogen of human immunodeficiency virus-infected patients. However, little is known about the characteristics of R. equi isolates from humans. This study characterized the plasmid content, expression of a virulence-associated antigen, and mouse virulence of 19 R. equi isolates from patients with and without AIDS. EcoRI digestion patterns and Southern, Western, and virulence analyses of these isolates with cryptic plasmids allowed definition of a new category of R. equi. Isolates from patients with AIDS tended either to be virulent and have 15- to 17-kDa antigens and an 85-kb plasmid (10(6) bacteria needed for lethality) or have intermediate virulence (10(7) bacteria needed for lethality) and one of four distinct large plasmids that share DNA homology and express a 20-kDa antigen. Most of the non-AIDS isolates were avirulent (> 10(8) bacteria needed for lethality) and did not express any of these antigens.

AIDS-Related Opportunistic Infections↗
PubMed2002

Analysis of the genome of the moderate halophile Halomonas eurihalina.

We have studied the genomic organization of Halomonas eurihalina, a moderately halophilic bacterium that produces an anionic exopolysaccharide with a potentially wide range of applications in industry. To estimate the genome size of strain F2-7, large restriction fragments of genomic DNA were separated by pulsed-field gel electrophoresis. According to the average size of the resolved restriction fragments, the genome size of H. eurihalina strain F2-7 was estimated to be around 2500 kb. The physical map of the chromosome for the endonuclease SwaI has been constructed. The F2-7 strain has two plasmids, pVE1 and pVE2, and in this study we have isolated three new plasmids, pVE3, pVE4, and pVE5, of 5.3, 16, and 6.5 kb, respectively, from strains H-1, H-217, and H-236 of H. eurihalina. We have characterized these plasmids and constructed their physical maps. Curing experiments produced no evidence for the involvement of these plasmids in the expression of the mucous phenotype.

Blotting, Southern↗
PubMed2006

Plasmids in the aphid endosymbiont Buchnera aphidicola with the smallest genomes. A puzzling evolutionary story.

Buchnera aphidicola, the primary endosymbiont of aphids, has undergone important genomic and biochemical changes as an adaptation to intracellular life. The most important structural changes include a drastic genome reduction and the amplification of genes encoding key enzymes for the biosynthesis of amino acids by their translocation to plasmids. Molecular characterization through different aphid subfamilies has revealed that the genes involved in leucine and tryptophan biosynthesis show a variable fate, since they can be located on plasmids or on the chromosome in different lineages. This versatility contrasts with the genomic stasis found in three distantly related B. aphidicola strains already sequenced. We present the analysis of three B. aphidicola strains (BTg, BCt and BCc) belonging to aphids from different tribes of the subfamily Lachninae, that was estimated to harbour the bacteria with the smallest genomes. The presence of both leucine and tryptophan plasmids in BTg, a chimerical leucine-tryptophan plasmid in BCt, and only a leucine plasmid in BCc, indicates the existence of many recombination events in a recA minus bacterium. In addition, these B. aphidicola plasmids are the simplest described in this species, indicating that plasmids are also involved in the genome shrinkage process.

Amino Acids↗
PubMed2003

DNA vaccination with plasmids containing various fragments of large segment genome of infectious bursal disease virus.

The present study was undertaken to determine the effectiveness in including VP2 gene of the large segment genome of infectious bursal disease virus (IBDV) in the DNA vaccine for protection of chickens against infectious bursal disease (IBD). Different fragments of the large segment gene of IBDV standard challenge (STC) strain were successfully amplified by reverse transcription-polymerase chain reaction (RT-PCR) followed by cloning into a eukaryotic expression plasmid vector (pCR3.1) as DNA vaccines. Transient expression of the encoded genes from various constructed plasmids was characterized in COS-7 cells by positive immunofluorescent staining with polyclonal or monoclonal antibody to IBDV. Chickens (1-day old) were intramuscularly injected with the individual plasmid three times at weekly intervals, challenged with IBDV strain STC at 21-day old, and observed for 10 days. Chickens receiving the plasmids containing VP2 genes, including VP243, VP24, or VP2 fragment, did not have clinical signs, mortality, and bursal atrophy and were effectively protected against IBDV infection. On the contrary, chickens receiving plasmids without containing VP2 genes, including VP4, VP3, or VP43 fragment, had marked bursal atrophy and were not protected against IBD. Antigen detection was correlated with protection; chickens protected from IBDV infection had undetectable IBDV antigen in bursae. Enzyme-linked immunosorbent assay (ELISA) antibody titer to IBDV was low or undetectable prior to or after challenge with IBDV in protected chickens receiving the plasmids containing the VP2 gene. The results indicate that inclusion of VP2 gene in the plasmid DNA is essential in achieving effective protection mediated by DNA vaccination against IBDV infection in chickens.

Animals↗
PubMed1998

A cryptic 19-kilobase plasmid associated with U.S. isolates of Yersinia pestis: a dimer of the 9.5-kilobase plasmid.

Yersinia pestis, the etiologic agent of plague, carries three prototypic plasmids with sizes of 110 kb (pFra, pTox), 70 kb (pLcr, pVW, pCad), and 9.5 kb (pPla, pPst). Studies suggest that geographic isolates of Y. pestis may be differentiated by plasmid profiles. Yersinia pestis isolated from the western United States harbor an additional plasmid, estimated to be approximately 19 kb in size. This cryptic plasmid was characterized by restriction endonuclease digestion, amplification and sequencing of the plasminogen activator gene segment, Southern blotting, and visualized by electron microscopy. Results revealed that this cryptic plasmid is a supercoiled DNA plasmid, 18.85+/-0.59 (mean+/-SD) kb in length, and is a dimer of the 9.5-kb plasmid. The genetic reason for the appearance of this form of the 9.5-kb plasmid in Y. pestis from Arizona, California, Colorado, New Mexico, and Texas is under study.

Animals↗
PubMed2001

Nucleotide sequence and characterization of the cell envelope proteinase plasmid in Lactococcus lactis subsp. cremoris HP.

AIMS: The major cell envelope proteinase (lactocepin; EC 3.4.21.96) produced by Lactococcus lactis cheese starter bacteria is required for starter growth and acid production in milk. The aim of this study was to characterize a lactocepin plasmid from a L. lactis subsp. cremoris cheese starter strain. METHODS AND RESULTS: A restriction map of the lactocepin plasmid pHP003 from strain HP was constructed, fragments were cloned in Escherichia coli vectors, and the complete DNA sequence (13,433 bp) was determined. Among 120 industrial L. lactis starter strains screened, five contained the same specificity-type lactocepin as pHP003. The lactocepin gene in these strains was invariably linked with a partially-deleted abiB gene. CONCLUSION: The lactocepin specificity type of strain HP, conferred by a known configuration of key residues, is relatively uncommon. The gene is invariably linked with a partially deleted abiB gene on each lactocepin plasmid. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first complete sequence reported for a lactocepin plasmid, and provides the basis for better understanding, or manipulation, of lactocepin production.

Animals↗
PubMed2001

A novel rolling-circle-replicating plasmid from Pseudomonas putida P8: molecular characterization and use as vector.

In Pseudomonas putida P8, three cryptic circular plasmids were detected, i.e. pPP8-1 (2.5 kbp), pPP8-2 (42 kbp) and pPP8-3 (approximately 100 kbp). Cloning and complete sequencing of pPP8-1 revealed a 2534 bp element harbouring four open reading frames (ORFs A, B, C and D). No function could be attributed to the latter three ORFs, whereas the predicted ORF A gene product is homologous to replication proteins known from small multicopy plasmids of Gram-positive bacteria and single-stranded (ss) phages, genetic elements replicating via a rolling circle (RC) mechanism involving characteristic ssDNA intermediates. Consistently, a double-strand origin of replication, highly conserved in rolling-circle-replicating (RCR) elements, was identified in pPP8-1, along with a putative single-strand origin. Beyond this, ss replication intermediates were confirmed by Southern analysis and mungbean-nuclease digestion. This being the first element of this type known in pseudomonads, a kanamycin-resistance gene was ligated into pPP8-1 and the resulting vector was successfully used for the transformation of both Escherichia coli and P. putida.

Amino Acid Sequence↗
PubMed1992

Organization and functional analysis of three T-DNAs from the vitopine Ti plasmid pTiS4.

The vitopine Ti plasmid pTiS4 of Agrobacterium vitis has an unusual T-DNA organization. The pTiS4 oncogenes, localized by screening selected pTiS4 clones for growth-inducing activity, are localized on three T-DNAs, whereas in all other characterized Ti plasmids one or two T-DNAs are found. The nucleotide sequences and predicted amino acid sequences of the pTiS4 oncogenes set them apart from the corresponding genes from other Ti or Ri plasmids. The oncogenes induce the same type of reaction on various test plants as the well-known pTiAch5 oncogenes but the pTiS4 ipt gene induces considerably more shoots than its Ach5 homologue. We have also identified the gene coding for vitopine synthase as well as a vitopine synthase pseudogene. Both sequences show homology to the octopine synthase gene. In terms of both nucleotide sequence and overall organization, the pTiS4 T-DNAs appear to be only distantly related to previously characterized T-DNAs.

Amino Acid Sequence↗
PubMed2005

Genetic organization of a plasmid from an industrial wastewater bioreactor.

Pseudomonas strain CT14 was isolated from activated sludge. Strain CT14 contained a 55, 216 bp plasmid that was characterized by sequence analysis. The plasmid had a modular structure with 51 open reading frames (ORFs) that were distributed between two clearly demarcated domains. Domain I primarily contained genes for plasmid-related functions and a novel origin of replication. Domain II bore evidence of extensive transposition and recombination. Domain II contained several genes from a meta-cleavage pathway for aromatic rings. These genes appeared to have been recruited from different hosts. This observation suggests that sequencing pCT14 may have revealed an intermediate stage in the evolution of a new assemblage of meta-cleavage pathway genes.

Base Sequence↗
PubMed1989

Evidence for unlinked rrn operons in the Planctomycete Pirellula marina.

Southern hybridization of rRNAs to chromosomal BamHI-digested DNA of the eubacterium Pirellula marina revealed the presence of two sets of 16S and 23S rRNA genes. The two copies of the 23S rRNA genes, located on 11- and about 13-kilobase (kb) inserts, were isolated from a lambda bacteriophage Charon 35 library. The 11-kb fragment was cloned directly into pBR322, while a 5.4-kb BamHI-PstI rDNA subfragment of the approximately 13-kb insert was cloned into pUC18. Both recombinant plasmids, pPI1100 and pPI540, were characterized by restriction enzyme mapping and Southern hybridization with the large rRNA species. Restriction fragments from both inserts were subcloned into phage M13 mp18 and mp19. Correlation of genomic hybridization data with physical characterization of recombinant plasmids showed that, in contrast to the general organization of rrn operons in eubacteria, the 16S rRNA genes of P. marina are separated by at least 8.5 (pPI540) and 4.4 (pPI1100) kb, respectively, from the closely linked 23S-5S rRNA genes. Comparison of the flanking regions from both 23S-5S rRNA genes with published consensus sequences of structural elements indicates the presence of putative transcription signals, i.e., a single Pribnow box, discriminator, antitermination boxes A, B, and C, and a Rho-independent terminator.

Base Sequence↗
PubMed1991

Characterization and comparative sequence analysis of replication origins from three large Bacillus thuringiensis plasmids.

The replication origins of three large Bacillus thuringiensis plasmids, derived from B. thuringiensis HD263 subsp. kurstaki, have been cloned in Escherichia coli and sequenced. The replication origins, designated ori 43, ori 44, and ori 60, were isolated from plasmids of 43, 44, and 60 MDa, respectively. Each cloned replication origin exhibits incompatibility with the resident B. thuringiensis plasmid from which it was derived. Recombinant plasmids containing the three replication origins varied in their ability to transform strains of B. thuringiensis, Bacillus megaterium, and Bacillus subtilis. Analysis of the derived nucleotide and amino acid sequences indicates that the replication origins are nonhomologous, implying independent derivations. No significant homology was found to published sequences of replication origins derived from the single-stranded DNA plasmids of gram-positive bacteria, and shuttle vectors containing the three replication origins do not appear to generate single-stranded DNA intermediates in B. thuringiensis. The replication origin regions of the large plasmids are each characterized by a single open reading frame whose product is essential for replication in B. thuringiensis. The putative replication protein of ori 60 exhibits partial homology to the RepA protein of the Bacillus stearothermophilus plasmid pTB19. The putative replication protein of ori 43 exhibits weak but extensive homology to the replication proteins of several streptococcal plasmids, including the open reading frame E replication protein of the conjugative plasmid pAM beta 1. The nucleotide sequence of ori 44 and the amino acid sequence of its putative replication protein appear to be nonhomologous to other published replication origin sequences.

Amino Acid Sequence↗
PubMed1990

IncN plasmid replicon. A deletion and subcloning analysis.

A DNA segment of approximately 2000 base-pairs bounded by restriction enzyme sites for PvuII and containing the minimal replicon of an N group plasmid was characterized. A natural derivative of this miniplasmid was found to have undergone a deletion within one of two tandem iteron families, the group I iterons. Further analysis showed that all plasmid-determined functions essential for stable maintenance in Escherichia coli were localized to a contiguous region of DNA of 1019 nucleotides that excludes entirely these iterons. However, the loss of these iterons led to an increase in plasmid copy number. This indicates that members of the group I iteron-family have a role in determining plasmid copy number perhaps by titrating a plasmid-specified trans-acting product. The 2000 base-pair segment contains six open reading frames of 40 or more amino acid residues. The essential segment contains a 368 nucleotide region that must be present in cis and within which there are three "GATC" sequences and a putative Escherichia coli DnaA protein-binding sequence (dnaA box). An interesting feature is that the cis-acting region is present entirely within a presumptive rep gene. The essential segment contains four open reading frames, only one of which has an Escherichia coli canonical ribosome-binding site. The 2000 base-pair miniplasmid has two separable regions determining N group plasmid incompatibility.

Bacterial Proteins↗
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↗
PubMed1994

Mitochondrial minicircle DNA supports plasmid replication and maintenance in nuclei of Trypanosoma brucei.

In a search for trypanosome DNA sequences that permit replication and stable maintenance of extrachromosomal elements, a 1-kilobase-pair (kbp) fragment from a mitochondrial kinetoplast DNA (kDNA) minicircle of Trypanosoma brucei was isolated and characterized. The plasmid pTbo-1, carrying the kDNA element, is maintained in T. brucei as a supercoiled concatemer containing approximately seven to nine pTbo-1 monomer units (5.6 kbp each) in a head-to-tail orientation. The concatemer is found in approximately one copy per cell when procyclic trypanosomes are cultured in the presence of 100 micrograms of hygromycin per ml; however, in the absence of continuous hygromycin selection, the plasmid is lost from the population with a t1/2 of approximately 8.7 days (17 cell generations). A second unrelated kDNA minicircle was also able to serve as an autonomously replicating sequence (ARS) element in T. brucei, suggesting that this is a general property of kDNA minicircles. Replication of mitochondrial DNA in the nucleus may be due to either a specific consensus sequence (such as in yeast ARS elements) or nonspecific sequence characteristics (such as the degree of A&T-richness or bent DNA).

Animals↗