Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Characterization of a small plasmid from Desulfovibrio desulfuricans and its use for shuttle vector construction.

ABSTRACT: A 2.3-kb plasmid present in about 20 copies per genome was identified in extracts of Desulfovibrio desulfuricans G100A and designated pBG1. It appears to be unable to replicate in Escherichia coli. Although composite plasmids of pBG1 inserted into pTZ18U are stable in E. coli, few if any pBG1-specific transcripts are detectable. The plasmid sequence reveals several features typical of the origin of replication of non-ColE1 enterobacterial plasmids as well as several potential open reading frames. This small replicon has been shown to support the replication of recombinant plasmids in D. desulfuricans G100A and Desulfovibrio fructosovorans. A conjugable shuttle vector has been constructed.

SUBMITTER: Wall JD

PROVIDER: S-EPMC204841 | biostudies-other | 1993 Jul

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Publications

Characterization of a small plasmid from Desulfovibrio desulfuricans and its use for shuttle vector construction.

Wall J D JD Rapp-Giles B J BJ Rousset M M

Journal of bacteriology 19930701 13

A 2.3-kb plasmid present in about 20 copies per genome was identified in extracts of Desulfovibrio desulfuricans G100A and designated pBG1. It appears to be unable to replicate in Escherichia coli. Although composite plasmids of pBG1 inserted into pTZ18U are stable in E. coli, few if any pBG1-specific transcripts are detectable. The plasmid sequence reveals several features typical of the origin of replication of non-ColE1 enterobacterial plasmids as well as several potential open reading frames ...[more]

PMID: 8320227

Similar Datasets

Construction and characterization of a highly efficient Francisella shuttle plasmid.

Project description:Francisella tularensis is a facultative intracellular pathogen that infects a wide variety of mammals and causes tularemia in humans. It is recognized as a potential agent of bioterrorism due to its low infectious dose and multiple routes of transmission. To date, genetic manipulation in Francisella spp. has been limited due to the inefficiency of DNA transformation, the relative lack of useful selective markers, and the lack of stably replicating plasmids. Therefore, the goal of this study was to develop an enhanced shuttle plasmid that could be utilized for a variety of genetic procedures in both Francisella and Escherichia coli. A hybrid plasmid, pFNLTP1, was isolated that was transformed by electroporation at frequencies of >1 x 10(7) CFU mug of DNA(-1) in F. tularensis LVS, Francisella novicida U112, and E. coli DH5alpha. Furthermore, this plasmid was stably maintained in F. tularensis LVS after passage in the absence of antibiotic selection in vitro and after 3 days of growth in J774A.1 macrophages. Importantly, F. tularensis LVS derivatives carrying pFNLTP1 were unaltered in their growth characteristics in laboratory medium and macrophages compared to wild-type LVS. We also constructed derivatives of pFNLTP1 containing expanded multiple cloning sites or temperature-sensitive mutations that failed to allow plasmid replication in F. tularensis LVS at the nonpermissive temperature. In addition, the utility of pFNLTP1 as a vehicle for gene expression, as well as complementation, was demonstrated. In summary, we describe construction of a Francisella shuttle plasmid that is transformed at high efficiency, is stably maintained, and does not alter the growth of Francisella in macrophages. This new tool should significantly enhance genetic manipulation and characterization of F. tularensis and other Francisella biotypes.

| S-EPMC535190 | biostudies-literature

Characterization of plasmid pOR1 from Ornithobacterium rhinotracheale and construction of a shuttle plasmid.

Project description:The bacterium Ornithobacterium rhinotracheale has been recognized as an emerging pathogen in poultry since about 10 years ago. Knowledge of this bacterium and its mechanisms of virulence is still very limited. Here we report the development of a transformation system that enables genetic modification of O. rhinotracheale. The system is based on a cryptic plasmid, pOR1, that was derived from an O. rhinotracheale strain of serotype K. Sequencing indicated that the plasmid consisted of 14,787 nucleotides. Sequence analysis revealed one replication origin and several rep genes that control plasmid replication and copy number, respectively. In addition, pOR1 contains genes with similarity to a heavy-metal-transporting ATPase, a TonB-linked siderophore receptor, and a laccase. Reverse transcription-PCR demonstrated that these genes were transcribed. Other putative open reading frames exhibited similarities with a virulence-associated protein in Actinobacillus actinomycetemcomitans and a number of genes coding for proteins with unknown function. An Escherichia coli-O. rhinotracheale shuttle plasmid (pOREC1) was constructed by cloning the replication origin and rep genes from pOR1 and the cfxA gene from Bacteroides vulgatus, which codes for resistance to the antibiotic cefoxitin, into plasmid pGEM7 by using E. coli as a host. pOREC1 was electroporated into O. rhinotracheale and yielded cefoxitin-resistant transformants. The pOREC1 isolated from these transformants was reintroduced into E. coli, demonstrating that pOREC1 acts as an independent replicon in both E. coli and O. rhinotracheale, fulfilling the criteria for a shuttle plasmid that can be used for transformation, targeted mutagenesis, and the construction of defined attenuated vaccine strains.

| S-EPMC522087 | biostudies-literature

Construction of a Shuttle Vector Using an Endogenous Plasmid From the Cyanobacterium Synechocystis sp. PCC6803.

Project description:To advance synthetic biology in the photosynthetic cyanobacterium Synechocystis sp. PCC6803 (Syn6803), we constructed a shuttle vector with some versatile features. This shuttle vector, pSCB-YFP, consists of a putative replicon identified on the plasmid pCC5.2, the origin of replication of pMB1 from E. coli, as well as the YFP reporter gene and a spectinomycin/streptomycin resistance cassette. pSCB-YFP is stably maintained in Syn6803M (a motile strain that lacks the endogenous pCC5.2) and expresses YFP. In addition, we engineered a fragment into pSCB-YFP that has multiple cloning sites and other features such that this plasmid can also be used as an expression vector (pSCBe). The shuttle vector pSCB-YFP can be stably maintained for at least 50 generations without antibiotic selection. It is a high copy number plasmid and can stably co-exist with the RSF1010-based pPMQAK1-GFP.

| S-EPMC6066503 | biostudies-literature

Characterization of a small cryptic plasmid from endophytic Pantoea agglomerans and its use in the construction of an expression vector.

Project description:A circular cryptic plasmid named pPAGA (2,734 bp) was isolated from Pantoea agglomerans strain EGE6 (an endophytic bacterial isolate from eucalyptus). Sequence analysis revealed that the plasmid has a G+C content of 51% and contains four potential ORFs, 238(A), 250(B), 131(C), and 129(D) amino acids in length without homology to known proteins. The shuttle vector pLGM1 was constructed by combining the pPAGA plasmid with pGFPmut3.0 (which harbors a gene encoding green fluorescent protein, GFP), and the resulting construct was used to over-express GFP in E. coli and P. agglomerans cells. GFP production was used to monitor the colonization of strain EGE6gfp in various plant tissues by fluorescence microscopy. Analysis of EGE6gfp colonization showed that 14 days after inoculation, the strain occupied the inner tissue of Eucalyptus grandis roots, preferentially colonizing the xylem vessels of the host plants.

| S-EPMC3085353 | biostudies-literature

Properties and construction of plasmid pFW213, a shuttle vector with the oral Streptococcus origin of replication.

Project description:Streptococcus parasanguinis is among the most successful colonizers of the human body. Strain FW213 harbors a 7.0-kb cryptic plasmid, pFW213, with a copy number at 5 to 10 per chromosome. Sequence and functional analyses of pFW213 revealed that the open reading frame (ORF) encoding the replication protein (Rep) is essential for the replication of pFW213, and the putative plasmid addiction system (RelB and RelE) and an ORF (ORF6) with no known function are required for its stability. The minimal replicon of pFW213 contains the rep gene and its 5'-flanking 390-bp region. Within the minimal replicon, an A/T-rich region followed by 5 contiguous 22-bp repeats was located 5' of the ATG of rep. No single-stranded replication intermediates were detected in the derivatives of pFW213, suggesting that pFW213 replicates via the theta replication mechanism. The minimal replicon was unstable in streptococcal hosts without selection, but the stability was greatly enhanced in derivatives containing the intact relBE genes. A Streptococcus-Escherichia coli shuttle vector, pCG1, was constructed with the pFW213 replicon. Plasmid pCG1 features a multiple cloning region and a spectinomycin resistance determinant that is expressed in both Streptococcus spp. and E. coli. Various streptococcal DNA fragments were cloned in pCG1, and the recombinant constructs were stably maintained in the streptococcal hosts. Since pCG1 is compatible with the most widely used streptococcal replicon, pVA380-1, pCG1 will provide a much needed tool allowing the cloning of two genes that work in concert in the same host.

| S-EPMC3131664 | biostudies-literature

Aneurysm Infection Caused by Desulfovibrio desulfuricans.

Project description:An 84-year-old man in Japan who had undergone endovascular aortic repair 9 years earlier had an infected aneurysm develop. We detected Desulfovibrio desulfuricans MB at the site. The patient recovered after surgical debridement, artificial vessel replacement, and appropriate antimicrobial therapy. Clinicians should suspect Desulfovibrio spp. infection in similar cases.

| S-EPMC10370862 | biostudies-literature

Desulfovibrio desulfuricans bacteremia in a dog.

Project description:Desulfovibrio desulfuricans was isolated from the blood of a dog presenting with fever, anorexia, and rear limb stiffness. The isolate was identified by 16S rRNA gene amplification and sequencing.

| S-EPMC86534 | biostudies-literature

Characterization of intracellular palladium nanoparticles synthesized by Desulfovibrio desulfuricans and Bacillus benzeovorans.

Project description:Early studies have focused on the synthesis of palladium nanoparticles within the periplasmic layer or on the outer membrane of Desulfovibrio desulfuricans and on the S-layer protein of Bacillus sphaericus. However, it has remained unclear whether the synthesis of palladium nanoparticles also takes place in the bacterial cell cytoplasm. This study reports the use of high-resolution scanning transmission electron microscopy with a high-angle annular dark field detector and energy dispersive X-ray spectrometry attachment to investigate the intracellular synthesis of palladium nanoparticles (Pd NPs). We show the intracellular synthesis of Pd NPs within cells of two anaerobic strains of D. desulfuricans and an aerobic strain of B. benzeovorans using hydrogen and formate as electron donors. The Pd nanoparticles were small and largely monodispersed, between 0.2 and 8 nm, occasionally from 9 to 12 nm with occasional larger nanoparticles. With D. desulfuricans NCIMB 8307 (but not D. desulfuricans NCIMB 8326) and with B. benzeovorans NCIMB 12555, the NPs were larger when made at the expense of formate, co-localizing with phosphate in the latter, and were crystalline, but were amorphous when made with H2, with no phosphorus association. The intracellular Pd nanoparticles were mainly icosahedrons with surfaces comprising {111} facets and about 5 % distortion when compared with that of bulk palladium. The particles were more concentrated in the cell cytoplasm than the cell wall, outer membrane, or periplasm. We provide new evidence for synthesis of palladium nanoparticles within the cytoplasm of bacteria, which were confirmed to maintain cellular integrity during this synthesis.

| S-EPMC4779138 | biostudies-literature

Cytochrome c(3) mutants of Desulfovibrio desulfuricans.

Project description:To explore the physiological role of tetraheme cytochrome c(3) in the sulfate-reducing bacterium Desulfovibrio desulfuricans G20, the gene encoding the preapoprotein was cloned, sequenced, and mutated by plasmid insertion. The physical analysis of the DNA from the strain carrying the integrated plasmid showed that the insertion was successful. The growth rate of the mutant on lactate with sulfate was comparable to that of the wild type; however, mutant cultures did not achieve the same cell densities. Pyruvate, the oxidation product of lactate, served as a poor electron source for the mutant. Unexpectedly, the mutant was able to grow on hydrogen-sulfate medium. These data support a role for tetraheme cytochrome c(3) in the electron transport pathway from pyruvate to sulfate or sulfite in D. desulfuricans G20.

| S-EPMC91879 | biostudies-literature