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Enterococcus faecalisblood isolates were probed for the serine protease activator of cytolysin (cylA) and aggregation substance (asa1), traits that generally reside on pheromone-responsive plasmids, to determine how commonly these genotypes were associated with disease. In dot blot assays, no significant difference was found in the frequency ofasa1for blood isolates [55 of 103 (54%)] and isolates...
The replication region of the lactococcal plasmid pJW563 was localized to a 2.3-kbEcoRI fragment. This DNA fragment was sequenced and a 1155-bp open reading frame,repB 563 , encoding a putative protein RepB 563 of 385 amino acids was found. An AT-rich noncoding region,repA 563 , was found upstream ofrepB 563 . This segment included several direct and inverted repeats...
Analysis of the 50-kb R-plasmid pTP10 from the clinical isolateCorynebacterium xerosisM82B revealed that the erythromycin resistance gene,ermCX,is located on a 4524-bp composite transposable element, Tn5432.The ends of Tn5432are identical, direct repeats of an insertion sequence, designated IS1249,encoding a putative transposase of the IS256family. IS1249consists of 1385 bp with 45/42 imperfect terminal...
The nucleotide sequence of the smallest plasmid ofErwinia stewartiiSW2 was determined. This plasmid, pSW100 (4272 bp), consists of a 702-bp region homologous to the origins of replication of plasmids p15A, ColE1, and ColA. Plasmid pSW100 also contains sequences homologous to thebomregion andmobCABDgenes of ColE1, except that a single-base deletion inmobAhas been detected. This deletion did not affect...
The plasmid pJ3356 confers high-level mupirocin resistance on a strain of Staphylococcus aureus isolated from a hospital. The plasmid also carries two copies of IS257. Recombination of an IS257-containing plasmid conferring erythromycin resistance, pOX7-IS, into either of the IS257s of pJ3356 has been observed. The co-integration of pJ3356 and a small plasmid, pOX7, is also reported and involves duplication...
Shuttle vectors useful for the genetic manipulation of several moderately halophilic bacteria have been constructed. These vectors are based on the minimal replicon of pCM1, a cryptic plasmid fromChromohalobacter marismortui,combined with the useful properties of pUC18 plasmid (i.e., small size, high copy number, multiple cloning sites,lacZfragment), as well as with the trimethoprim resistance gene...
pIP501 is a broad-host-range gram-positive conjugative plasmid. Previous transposon mutagenesis of a pIP501 derivative, pVA1702, identified two regions, A and B, that were involved in conjugation proficiency. Recently, we reported that the A region contained a functionaloriTsite similar to those found on plasmids of gram-negative origin (Wang and Macrina, 1995). We report here the nucleotide sequence...
pTIM3 is a suicide plasmid vector for the delivery of a transposition defective derivative of Tn5, expressing inducible mercury resistance (Hg R ) and catechol 2,3-dioxygenase (C23O) activity, to a range of gram-negative microorganisms. pTIM3 was constructed by a four-stage process from pNMM1, a derivative of pSUP5011 containing a modified Tn5where antibiotic-resistance determinants have been...
Temperature-sensitive Saccharomyces cerevisiae cdc6 mutants, under restrictive conditions, show an increase in recombination frequency, as well as chromosome and circular minichromosome loss. The role of the essential CDC6 gene was tested in trans and in cis to study circular plasmid stability. It was possible to demonstrate that the product of the CDC6 gene, acting in trans, is important for centromeric,...
A cosmid library was generated to the 200-kb self-transmissiblenifplasmid pEA9 isolated fromEnterobacter agglomerans339. The cosmid clone identified to contain the completenifcluster was used to determine thenifgene organization and the physical map. The restriction pattern andnifgene organization of thisnifcluster showed remarkable similarities to thenifcluster identified on the 110-kb plasmid pEA3...
A naphthalene-degrading strain of corynebacteria, Corynebacterium renale, harbors multiple small plasmids designated pCR1, pCR2, pCR3, and pCR4 with sizes of 1.4, 3.2, 4.4, and 5.7 kb, respectively. Plasmid pCR1 of 1.4 kb is the smallest plasmid reported in this group of bacteria and is present in high copy number. Attempts to clone whole pCR1 in Escherichia coli were unsuccessful but two of its fragments...
The nucleotide sequence and genetic organization of theBacteroidesplasmid pBI143 were determined. The plasmid was 2747 base pairs (bp) and had a G+C content of 41% (GenBank Accession No. U30316). There were two open reading frames greater than 50 codons and these were designatedmobAandrepA.A 56-bp inverted repeat divided pBI143 into modules withrepAandmobAin separate regions. There was a marked difference...
The nucleotide sequence of a small cryptic plasmid, pCL2.1, fromLactococcus lactisssp.lactisML 8 was determined. Sequence analysis of the pCL2.1 revealed that it contained 2112 bp, 33.9% GC, and two open reading frames that encoded polypeptides of 26 and 14 kDa.In vitrotranscription–translation of the pCL2.1 confirmed the existence of two polypeptides. Based on sequence homology, it is deduced...
The relaxase of RP4 nicks the double-stranded plasmid at the oriT site and binds covalently to DNA at the 5′ end of the nick. The 80-kDa relaxase (TraI) is encoded on an operon with several overlapping open reading frames (ORFs). The importance in conjugation of a short ORF (traX) with a start site overlapping the 5′ terminus of traI was investigated, as well as the effects of specific mutations in...
Recently an insertional mutagenesis procedure has been developed to permit cloning of genes affected in developmental mutants ofDictyostelium discoideum(Kuspa and Loomis,Proc. Natl. Acad. Sci. USA89,8803–8807, 1992). In this procedure a plasmid bearing the URA (pyr5-6) gene is linearized with a restriction enzyme and electroporated into URA − amoebae (auxotrophic for uracil) together with...
We describe several new cloning vectors for mutagenesis and allele replacement experiments. These plasmids have the R6Kγ DNA replication origin (oriR R6Kγ ) so they replicate only in bacteria supplying the Π replication protein (encoded bypir), and they can be maintained at low or high plasmid copy number by usingEscherichia colistrains encoding either wild-type or mutant forms of Π. They...
A system for the positive selection of structural plasmid rearrangements inBacillus subtiliswas developed. Random deletions removing a transcription terminator structure in the assay plasmid, designated pGP100, resulted in expression of thecat-86gene, under control of a constitutive bacteriophage promoter. The resulting chlorampenicol-resistant colonies were analyzed for plasmid contents and were...
The essential replicon region of plasmid pCU1 has, within 1.2 kb, two origins of replication that can function in the absence ofEscherichia coliDNA polymerase I and one that requires this polymerase. To isolate mutants in the replicon pathway that uses the PolI-dependent origin in the presence of the two other origins, we examined the feasibility of exploitingE. colistrains carrying a polymerase c...
Southern hybridization analysis of the IncC plasmid pDGO100 showed that, in addition to the well-characterized integron In7, there is a second integron which is located on a 3.6-kbBamHI fragment. This integron also possesses theqacEΔ1andsulIgenes typically found as part of the 3′-conserved segment of integrons. The 3.6-kbBamHI fragment, when cloned into pUC19 to form pDGO301, conferred resistance...
Highly DNA-restrictiveCorynebacteriacan be transformed with DNA madein vitroby PCR amplification of a sequence that contains the replication origin of pBL1, a plasmid common to manyCorynebacteria.In all strains examined, the transformation efficiencies of PCR-synthetized DNA equal or improve the performances of heterologous DNA extracted from wild-type anddam − -dcm − strains ofEscherichia...
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