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The complete sequence of the plasmid MccC7-H22 encoding microcin C7, isolated from probiotic E. coli H22, was determined and analyzed. DNA of pMccC7-H22 comprises 32,014bp and contains 39 predicted ORFs. Two main gene clusters, i.e., genes involved in plasmid replication and maintenance and genes encoding microcin C7 synthesis, are separated by several ORFs homologous to ORFs present in IS (insertion...
Translation initiation of mRNA encoding the plasmid-specified initiator protein (Rep) required for initiation of the ColE2 plasmid DNA replication is fairly efficient in Escherichia coli despite the absence of a canonical Shine–Dalgarno sequence. Although a GA cluster sequence exists upstream the initiation codon, its activity as the SD sequence has been shown to be very inefficient. Deletion analyses...
Condensin activity establishes and maintains mitotic chromosome condensation, however the mechanisms of condensin recognition of specific chromosomal sites remain unknown. rDNA is the chief condensin binding locus in Saccharomyces cerevisiae, and the level of nucleolar transcription is one of the key factors determining condensin loading to the nucleolar organizer. A new aspect of this transcriptional...
The advent of recombineering technology in Escherichia coli has revolutionized the way recombinant DNA molecules are constructed. We present a novel application of recombineering to linearize DNA by capping their ends with individual telomeres derived from bacteriophage N15, which exists as a linear prophage in E. coli. The N15 telomerase occupancy site was recombined into circular DNA and resolved...
Tandem affinity purification has become a valuable tool for the isolation of protein complexes. Here we describe the construction and use of a series of plasmid vectors for Gram positive bacteria. The vectors utilize the SPA tag as well as variants containing a 3C rather than the TEV protease site as 3C protease has been shown to work efficiently at the low temperatures (4°C) used to isolate protein...
The TraM protein encoded by plasmid R1 has three functions in conjugative transfer: (i) it positively controls transfer gene expression, (ii) it stimulates efficient site-specific ssDNA cleavage at the oriT in vivo and (iii) it couples the relaxosome to the envelope-bound transport complex. Plasmid R1 contains two binding regions for TraM, sbmA and sbmB, either of which comprises several minimal TraM...
The complete nucleotide sequence of plasmid pSMA23 isolated from Lactobacillus casei A23 was determined. Plasmid pSMA23 is a 3497bp circular molecule with a G+C content of 38.18%. Four putative open reading frames were identified. Based on homology, two orfs were identified as genes encoding replication initiation (Rep) and mobilisation (Mob) protein, respectively. Various regulatory regions like...
Without prior knowledge of the promoters of various genes in bacteria, it can be difficult to study gene regulation using reporter-gene fusions. Regulation studies of promoters are ideal at their native locus, which do not require prior knowledge of promoter regions. Based on a previous study with FRT–lacZ–Km R constructs, we constructed two novel FRT–lacZ–Gm R plasmids. This allows...
Conjugative plasmids have evolved entry exclusion mechanisms to inhibit redundant DNA transfer from donor cells into recipients harboring isogenic or closely related plasmids. This exclusion phenomenon has been documented in the incompatibility H group (IncH) plasmid R27. A cosmid library representing the majority of the large (180kb) R27 plasmid was transformed into recipient cells and a conjugation...
Replication of the ColE2 plasmid requires a plasmid-coded initiator protein (Rep). Rep expression is controlled by antisense RNA (RNAI) against the Rep mRNA at a translational step. In this paper, we examined the effects of host RNA degradation enzymes on the degradation process of the Rep mRNA and its degradation intermediates especially those carrying the 5′ untranslated region. We showed that the...
Recent characterisations of plasmids related to the anthrax virulence plasmids pXO1 and pXO2 in clinical isolates of Bacillus cereus and Bacillus thuringiensis have contributed to the emerging picture of a virulence-associated plasmid pool in the B. cereus sensu lato group. The family of pXO2-like plasmids includes the conjugative plasmid pAW63 from the biopesticide strain B. thuringiensis subsp....
The complete nucleotide sequences of six Actinobacillus porcitonsillarum plasmids pKMA202 (13.425-kb), pKMA1467 (11.115-kb), pKMA5 (9.549-kb), pIMD50 (8.751-kb), pKMA505 (8.632-kb) and pKMA757 (4.556-kb) and three Actinobacillus pleuropneumoniae plasmids pPSAS1522 (4.244-kb), pARD3079 (3.884-kb) and pKMA2425 (3.156-kb) were determined. All the plasmids contain the sulfonamide resistance gene sul2...
A 10.3kb linear mitochondrial DNA plasmid designated pFP1 was isolated from Fusarium proliferatum. The DNA sequence of the plasmid consists of 10,336bp with perfect terminal inverted repeats of 400bp. Two major, non-overlapping ORFs were identified on opposite strands, encoding a phage-type RNA polymerase and a family B type DNA polymerase, respectively. One additional minor ORF encoding a putative...
In the lysogenic state, bacteriophage P1 is maintained as a low copy-number circular plasmid. Site-specific recombination at loxP by the phage-encoded Cre protein keeps P1 monomeric, thus helping to ensure stable plasmid inheritance. Two Escherichia coli DNA-binding proteins, PepA and ArgR, were recently reported to be necessary for maintenance or establishment of P1 lysogeny. PepA and ArgR bind to...
Despite the prevalence of Arthrobacter in the environment little is known about their plasmids, or the capacity of Arthrobacter plasmids to mediate horizontal gene transfer. In this study, we compared eight plasmids from five Arthrobacter strains in order to identify putative core maintenance genes for replication, segregation, and conjugation. Iteron like sequences were identified on some of the...
The members of the Bacillus cereus group, Bacillus anthracis, Bacillus thuringiensis, and B. cereus senso stricto, are largely defined by their content of large plasmids, which encode major virulence factors. Here we offer an easy, fast, and reliable protocol for the isolation and detection of large plasmids up to the size of at least 350kb. Furthermore, using this method, we report that Bacillus...
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