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The exact nature of the sequence differences between the medically important family of gonococcal penicillinase-producing plasmids has been ascertained. The entire DNA sequence of the Asia-type plasmid, pJD4, demonstrated that it is 7426 bp and contains two direct repeats (DR30) that are implicated in the formation of deletion variant plasmids, such as the Africa-type plasmid. We have identified putative...
A useful method for inserting any DNA fragment into the chromosome of Neisseriae has been developed. The method relies on recombination-proficient vector plasmid pNLE1, a pUC19 derivative containing (1) genes conferring resistance to ampicillin and erythromycin, as selectable markers; (2) a chromosomal region necessary for its integration into the Neisseria chromosome; (3) a specific uptake sequence...
Bacillus thuringiensis, the entomopathogenic bacteria from the Bacillus cereus group, harbors numerous extrachromosomal molecules whose sizes vary from 2 to more than 200kb. Apart from the genes coding for the biopesticide δ-endotoxins located on large plasmids, little information has been obtained on these plasmids and their contribution to the biology of their host. In this paper, we embarked on...
Allelic exchange experiments allow investigation of the functions of many unknown genes identified during the sequencing of entire genomes. Isogenic strains differing by only specific mutations can be constructed. Among other tools, suicide plasmids are widely used for this task. They present many advantages because they leave no scars on the chromosome, and therefore allow combining several mutations...
The recA gene of the recombination deficient Enterococcus faecalis strain UV202 was sequenced and found to encode a glycine to aspartic acid mutation at amino acid 265. Both the UV sensitive and recombination deficient phenotypes of the UV202 strain were complemented by expression of the wild-type recA gene cloned under the control of the nisin-inducible promoter of an expression vector.
A plasmid containing multidrug resistance genes has been discovered from a clinical Actinobacillus pleuropneumoniae strain isolated in China. The complete 15079kb sequence of this plasmid, designated pHB0503, was analyzed with regard to the organization and evolution of multidrug resistance genes. The deduced amino acid sequences from seven open reading frames (sul2 catA3, aacC2, strA, truncated strB...
During the last 20years, two-dimensional agarose gel electrophoresis combined with other techniques such as Polymerase Chain Reaction, helicase assay and electron microscopy, helped to characterize plasmid DNA replication and topology. Here we describe some of the most important findings that were made using this method including the characterization of uni-directional replication, replication origin...
A streptomycin and tetracycline resistance plasmid R64 isolated from Salmonella enterica serovar Typhimurium belongs to the incompatibility group I1 (IncI1). The DNA sequence of the R64 conjugative transfer region was described previously (Komano et al., 2000). Here, we report the complete genome sequence of R64. In the circular double-stranded R64 genome with 120,826bp, 126 complete ORFs are predicted...
Error-prone rolling circle amplification (RCA) is a promising alternative to error-prone PCR for random mutagenesis. The main disadvantage of error-prone RCA is the low transformation efficiency of the DNA concatemer produced in the amplification reaction. We improved the method by introducing loxP recombination site of bacteriophage P1 Cre recombinase into the target plasmid and reducing the concatemer...
In this study we determined the complete nucleotide sequence of multidrug-resistance plasmid p9134, and its variants p9134dT and p9134dAT which spontaneously lost either tetracycline or both tetracycline and ampicillin resistance, respectively. The plasmids were 133,802 bp, 109,512 bp and 127,291 bp in size, respectively, and their basic backbone was similar to that of IncI plasmids. Genes coding...
The blaCTX-M-15 gene, encoding the globally dominant CTX-M-15 extended-spectrum β-lactamase, has generally been found in a 2.971-kb ISEcp1-blaCTX-M-15-orf477Δ transposition unit, with ISEcp1 providing a promoter. In available IncF plasmid sequences from Escherichia coli, this transposition unit interrupts a truncated copy of transposon Tn2 that lies within larger multiresistance regions. In E. coli,...
Integrative and conjugative elements (ICEs) are generally regarded as regions of contiguous DNA integrated within a bacterial genome that are capable of excision and horizontal transfer via conjugation. We recently characterized a unique group of ICEs present in Mesorhizobium spp., which exist as three entirely separate but inextricably linked chromosomal regions termed α, β and γ. These regions occupy...
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