PubMed Health⌕ Search

Biomedical subjects

L Jannière

Publications and source records attributed to L Jannière.

11 recordsLinked to original sources
PubMed1991

Unidirectional theta replication of the structurally stable Enterococcus faecalis plasmid pAM beta 1.

Numerous bacterial replicons remain poorly characterized due to difficulties in localization of the replication origin. We have circumvented this problem in the characterization and fine mapping of the origin of plasmid pAM beta 1 by exploiting the Bacillus subtilis termination signal, terC. In terC-containing derivatives, theta-form molecules with two invariant endpoints accumulate. The endpoints, which correspond to plasmid origin and terC, were mapped with single-nucleotide precision. Analysis of the replication intermediates of wild-type molecules by two-dimensional gel electrophoresis confirmed the location of the plasmid origin. Our results demonstrate that pAM beta 1 replication proceeds unidirectionally by a theta mechanism. This work confirms the use of termination signals to localize origins, suggests that termination in B. subtilis occurs by a mechanism similar to that of Escherichia coli and establishes that in addition to rolling circle replicating plasmids, Gram positive bacteria harbour plasmids which replicate by a theta mechanism.

Base Sequence↗
PubMed1991

Characterization of a region of the Enterococcus faecalis plasmid pAM beta 1 which enhances the segregational stability of pAM beta 1-derived cloning vectors in Bacillus subtilis.

The nucleotide sequence of a 2.13-kb EcoRI-HindIII, pAM beta 1-derived fragment, isolated from the gram-positive cloning vector pHV1431, has been determined and shown to encode two ORFs. ORF H encodes for a protein of 23,930 Da which exhibits substantial homology to bacterial site-specific recombinases, particularly the resolvases of the gram-positive transposons Tn917 (30.3% identity) and Tn552 (31.6% identity) and the clostridial plasmid pIP404 (27.1% identity). The second ORF (I) is incomplete and encodes a polypeptide which has significant homology with Escherichia coli topoisomerase I (26.0% identity). Insertion of either the entire 2.13-kb EcoRI-HindIII fragment or a 0.73-kb EcoRI-DraI subfragment encoding only the resolvase into the pAM beta 1-based cloning vector pMTL500E causes a significant enhancement of segregational stability (from 6.5 X 10(-2) to 3.0-4.0 X 10(-3) plasmid loss per cell per generation). Improved segregational stability is mirrored by a reduction in plasmid polymerization. The introduction of a stop codon into the resolvase coding region negates its ability to promote segregational stability. It is proposed that the identified determinant stabilizes pAM beta 1-based vectors in Bacillus subtilis by maintaining the plasmid population in the monomeric state, thereby reducing the chances of producing plasmid-free segregants.

Amino Acid Sequence↗
PubMed1990

Structurally stable Bacillus subtilis cloning vectors.

Cloning of long DNA segments (greater than 5 kb) in Bacillus subtilis is often unsuccessful when naturally occurring small (less than 10 kb) plasmids are used as vectors. In this work we show that vectors derived from the large (26.5 kb) plasmids pAM beta 1 and pTB19 allow efficient cloning and stable maintenance of long DNA segments (up to 33 kb). The two large plasmids differ from the small ones in several ways. First, replication of the large plasmids does not lead to accumulation of detectable amounts of ss DNA, whereas the rolling-circle replication typical for small plasmids does. In addition, the replication regions of the two large plasmids share no sequence homology with the corresponding regions of the known small plasmids, which are highly conserved. Taken together, these observations suggest that the mode of replication of the large plasmids is different from that of small plasmids. Second, short repeated sequences recombine much less frequently when carried on large than on small plasmids. This indicates that large plasmids are structurally much more stable than small ones. We suggest that the high structural stability of large plasmids is a consequence of their mode of replication and that plasmids which do not replicate as rolling circles should be used whenever it is necessary to clone and maintain long DNA segments in any organism.

Bacillus subtilis↗
PubMed1990

Tn10-derived transposons active in Bacillus subtilis.

Small derivatives of the Escherichia coli transposon Tn10, comprising IS10 ends and a chloramphenicol resistance gene, were introduced in Bacillus subtilis on a thermosensitive plasmid, pE194. In the presence of the Tn10 transposase gene fused to signals functional in B. subtilis, these derivatives transposed with a frequency of 10(-6) per element per generation. They had no highly preferred insertion site or region, as judged by restriction analysis of the chromosomal DNA, and generated auxotrophic and sporulation-deficient mutants with a frequency of about 1%. These results suggest that Tn10 derivatives might be a useful genetic tool in B. subtilis and possibly other gram-positive microorganisms.

Bacillus subtilis↗
PubMed1988

Restriction and modification in Bacillus subtilis Marburg 168: target sites and effects on plasmid transformation.

The effects of the restriction system of Bacillus subtilis strain M on plasmid transformation were studied. Plasmid pHV1401 DNA prepared from B. subtilis transformed the restriction-proficient M strain 100 times more efficiently than the DNA prepared from Escherichia coli, while the two DNA preparations transformed restriction-deficient derivatives of that strain with similar efficiencies. This indicates that transformation with pHV1401 is sensitive to the M restriction system. pHV1401 contains three CTCGAG (XhoI sites). Successive removal of these abolished the effect of restriction. This indicates that the XhoI sites are the targets for the M restriction system.

Bacillus subtilis↗
PubMed1987

Recombination between short repeated sequences is more frequent in plasmids than in the chromosome of Bacillus subtilis.

We measured in Bacillus subtilis the recombination frequency between directly repeated 9 bp sequences flanking the transposon Tn5. Four different repeats were analyzed. They recombined with frequencies between 10(-7) and 10(-6) when they were carried on plasmids, and 150-1500 times less frequently when they were carried on the chromosome. We propose that this difference is due to the generation of single stranded DNA during plasmid replication.

Bacillus subtilis↗
PubMed1985

Stable gene amplification in the chromosome of Bacillus subtilis.

We constructed five different structures, consisting of a genetic marker flanked by directly repeated sequences 2-4 kb long, in the Bacillus subtilis chromosome. When a selective pressure was applied amplification of the marker and one of the repeats was observed in all cases. Amplification was not detected with two markers which were not flanked by the repeated sequences. The maximum amplification level observed with the different structures varied between 5 and 50. The size of the most amplified structure corresponded to 7.5% of the chromosome. Amplification was stable upon growth of cells under non-selective conditions. Each copy of an amplified gene was expressed with equal efficiency. These results indicate that chromosomal gene amplification may be useful for constructing genetically engineered B. subtilis strains.

Bacillus subtilis↗
PubMed1985

Integration of linear, heterologous DNA molecules into the Bacillus subtilis chromosome: mechanism and use in induction of predictable rearrangements.

Linear DNA molecules composed of a central region nonhomologous with the Bacillus subtilis chromosome and two flanking regions homologous with the chromosome can integrate into the chromosome, provided that the homologous regions have the same relative orientation. The resulting chromosome can be maintained in a haploid or in a merodiploid cell together with a parental chromosome. This can most easily be explained by supposing that the integration occurs by crossing over at each homologous region and that a part of the chromosome between these regions is deleted and replaced by the central nonhomologous region of the integrating molecule. If no essential genes were replaced during that process a haploid cell would be obtained; if essential genes were replaced a merodiploid cell would be obtained. The use of appropriate linear molecules therefore should allow the induction of deletions, extending from a given chromosomal site in a predetermined direction, and defined duplications in the B. subtilis chromosome.

Bacillus subtilis↗