Plasmid replication stimulates DNA recombination in Bacillus subtilis.

The effects of plasmid replication on the frequency of homologous recombination have been investigated. For that purpose Bacillus subtilis strains that carry in their chromosome directly repeated DNA sequences, and an integrated copy of plasmid pE194 either proximal or distal to the repeats, were constructed. The repeat consists either of 3.9 X 10(3) base pBR322 sequences or 2.1 X 10(3) base B. subtilis chromosomal sequences. As plasmid pE194 is naturally thermosensitive for replication, the activity of the replicon could be regulated. Recombination between the repeated sequences was infrequent (about 10(-4) per generation) when the integrated plasmid did not replicate. It was 20 to 450 times higher when the plasmid was allowed to replicate, provided that the repeats were in the proximity of the plasmid. These results show that plasmid replication stimulates DNA recombination.

Mechanisms of illegitimate recombination.

Illegitimate recombination, which is one of the major causes of genome rearrangements, can occur in a number of ways. These might involve enzymes which cut and join DNA or enzymes which replicate DNA, as illustrated by two examples: (i) formation of deletions at the replication origin (ori) of an Escherichia coli bacteriophage, M13; and (ii) excision of E. coli transposon Tn10. It is proposed that a common theme to various ways by which illegitimate recombination can occur might be the capacity to create ends in the DNA molecule and to make the ends meet.

DNA strand invasion promoted by Escherichia coli RecT protein.

The RecT protein of Escherichia coli is a DNA-pairing protein required for the RecA-independent recombination events promoted by the RecE pathway. The RecT protein was found to bind to both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) in the absence of Mg2+. In the presence of Mg2+, RecT binding to dsDNA was inhibited drastically, whereas binding to ssDNA was inhibited only to a small extent. RecT promoted the transfer of a single-stranded oligonucleotide into a supercoiled homologous duplex to form a D (displacement)-loop. D-loop formation occurred in the absence of Mg2+ and at 1 mM Mg2+ but was inhibited by increasing concentrations of Mg2+ and did not require a high energy cofactor. Strand transfer was mediated by a RecT-ssDNA nucleoprotein complex reacting with a naked duplex DNA and was prevented by the formation of RecT-dsDNA nucleoprotein complexes. Finally, RecT mediated the formation of joint molecules between a supercoiled DNA and a linear dsDNA substrate with homologous 3'-single-stranded tails. Together these results indicate that RecT is not a helix-destabilizing protein promoting a reannealing reaction but rather is a novel type of pairing protein capable of promoting recombination by a DNA strand invasion mechanism. These results are consistent with the observation that RecE (exonuclease VIII) and RecT can promote RecA-independent double-strand break repair in E. coli.

[Nutritional, cardiac and adrenergic changes induced by swimming training in rats (author's transl)]. / Etude chez le rat des modifications nutritionnelles, cardiaques et adrénergiques induites par l'entraînement.

The effects of training were investigated in male Sprague Dawley rats group (N), fed ad libitum, by measuring the weight increase and food intake of the animals, biochemical parameters (myocardial triglycerides and glycogen levels), mechanical and metabolic properties of the heart, and adrenergic reactions to swimming stress. An other group of rats remaining sedentary served as control (T). Conditioned rats had been submitted to a one hour test swim 6 days a week for 9 weeks. Gradually additional weights were fixed to the thorax. At the end of training, the load reached 6% of the body weight. Both groups were sacrified by decapitation at the same time, thirty hours after the last swimming session. The study of mechanical performances and metabolic properties was achieved with isolated working heart preparation. Adrenergic reactions of swimming stress were evaluated from plasma, heart and adrenal catecholamine concentrations.

A five-nucleotide sequence protects DNA from exonucleolytic degradation by AddAB, the RecBCD analogue of Bacillus subtilis.

Homologous recombination in Bacillus subtilis requires the product of the addA and addB genes, the AddAB enzyme. This enzyme, which is both a helicase and a powerful nuclease, is thought to be the counterpart of the Escherichia coli RecBCD enzyme. From this analogy, it is expected that the nuclease activity of AddAB can be downregulated by a specific DNA sequence, which would correspond to the chi site in E. coli. Using protection of linear double-stranded DNA as a criterion, we identified the five-nucleotide sequence 5'-AGCGG-3', or its complement 5'-CCGCT-3', as being sufficient for AddAB nuclease attenuation. We have shown further that this attenuation occurs only if the sequence is properly oriented with respect to the translocating AddAB enzyme. Finally, inspection of the complete B. subtilis genome revealed that this five-nucleotide sequence is over-represented and is, in a majority of cases, co-oriented with DNA replication. Based on these observations, we propose that 5'-AGCGG-3', or its complement, is the B. subtilis analogue of the E. coli chi sequence.

SPiD: a subtilis protein interaction database.

MOTIVATION: Protein-protein interactions are a potential source of valuable clues in determining the functional role of as yet uncharacterized gene products in metabolic pathways. Graph-like structures emerging from the accumulation of interaction data make it difficult to maintain a consistent and global overview by hand. Bioinformatics tools are needed to perform this graph visualization while maintaining a link to the experimental data. RESULTS: "SPiD" is an online database for exploring networks of interacting proteins in Bacillus subtilis characterized by the two-hybrid system. Graphical displays of interaction networks are created dynamically as users interactively navigate through these networks. Third party applications can interface the database through a Common Object Request Broker Architecture (CORBA) tier. AVAILABILITY: SPiD is available through its web site at http://www-mig.versailles.inra.fr/bdsi/SPiD, and through an Interoperable Object Reference (IOR) and its associated Interface Definition Language (IDL). CONTACT: hoebeke@versailles.inra.fr

Apparent and real recombination frequencies in multicopy plasmids: the need for a novel approach in frequency determination.

Recombination studies of bacteria are often carried out with multicopy plasmids, and recombination frequencies are often deduced from the proportion of cells in the population that express a recombinant phenotype. These frequencies should however be called apparent frequencies, since detection of the recombinant cells requires not only the formation of a rearranged plasmid but also its establishment in the cell. The establishment of the recombinant plasmid can possibly be affected by its interaction with the parental plasmids. To test this hypothesis, we have used a plasmid system enabling the study of deletion formation between short direct repeats (18 bp) in Bacillus subtilis and developed a method by which deletion frequencies are measured under conditions under which interaction is abolished. Real deletion frequencies were thus determined and compared with apparent deletion frequencies. Real frequencies were underestimated by a factor ranging from 4- to 500-fold, depending upon the plasmid under study. This implies that a large majority of the recombinant molecules that are formed are generally not detected. We show that apparent deletion frequencies strongly depend upon (i) the parental plasmid copy number, (ii) the ability of the recombinant molecules to form heterodimeric plasmids, and (iii) the fitness of the recombinant molecules relative to that of parental molecules. Finally, we show that under conditions under which all recombinant molecules are scored, transcription can inhibit the deletion process 10-fold.

Initiation of rolling-circle replication in pT181 plasmid: initiator protein enhances cruciform extrusion at the origin.

Plasmid pT181 DNA secondary structures have been analyzed in vitro by nuclease S1 digestion and in vivo by bromoacetaldehyde treatment. A cruciform structure occurring at the pT181 replication origin in vitro is greatly enhanced by the binding of the plasmid-encoded initiator protein RepC. In vivo a DNA secondary structure also existed in the replication origin. Its frequency of formation was correlated with efficiency of RepC utilization. These data suggest that cruciform extrusion at the origin is involved in initiation of pT181 rolling-circle replication. A neighboring DNA structure influences the conformation of the origin in vivo.

[Influence of the GABAergic system on eating behavior induced by the tail pinching technic in rats]. / Influence du système GABAergique sur le copmportement alimentaire induit par la technique de pincement de la queue (tail-pinching ou T.P.) chez le rat.

The present experiment was undertaken to investigate the possible involvement of GABAergic neurons in the regulation of the feeding behavior induced by tail pinching. Injections before the tail pinching of gamma-aminobutyric acid (GABA) agonist (Muscimol: 570 micrograms.kg-1 i.p. 20 min before testing), GABA antagonist (Picrotoxine: 1,5 mg.kg-1 i.p. 30 min before testing) and a GABA transaminase inhibitor (Acetate dipropyle: 200 mg.kg-1 i.p. 40 min before testing) have no effects on the ingestion of sweet milk. These preliminary results provide support for the hypothesis that the feeding behavior induced by TP is not mediated through GABAergic system.

Frequency of deletion formation decreases exponentially with distance between short direct repeats.

The effect of distance between 18 bp direct repeats on deletion formation has been examined in Bacillus subtilis. The deletion frequency decreased exponentially by more than 1000-fold as the distance increased from 33 to 2313 bp. This decrease occurred in two distinct phases, which may be determined by DNA-duplex flexibility. A similar relationship between deletion formation and distance was observed in a theta-replicating plasmid and in the chromosome, indicating that this relationship might have a general validity.

[Evaluation of food intake and adrenergic response in rats during the tail pinch test]. / Evaluation de la consommation alimentaire et des réactions adrénergiques du rat soumis à l'épreuve du pincement de la queue.

The urinary catecholamines output and the food intake were investigated in two groups of rats: a control group and a group where Tail Pinches (T.P.) were performed. Our results have shown, as these of antelman and al., that the Tail Pinch induced a high concentrate milk consumption, in spite of an increase of adrenergic activity which is known to have an inhibitory effect on caloric intake. We suggest that the role of various stress on food intake dependes on the balance of these two antagonist effects.

Induction of DNA amplification in the Bacillus subtilis chromosome.

A system allowing the induction of DNA amplification in Bacillus subtilis was developed, based on a thermosensitive plasmid, pE194, stably integrated in the bacterial chromosome. An amplification unit, comprising an antibiotic resistance marker flanked by directly repeated sequences, was placed next to the integrated plasmid. Activation of pE194 replication led to DNA amplification. Two different amplification processes appeared to take place: one increased the copy number of all sequences in the vicinity of the integrated plasmid and was possibly of the onion skin type, while the other increased the copy number of the amplification unit only and generated long arrays of amplification units. These arrays were purified and shown to consist mainly of directly repeated amplification units but to also contain non-linear regions, such as replication forks and recombination intermediates. They were attached to the chromosome at one end only, and were, in general, not stably inherited, which suggests that they are early amplification intermediates. Longer arrays were detected before the shorter ones during amplification. When the parental amplification unit contained repeats which differed by a restriction site the arrays which derived thereof contained in a majority of cases only a single type of repeat. We propose that the amplified DNA is generated by rolling circle replication, and that such a process might underlie a number of amplification events.

Copy-choice illegitimate DNA recombination revisited.

Nearly precise excision of a transposon related to Tn10 from an Escherichia coli plasmid was used as a model to study illegitimate DNA recombination between short direct repeats. The excision was stimulated 100-1000 times by induction of plasmid single-stranded DNA synthesis and did not involve transfer of DNA from the parental to the progeny molecule. We conclude that it occurred by copy-choice DNA recombination, and propose that other events of recombination between short direct repeats might be a result of the same process.

[Water deprivation. An uncommon form of child abuse]. / Privation d'eau. Une forme inhabituelle de maltraitance.

The authors report a case of hypernatremic dehydration following chronic water deprivation and very likely salt load as a form of child abuse in a 2 11/12 year-old girl. This uncommon picture often expresses a severely disturbed relationship between parents and child which has to be considered in the family management.

The beta-propeller protein YxaL increases the processivity of the PcrA helicase.

The DNA helicase PcrA is found in gram-positive bacteria and belongs to the superfamily 1 (SF1) of helicases, together with Rep and UvrD helicases from Escherichia coli. These helicases have been extensively studied in vitro and their mode of unwinding are well characterised. However, little is known about the putative cellular partners of such helicases. To identify PcrA-interacting factors, PcrA was used as a bait in a genome-wide yeast two-hybrid screen of a Bacillus subtilis library. Three proteins with unknown functions - YxaL, YwhK and YerB - were found to interact specifically with PcrA. The yxaL gene was cloned, the product was overexpressed and purified, and its effect on the PcrA activity was investigated in vitro. YxaL enhanced the processivity of the PcrA helicase. A comparison of the amino acid sequence of YxaL with other proteins from data banks suggests that YxaL belongs to a family of proteins with a repeated domain, which adopt a typical three-dimensional structure designated as a "beta-propeller". This raises the possibility that YxaL acts as a connector protein between PcrA and another cellular component.