The carboxy‐terminal C 2 ‐like domain of the α‐toxin from Clostridium perfringens mediates calcium‐dependent membrane recognition

International audienceThe lethal, cytolytic alpha-toxin (phospholipase C) of Clostridium perfringens consists of two distinct modules: the larger N-terminal domain catalyses phospholipid hydrolysis, and its activity is potentiated by a smaller C-terminal domain. Calcium ions are essential for the binding of alpha-toxin to lipid films. Sixteen alpha-toxin variants with single amino acid substitutions in the C-terminal region were obtained using site-directed mutagenesis and T7 expression technology. Five of these variants showed reduced phospholipase C activity and were considerably less active than native alpha-toxin under calcium-limiting conditions. Replacement of Thr-272 by Pro diminished phospholipase C activity, severely affected haemolysis and platelet aggregation and perturbed a surface-exposed conformational epitope. The results of sequence comparisons and molecular modelling indicate that the C-terminal region probably belongs to the growing family of C2 beta-barrel domains, which are often involved in membrane interactions, and that the functionally important substitutions are clustered at one extremity of the domain. The combined findings suggest that the C-terminal region of alpha-toxin mediates interactions with membrane phospholipids in a calcium-dependent manner. Mutations to this domain may account for the natural lack of toxicity of the alpha-toxin homologue, phospholipase C of Clostridium bifermentans

Regulation of the Bacillus subtilis ytmI Operon, Involved in Sulfur Metabolism

The YtlI regulator of Bacillus subtilis activates the transcription of the ytmI operon encoding an l-cystine ABC transporter, a riboflavin kinase, and proteins of unknown function. The expression of the ytlI gene and the ytmI operon was high with methionine and reduced with sulfate. Using deletions and site-directed mutagenesis, a cis-acting DNA sequence important for YtlI-dependent regulation was identified upstream from the −35 box of ytmI. Gel mobility shift assays confirmed that YtlI specifically interacted with this sequence. The replacement of the sulfur-regulated ytlI promoter by the xylA promoter led to constitutive expression of a ytmI′-lacZ fusion in a ytlI mutant, suggesting that the repression of ytmI expression by sulfate was mainly at the level of YtlI synthesis. We further showed that the YrzC regulator negatively controlled ytlI expression while this repressor also acted on ytmI expression via YtlI. The cascade of regulation observed in B. subtilis is conserved in Listeria spp. Both a YtlI-like regulator and a ytmI-type operon are present in Listeria spp. Indeed, the Lmo2352 protein from Listeria monocytogenes was able to replace YtlI for the activation of ytmI expression and a lmo2352′-lacZ fusion was repressed in the presence of sulfate via YrzC in B. subtilis. A common motif, AT(A/T)ATTCCTAT, was found in the promoter region of the ytlI and lmo2352 genes. Deletion of part of this motif or the introduction of point mutations in this sequence confirmed its involvement in ytlI regulation

Identification of Bacillus subtilis CysL, a Regulator of the cysJI Operon, Which Encodes Sulfite Reductase

The way in which the genes involved in cysteine biosynthesis are regulated is poorly characterized in Bacillus subtilis. We showed that CysL (formerly YwfK), a LysR-type transcriptional regulator, activates the transcription of the cysJI operon, which encodes sulfite reductase. We demonstrated that a cysL mutant and a cysJI mutant have similar phenotypes. Both are unable to grow using sulfate or sulfite as the sulfur source. The level of expression of the cysJI operon is higher in the presence of sulfate, sulfite, or thiosulfate than in the presence of cysteine. Conversely, the transcription of the cysH and cysK genes is not regulated by these sulfur sources. In the presence of thiosulfate, the expression of the cysJI operon was reduced 11-fold, whereas the expression of the cysH and cysK genes was increased, in a cysL mutant. A cis-acting DNA sequence located upstream of the transcriptional start site of the cysJI operon (positions −76 to −70) was shown to be necessary for sulfur source- and CysL-dependent regulation. CysL also negatively regulates its own transcription, a common characteristic of the LysR-type regulators. Gel mobility shift assays and DNase I footprint experiments showed that the CysL protein specifically binds to cysJ and cysL promoter regions. This is the first report of a regulator of some of the genes involved in cysteine biosynthesis in B. subtilis

Tolérance et réponse adaptative au stress acide chez Lactobacillus delbrueckii ssp. bulgaricus

International audienceTolerance and adaptative acid stress response of Lactobacillus delbrueckii ssp. bulgaricus. Using several approaches, we studied the acid stress response of different strains of Lactobacillus bulgaricus. We showed that after an acid shock, the cell survival varied considerably between strains and that all the studied strains were able to develop an acid adaptive response. For one of these strains, we characterized, using two-dimensional electrophoresis, the modifications in the protein pattern induced by acid adaptation. Out of the 50 proteins up-regulated under the adaptive conditions, 21 were identified after N-terminal sequencing. They belong to different functional categories including stress response and general metabolism. To allow a larger genetic exploration of L. bulgaricus, the genome of the type strain ATCC 11842 was sequenced in collaboration with the Genoscope. All together, these genomic and post-genomic data should lead to a better understanding of L. bulgaricus physiology and its environmental adaptability.En utilisant plusieurs approches, nous avons étudié le comportement de différentes souches de Lactobacillus bulgaricus face à un stress acide. Nous avons observé que la survie des cellules à un choc acide variait considérablement entre les souches et que toutes les souches étudiées étaient capables d'initier une réponse adaptative à l'acidité. Pour l'une des souches, nous avons caractérisé, par électrophorèse bidimensionnelle, les changements protéiques intervenant dans la réponse adaptative à l'acidité. Parmi les 50 protéines induites pendant l'adaptation, 21 ont pu être identifiées après séquençage N-terminal. Elles appartiennent à différentes catégories fonctionnelles dont celles des réponses aux stress et du métabolisme général. Afin de permettre une exploration génétique plus large de L. bulgaricus, le génome complet de la souche type ATCC 11842 a été séquencé en collaboration avec le Génoscope. Toutes ces données de génomique et de post-génomique nous conduiront à une meilleure compréhension de la physiologie de L. bulgaricus et notamment de son adaptabilité environnementale

Postgenomic Analysis of Streptococcus thermophilus Cocultivated in Milk with Lactobacillus delbrueckii subsp. bulgaricus: Involvement of Nitrogen, Purine, and Iron Metabolism▿ †

Streptococcus thermophilus is one of the most widely used lactic acid bacteria in the dairy industry, in particular in yoghurt manufacture, where it is associated with Lactobacillus delbrueckii subsp. bulgaricus. This bacterial association, known as a proto-cooperation, is poorly documented at the molecular and regulatory levels. We thus investigate the kinetics of the transcriptomic and proteomic modifications of S. thermophilus LMG 18311 in response to the presence of L. delbrueckii subsp. bulgaricus ATCC 11842 during growth in milk at two growth stages. Seventy-seven different genes or proteins (4.1% of total coding sequences), implicated mainly in the metabolism of nitrogen (24%), nucleotide base (21%), and iron (20%), varied specifically in coculture. One of the most unpredicted results was a significant decrease of most of the transcripts and enzymes involved in purine biosynthesis. Interestingly, the expression of nearly all genes potentially encoding iron transporters of S. thermophilus decreased, whereas that of iron-chelating dpr as well as that of the fur (perR) regulator genes increased, suggesting a reduction in the intracellular iron concentration, probably in response to H2O2 production by L. bulgaricus. The present study reveals undocumented nutritional exchanges and regulatory relationships between the two yoghurt bacteria, which provide new molecular clues for the understanding of their associative behavior