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International audience; Cyclic di-AMP is an essential signaling molecule in Gram-positive bacteria. This second messenger regulates the osmotic pressure of the cell by interacting directly with the regulatory domains, either RCK_C or CBS domains, of several potassium and osmolyte uptake membrane protein systems. Cyclic di-AMP also targets stand-alone CBS domain proteins such as DarB in Bacillus subtilis and CbpB in Listeria monocytogenes. We show here that the CbpB protein of Group B Streptococcus binds c-di-AMP with a very high affinity. Crystal structures of CbpB reveal the determinants of binding specificity and significant conformational changes occurring upon c-di-AMP binding. Deletion of the cbpB gene alters bacterial growth in low potassium conditions most likely due to a decrease in the amount of ppGpp caused by a loss of interaction between CbpB and Rel, the GTP/GDP pyrophosphokinase.

The molecular mechanisms and forces involved in the translocation of bacterial toxins into host cells are still a matter of intense research. The adenylate cyclase (CyaA) toxin from Bordetella pertussis displays a unique intoxication pathway in which its catalytic domain is directly translocated across target cell membranes. The CyaA translocation region contains a segment, P454 (residues 454–484), which exhibits membrane‐active properties related to antimicrobial peptides. Herein, the results show that this peptide is able to translocate across membranes and to interact with calmodulin (CaM). Structural and biophysical analyses reveal the key residues of P454 involved in membrane destabilization and calmodulin binding. Mutational analysis demonstrates that these residues play a crucial role in CyaA translocation into target cells. In addition, calmidazolium, a calmodulin inhibitor, efficiently blocks CyaA internalization. It is proposed that after CyaA binding to target cells, the P454 segment destabilizes the plasma membrane, translocates across the lipid bilayer and binds calmodulin. Trapping of CyaA by the CaM:P454 interaction in the cytosol may assist the entry of the N‐terminal catalytic domain by converting the stochastic motion of the polypeptide chain through the membrane into an efficient vectorial chain translocation into host cells., The mechanism of cell invasion by bacterial toxins is still poorly understood. The adenylate cyclase (CyaA) toxin from Bordetella pertussis, the causative agent of whooping cough, directly translocates its catalytic domain across plasma membranes. The membrane‐permeabilizing segment (yellow) of CyaA translocates across the plasma membrane and binds calmodulin (red), which assists the entry of the catalytic domain (blue) into host cells while the hydrophobic and acylation domains (green) interact with the membrane and the C‐terminal Repeat‐in‐Toxin domain (grey) remains in the extra‐cellular milieu.

rv1098c, an essential gene in Mycobacterium tuberculosis, codes for a class II fumarase. We describe here the crystal structure of Rv1098c in complex with l -malate, fumarate or the competitive inhibitor meso-tartrate. The models reveal that substrate binding promotes the closure of the active site through conformational changes involving the catalytic SS-loop and the C-terminal domain, which likely represents a general feature of this enzyme superfamily. Analysis of ligand–enzyme interactions as well as site-directed mutagenesis suggest Ser318 as one of the two acid–base catalysts. Structured summary of protein interactions Rv1098c and Rv1098c bind by X-ray crystallography ( View interaction )

International audience; Dorsal root ganglia (DRG) neurons exhibit a wide molecular heterogeneity in relation to the various sensory modalities (mechanoception, thermoception, nociception) that they subserve. Finding markers of subpopulations is an important step in understanding how these neurons convey specific information. We identified fibroblast growth factor homologous factor 1 (FHF1) in a search for markers of subpopulations of DRG neurons. FHFs constitute a family of four factors that share some structural properties with fibroblast growth factors (FGFs) but are functionally distinct. They are expressed in specific subsets of neurons and are involved in the modulation of sodium channel activity. The pattern of expression of FHF1 in the DRG was determined during development, in the adult and after axotomy. We show that in the adult, FHF1 is expressed in two populations, one composed of nociceptors and another in which no neurotrophic factor receptors were detected (panTrk-/c-Ret-). Interestingly, in the nociceptors, FHF1 expression was restricted to a subset of TrkA+/calcitonin gene-related peptide (CGRP)-positive neurons. Neurofilament 200 (NF-200) and peripherin labeling indicates that 70% of the FHF1-expressing neurons contribute to A-fibers and 30% to C-fibers. FHF1 interacts with the Na(v)1.9 sodium channel isoform, which is strongly expressed in cRet+/isolectin-B4 binding neurons, but we show that FHF1 is not expressed in the cRet+/IB4+ subclass and that it does not colocalize with Na(v)1.9. Our results argue strongly against the possibility that FHF1 has a modulatory effect on this channel in cRet+/IB4+ neurons, but FHF1 could play a role in a distinct subset of TrkA+/CGRP+ nociceptors.

Ca2+ influx through voltage-gated calcium channels probably influences neuronal ontogenesis. Many developing neurones transiently express T-type/Cav3 calcium channels that contribute to their electrical activity and potentially to their morphological differentiation. Here we have characterized the electrophysiological properties and the functional role of a large T-type calcium current that is present in mouse developing primary vestibular neurones at embryonic day E17. This T-type current showed fast activation and inactivation, as well as slow deactivation kinetics. The overlap of activation and inactivation parameters produced a window current between −65 and −45 mV. Recovery from short-term inactivation was slow suggesting the presence of the Cav3.2 subunit. This T-type current was blocked by micromolar concentrations of Ni2+ and was inhibited by fast perfusion velocities in a similar fashion to recombinant Cav3.2 T-type channels expressed in HEK-293 cells. More importantly, current clamp experiments have revealed that the T-current could elicit afterdepolarization potentials during the repolarization phase of action potentials, and occasionally generate calcium spikes. Taken together, we demonstrate that the Cav3.2 subunit is likely to be the main T-type calcium channel subunit expressed in embryonic vestibular neurones and should play a key role in the excitability of these neurones during the ontogenesis of vestibular afferentation.

Cellulosomes are multi-enzyme complexes that orchestrate the efficient degradation of cellulose and related plant cell wall polysaccharides. The complex is maintained by the high-affinity protein-protein interaction between two complementary modules: the cohesin and the dockerin. In order to characterize the interaction between different cohesins and dockerins, we have developed matching fusion-protein systems, which harbor either the cohesin or the dockerin component. For this purpose, corresponding plasmid cassettes were designed, which encoded for the following carrier proteins: (i) a thermostable xylanase with an appended His-tag; and (ii) a highly stable cellulose-binding module (CBM). The resultant xylanase-dockerin and CBM-cohesin fusion products exhibited high expression levels of soluble protein. The expressed, affinity-purified proteins were extremely stable, and the functionality of the cohesin or dockerin component was retained. The fusion protein system was used to establish a sensitive and reliable, semi-quantitative enzyme-linked affinity assay for determining multiple samples of cohesin-dockerin interactions in microtiter plates. A variety of cohesin-dockerin systems, which had been examined previously using other methodologies, were revisited applying the affinity-based enzyme assay, the results of which served to verify the validity of the approach.

The mammalian utricular sensory receptors are commonly believed to be non-spiking cells with electrical activity limited to graded membrane potential changes. Here we provide evidence that during the first post-natal week, the sensory hair cells of the rat utricle express a tetrodotoxin (TTX)-sensitive voltage-gated Na+ current that displays most of the biophysical and pharmacological characteristics of neuronal Na+ current. Single-cell RT-PCR reveals that several alpha-subunit isoforms of the Na+ channels are co-expressed within a single hair cell, with a major expression of Nav1.2 and Nav1.6 subunits. In neonatal hair cells, 30 % of the Na+ channels are available for activation at the resting potential. Depolarizing current injections in the range of the transduction currents are able to trigger TTX-sensitive action potentials. We also provide evidence of a TTX-sensitive activity-dependent brain-derived neurotrophic factor (BDNF) release by early post-natal utricle explants. Developmental analysis shows that Na+ currents decrease dramatically from post-natal day 0 (P0) to P8 and become almost undetectable at P21. Concomitantly, depolarizing stimuli fail to induce both action potential and BDNF release at P20. The present findings reveal that vestibular hair cells express neuronal-like TTX-sensitive Na+ channels able to generate Na+-driven action potentials only during the early post-natal period of development. During the same period an activity-dependent BDNF secretion by utricular explants has been demonstrated. This could be an important mechanism involved in vestibular sensory system differentiation and synaptogenesis.

The cohesin-dockerin interaction provides the basis for incorporation of the indi- vidual enzymatic subunits into the cellulosome com- plex. In a previous article (Pages et al., Proteins 1997;29:517-527) we predicted that four amino acid residues of the ;70-residue dockerin domain would serve as recognition codes for binding to the cohesin domain. The validity of the prediction was exam- ined by site-directed mutagenesis of the suspected residues, whereby the species-specificity of the cohe- sin-dockerin interaction was altered. The results support the premise that the four residues indeed play a role in biorecognition, while additional resi- dues may also contribute to the specificity of the interaction. Proteins 2000;39:170 -177.

4-Nitrophenyl-β-xylobioside was synthesized by an improved short chemical-enzymatic method, based on the use of xylobiose as a starting material. Xylobiose was prepared following extensive enzymatic digestion of birchwood xylan with xylanase T-6. The resulting digest, containing mainly xylobiose and xylose, was directly subjected to an acetylation step, which after silica gel chromatography, provided highly pure hexaacetate of xylobiose. Bromination with HBr in acetic acid gave in quantitative yields the corresponding bromide, which after the coupling and deprotection steps, afforded the target 4-nitrophenyl-β-xylobioside.

International audience; Spirochetes can be distinguished from other bacteria by their spiral-shaped morphology and subpolar periplasmic flagella. This study focused on FlhF and FlhG, which control the spatial and numerical regulation of flagella in many exoflagellated bacteria, in the spirochete Leptospira. In contrast to flhF which seems to be essential in Leptospira, we demonstrated that flhG- mutants in both the saprophyte L. biflexa and the pathogen L. interrogans were less motile than the wild-type strains in gel-like environments but not hyperflagellated as reported previously in other bacteria. Cryo-electron tomography revealed that the distance between the flagellar basal body and the tip of the cell decreased significantly in the flhG- mutant in comparison to wild-type and complemented strains. Additionally, comparative transcriptome analyses of L. biflexa flhG- and wild-type strains showed that FlhG acts as a negative regulator of transcription of some flagellar genes. We found that the L. interrogans flhG- mutant was attenuated for virulence in the hamster model. Cross-species complementation also showed that flhG is not interchangeable between species. Our results indicate that FlhF and FlhG in Leptospira contribute to governing cell motility but our data support the hypothesis that FlhF and FlhG function differently in each bacterial species, including among spirochetes.