No description available.

Carnocyclin A (CclA) is a remarkably stable, potent bacteriocin produced by Carnobacterium maltaromaticum UAL307. Elucidation of the amino acid and genetic sequences revealed that CclA is a circular bacteriocin. Preliminary structural studies (dynamic light scattering, NMR, circular dichroism, stereochemical analysis) indicated that CclA is monomeric and alpha-helical in aqueous conditions and composed of L-residues. The 3D structure of [13C,15N]CclA was solved by NMR, revealing a compact arrangement of four helices. To examine the structure of the precursor peptide (pCclA) several fusion proteins were constructed and overexpressed; however, pCclA could not be isolated. To investigate the requirements for cyclization, several internally hexahistidine-tagged (His6) pCclA mutants were constructed. Expression conditions are underway. PisI was heterologously expressed and confirmed to impart protection against piscicolin 126 (PisA). Labeled and unlabeled PisA and PisI were purified following overexpression as maltose-binding protein fusions (MalE-fusions) and Factor Xa cleavage. NMR studies indicated that PisI and PisA do not physically interact. The 3D structure of PisI was solved by NMR, confirming that the four-helix bundle is a conserved motif for the immunity proteins of type IIa bacteriocins. The putative receptor proteins for these bacteriocins were cloned and overexpressed as His6-fusion proteins. Experiments are underway to optimize the expression and purification of these membrane proteins. The peptidase domain of the ABC-transporter protein (CbnTP) for carnobacteriocin B2 (CbnB2) was overexpressed as a His6-fusion protein. Active protease could not be purified from inclusion bodies, but was obtained as soluble protein following low-temperature overexpression. The CbnB2 precursor pCbnB2 (and a truncated derivative pCbnB2-RP) was purified following overexpression as a MalE-fusion and Factor Xa cleavage. pCbnB2 was incubated with CbnTP and MALDI-TOF and activity testing confirmed that CbnTP cleaved the leader peptide from pCbnB2. Five CysSer CbnTP mutants were constructed. Crystallographic studies of CbnTP are underway. Six bacteriocins (nisin, gallidermin, lacticin 3147, CclA, PisA, enterocin 710C) were tested against Gram-negative bacteria (E. coli DH5, Pseudomonas aeruginosa ATCC 14207, Salmonella typhimurium ATCC 23564) in the absence and presence of EDTA. PisA and lacticin 3147 exhibited minimal activity, whereas the other bacteriocins killed at least one strain, in the presence of EDTA.

No description available.

Chez les bactéries à Gram-négatif, deux systèmes très bien conservés et essentiels à la survie de la cellule bactérienne ont été identifiés : les systèmes Tol et TonB. Ces deux systèmes utilisent la force proton motrice, issue de la membrane interne et transfert l’énergie associée pour le transport actif de molécules (TonB) ou nécessaire au maintien de l’intégrité membranaire (Tol). Ces 2 systèmes ont été détournés de leurs fonctions initiales et parasités par les colicines, leur conférant un rôle primordial dans le mécanisme d’import de la colicine. Une colicine est une bactériocine (toxine) produite par Escherichia coli pour tuer des souches apparentées. Ce sont des toxines spécifiques et hautement actives. Cependant E.coli a développé des mécanismes de protection afin de résister à l’action cytotoxique des colicines. Ces mécanismes de résistance consistent essentiellement à produire des protéines d’immunité, qui vont pour la plupart se fixer sur le domaine catalytique de la colicine et l’empêcher d’exercer son action létale. La bactérie Yersinia pestis, agent de la peste, possède une colicin-like bactériocine, la pesticine, dont l’activité est de dégrader le peptidoglycane. L’action de la pesticine est inhibée par une protéine d’immunité, Pim, localisée dans le périplasme. Le principal objectif de ce projet est de comprendre les mécanismes d’inhibition de la pesticine par sa protéine d’immunité, grâce à des données biochimiques et structurales, mais aussi d’apporter des solutions pour contourner ce problème de résistance. La structure de la pesticine révèle des homologies structurales avec le T4 lysozyme du bactériophage T4. Pour contourner le problème de la résistance bactérienne liée à la protéine d’immunité, une solution a été de fusionner le domaine de réception/translocation de la pesticine avec le T4 lysozyme. Nous avons ainsi pu créer et résoudre la structure tridimensionnelle d’une protéine chimère fonctionnelle, capable de se fixer sur FyuA (récepteur de la pesticine) et tuer une souche exprimant ce récepteur et dont l’activité létale n’est pas inhibée par Pim. / In Gram-negative bacteria, two essential systems for cell survival have been characterized: the Tol and TonB system. Both Ton and Tol systems are very well conserved in Gram-negative bacteria and coupled to the proton motive force across the inner membrane, acting as energy transducers for active transport (Ton) or maintenance of outer envelope integrity (Tol). Both systems have been embezzled from their primary function and hijacked by colicins as part of the colicin killing pathway. Colicin is a bacteriocin (toxin) produced by and toxic to some strains of Escherichia coli. Colicins are highly effective toxins. However E.coli could develop protective mechanisms to resist to colicin cytotoxic effect. These mechanisms essentially consist to produce an immunity protein. These proteins bind to colicin catalytic domain and inhibit its lethal activity. Yersinia pestis, plague agent, possesses its own colicin-like bacteriocin, Pesticin, which degrades murein. Pesticin activity is inhibited by an immunity protein, Pim, localized in the periplasm. The main goal of this project is to understand inhibition mechanisms between Pim and Pesticin by biochemical and structural data and to provide solution to overcome the resistance issue, since Pesticin was thought to be used as antimicrobial agent. The Pesticin structure has revealed that Pesticin share structural homologies with the T4 lysozyme from the bacteriophage T4. To overcome the resistance issue due to the immunity protein, one solution has been to fuse the Pesticin binding/translocation domain with the T4 lysozyme. Thus, we could engineered and solved the three-dimensional structure of a chimera protein, able to bind FyuA (Pesticin physiological receptor) and kill a FyuA expressing strain, in which the lethal activity is not affected by Pim.