In previous studies (Malissard et al., FEMS Microbiol. Lett. (1993) 110, 101-106), the alginate lyase AlxM of the marine bacterium ATCC 433367 was produced in Escherichia coli TC4/pAL-A3 with a yield of 50 micrograms per litre of culture. The polypeptide chain was cleaved between two cysteine residues, C169 and C183, themselves linked by a disulphide bridge. AlxM has now been overproduced in E. coli BL21(DE3)/pAL-Sur/pLysS. Under conditions in which formation of inclusion bodies can be avoided, the enzyme is synthesized as a catalytically active, water-soluble, unnicked polypeptide with a yield of 32 mg per litre of culture. It has been purified to protein homogeneity using a one-step procedure. The nicked AlxMA and unnicked AlxMB alginate lyases have identical alginate-degrading activities at high salt concentrations.
Comparative analysis of partial tuf sequences was evaluated for the identification and differentiation of lactobacilli. Comparison of the amino acid sequences allowed differentiation between species and also between the subspecies of Lactobacillus delbrueckii. The nucleotide sequence comparison allowed differentiation between other subspecies and between some strains. Lactobacilli from several collections and isolates from dairy samples were clearly identified by comparison of short tuf sequences with those of the type strains. In evaluating the taxonomy of the Lactobacillus casei-related taxa, different tuf amino acid signatures are in favour of a classification into three distinct species. The type strain designation for the L. casei species is discussed.
L'alginate, polymere d'acides mannronique et guluronique, est un facteur de virulence de pseudomonas aeruginosa. Il protege la bacterie de la phagocytose et de l'action des antibiotiques. Le but de mon travail etait d'obtenir des anticorps anti-alginate et de produire en grande quantite des alginate lyases actives sur l'alginate de p. Aeruginosa. Des anticorps monoclonaux igm anti-alginate ont ete obtenus par immunisation de souris avec un conjugue bsa/alginate de p. Aeruginosa. Ils ont ete purifies puis reduits en monomeres pour un couplage avec la peroxydase. Le conjugue fonctionnel obtenu a permis de valider la methode pour la synthese du conjugue alginate lyase/anticorps anti-alginate. L'alginate lyase alxm de la bacterie marine atcc 433367 a ete surproduite chez e. Coli et purifiee en une seule etape: sa masse est de 30 kda, le rendement de purification est de 90% (32 mg/l de culture). Les conditions optimales d'activite de alxm ont ete determinees, puis utilisees pour l'etude de sa specificite. Alxm est une mannuronate lyase, elle produit, par un mecanisme de -elimination avec retention de configuration, un oligomere de dp3 quel que soit le substrat. Ses parametres cinetiques ont ete determines pour des substrats oligomeriques de structure definie. Le gene algi codant pour l'alginate lyase produite par pseudomonas alginovora a ete amplifie par pcr directe et inverse, sequence puis clone dans le vecteur de surexpression pet-22b. L'enzyme surproduite a 18c dans e. Coli, algisur, a ete purifiee en une seule etape par chromatographie d'affinite avec un rendement de 67% (5 mg/l de culture). Conformement a la strategie de surexpression, sa masse de 24635 da correspond a celle de algi additionnee de la sequence poly(his). Algi et algisur ont le meme profil d'activite avec une specificite de type mannuronate lyase. Une etude par hca sur alxm, algi et quatre autres alginate lyases de structures primaires connues a permis de classer ces enzymes deux par deux en trois familles
A gene of Pseudomonas alginovora, called aly, has been cloned in Escherichia coli using a battery of PCR techniques and sequenced. It encodes a 210-amino-acid alginate lyase (EC 4.2.2.3), Aly, in the form of a 233-amino-acid precursor. P. alginovora Aly has been overproduced in E. coli with a His-tag sequence fused at the C-terminal end under conditions in which the formation of inclusion bodies is avoided. His-tagged P. alginovora Aly has the same enzymic properties as the wild-type enzyme and has the specificity of a mannuronate lyase. It can be purified in a one-step procedure by affinity chromatography on Ni2+-nitriloacetate resin. The yield is of 5 mg of enzyme per litre of culture. The amplification factor is 12.5 compared with the level of production by wild-type P. alginovora. The six alginate lyases of known primary structure fall into three distinct classes, one of which comprises the pair P. alginovora Aly and Klebsiella pneumoniae Aly.
Comparative analysis of partial tuf sequences was evaluated for the identification and differentiation of lactobacilli. Comparison of the amino acid sequences allowed differentiation between species and also between the subspecies of Lactobacillus delbrueckii. The nucleotide sequence comparison allowed differentiation between other subspecies and between some strains. Lactobacilli from several collections and isolates from dairy samples were clearly identified by comparison of short tuf sequences with those of the type strains. In evaluating the taxonomy of the Lactobacillus casei-related taxa, different tuf amino acid signatures are in favour of a classification into three distinct species. The type strain designation for the L. casei species is discussed.
The oligopeptidase PepO from Streptococcus thermophilus A was purified to protein homogeneity by a five-step chromatography procedure. It was estimated to be a serine metallopeptidase of 70 kDa, with maximal activity at pH 6.5 and 41°C. PepO has endopeptidase activity on oligopeptides composed of between five and 30 amino acids. PepO was demonstrated to be active and stable at the pH, temperature and salt concentrations found in Swiss-type cheese during ripening. Using a battery of PCR techniques, the pepO gene was amplified, subcloned and sequenced, revealing an open reading frame of 1893 nucleotides. The amino acid sequence analysis of the pepO gene-translation product shows homology with PepO enzymes from other lactic acid bacteria and contains the signature sequence of the metallopeptidase family.
This study evaluated, in vitro, the role of different Pseudomonas aeruginosa exopolysaccharides (EPS) in mediating adherence to human respiratory epithelial cells. Two mucoid and non-mucoid isogenic pairs of P. aeruginosa strains isolated from patients with cystic fibrosis (CF) and bronchiectasis were used. Adherence was tested with human tracheal epithelial cell lines from CF and normal fetuses. The CF cells bound significantly more bacteria than the normal cells. The strain from the bronchiectasis patient was significantly more adherent than that from the CF patient and this difference was consistently most marked with the non-mucoid variant and with normal epithelial cells. The differing behaviour of mucoid CF and non-mucoid bronchiectasis strains reflected the chemical composition of their EPS: mainly alginate in the former and neutral polysaccharides in the latter. Additive inhibition experiments with chemically characterised EPS indicated that neutral polysaccharides associated with alginate may act as ligands for the adherence of P. aeruginosa to CF epithelial cells.
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