Comparative analysis of binding of intact glucose-grown Fibrobacter succinogenes strain S85 cells and adhesion-defective mutants AD1 and AD4 to crystalline and acid-swollen (amorphous) cellulose showed that strain S85 bound efficiently to both forms of cellulose while mutant Ad1 bound to acid-swollen cellulose, but not to crystalline cellulose, and mutant Ad4 did not bind to either. One-and two-dimensional electrophoresis (2-DE) of outer membrane cellulose binding proteins and of outer membranes, respectively, of strain S85 and adhesion-defective mutant strains in conjunction with mass spectrometry analysis of tryptic peptides was used to identify proteins with roles in adhesion to and digestion of cellulose. Examination of the binding to cellulose of detergent-solubilized outer membrane proteins from S85 and mutant strains revealed six proteins in S85 that bound to crystalline cellulose that were absent from the mutants and five proteins in Ad1 that bound to acid-swollen cellulose that were absent from Ad4. Twenty-five proteins from the outer membrane fraction of cellulose-grown F. succinogenes were identified by 2-DE, and 16 of these were up-regulated by growth on cellulose compared to results with growth on glucose. A protein identified as a Cl-stimulated cellobiosidase was repressed in S85 cells growing on glucose and further repressed in the mutants, while a cellulose-binding protein identified as pilin was unchanged in S85 grown on glucose but was not produced by the mutants. The candidate differential cellulose binding proteins of S85 and the mutants and the proteins induced by growth of S85 on cellulose provide the basis for dissecting essential components of the cellulase system of F. succinogenes.Fibrobacter succinogenes S85 is a rod-shaped, gram-negative, strictly anaerobic ruminal bacterium that is one of the major cellulolytic bacteria within the rumen (2, 24). Because of the ability of this bacterium to efficiently adhere to and degrade plant cell walls, it has been extensively studied to elucidate the mechanism involved in adhesion and digestion of plant cell walls. As a result, five endoglucanases (31, 33-35), one cellobiosidase (17), one cellodextrinase (15), four xylanases (21, 52), two acetyl xylan esterases (22), and two cellulose-binding proteins (11) have been purified or cloned and purified and the catalytic properties of the enzymes determined. Although those studies have provided valuable information about many of the cellulases and hemicellulases of F. succinogenes, the mechanism of binding of cells to cellulose and hydrolysis of cellulose has not been solved.Similar to the case with other anaerobic cellulolytic bacteria, adhesion of F. succinogenes cells to cellulose appears to be a prerequisite for rapid and efficient cellulose hydrolysis by this organism (30). This claim is supported by the observation that carboxymethylcellulose, which blocks adhesion to cellulose, also blocked cellulose degradation (25). Gong and Forsberg (12) isolated nonadherent mutants of F. succinogenes S85 tha...

The polysaccharides from the outer membrane of the Gramnegative ruminal bacterium Fibrobacter succinogenes were isolated by phenol/water extraction and separated by sizeexclusion chromatography in the presence of deoxycholate detergent into a lower-molecular-mass fraction designated glycolipid' and a high-molecular-mass`capsular polysaccharide' fraction. Both fractions lacked typical lipopolysaccharide components including 2-keto-3-deoxyoctulosonic acid and 3-hydroxy fatty acids. Carbohydrate components of these fractions were represented by two polysaccharides and one oligosaccharide (possibly glycolipid) with the following structures:where HEAEP is N-(2-hydroxyethyl)-2-aminoethylphosphonic acid, found for the first time in natural compounds. The polysaccharides contained pentadecanoic acid and anteisopentadecanoic acid, possibly present as the acyl components. All constituent monosaccharides except l-rhamnose had a d-configuration.In addition to having a structural role in the outer membrane, these polysaccharides may provide protection for this lipopolysaccharide-less bacterium in the highly competitive ruminal environment, as phosphonic acids covalently linked to membrane polymers have in the past been attributed the function of stabilizing membranes in the presence of phosphatases and lipases.Keywords: Fibrobacter succinogenes; glycolipid; NMR; polysaccharide; structure.The outer membrane (OM) of Gram-negative bacteria serves as a permeability barrier to a wide variety of compounds [1]. Types of compounds excluded are hydrophobic antibiotics (e.g. macrolides, novobiocins, rifamycins, actinomycin D, fusidic acid), detergents (e.g. bile salts, SDS, Triton X-100), and hydrophobic dyes (e.g. eosin, methylene blue, brilliant green acridine dyes).Lipopolysaccharide molecules are located on the outer leaflet of the OM and provide the key barrier function, low fluidity, and tight architecture of the OM [1,2]. In addition to barrier functions, lipopolysaccharide has a role in the adhesion of bacteria to various mammalian cell types [3]. Gram-negative ruminal bacteria live in the highly competitive intestinal environment containing varying concentrations of fatty acids, bacteriocins [4] and other types of inhibitory molecules [5,6].Fibrobacter succinogenes S85 is a highly cellulolytic Gram-negative ruminal bacterium. It has been shown to adhere to substrates by means of extracellular polymeric material (glycocalyx) [7]. The nature and composition of this coat has never been investigated. However, being a Gram-negative organism, it may be composed of cell surface capsular polysaccharide (CPS), lipopolysaccharide, or a combination of the two. Information on the structure and biology of the surface polysaccharides from this bacterium will contribute to our understanding of the role of these exopolysaccharides.In this study, we isolated and purified the polysaccharides associated with the OM of F. succinogenes S85 and characterized their structures. Eur. J. Biochem. 268, 3566±3576 (2001) ); Kdo, 3-deoxy-d-manno-oct-2-u...

Fibrobacter succinogenes possesses seven cellulose-binding proteins (CBPs) of 40,45,50, 120, 180,220, and 240 kDa. The 120-, 180-, 220-, and 240-kDa proteins were present in the outer membrane (OM), while the 40-, 45-, 50-, and 120-kDa proteins were either periplasmic or peripheral membrane proteins. The 120-kDa CBP, which was identified as endoglucanase 2, was a major component in both the OM and periplasm. Zymogram analysis for glucanases showed that the major membrane-associated CBPs, with the exception of endoglucanase 2, lacked endoglucanase activity. Affinitypurified antibodies against the 180-kDa CBP cross-reacted strongly with numerous cell envelope proteins of higher and lower molecular mass, including the previously characterized chloride-stimulated cellobiosidase. Treatment of the 180-kDa CBP and cell envelope proteins with periodate resulted in almost complete loss of antibody binding, suggesting that they possessed a common epitope that was carbohydrate in nature. Immunogold labelling of whole cells using antibodies against the 180-kDa CBP demonstrated that either the 180-kDa CBP or related proteins with a cross-reactive epitope were located at the cell surface. These epitopes were distributed uniformly on cells not bound to cellulose but congregated on the cell surface at sites of adhesion of cells to cellulose. Antibodies to the 180-kDa protein caused 62% inhibition of binding of F. succinogenes to crystalline cellulose, which provides evidence that either the 180-kDa CBP and (or) other related cross-reactive surface proteins have a role in adhesion to cellulose.Rksumk : Fibrobacter succinogenes posskde sept protCines se liant 8 la cellulose (CBPs) de 40,45,50, 120, 180,220 et 240 kDa. Les protkines de 120, 180,220 et 240 kDa Ctaient prCsentes dans la paroi exteme alors que les protCines de 40,45, 50 et 120 kDa Ctaient pCriplasmiques ou sit6es 8 la pCriphCrie de la membrane. La CBP de 120 kDa, qui a Ct C identifiCe comme l'endoglucanase 2, Ctait une composante majeure de la paroi exteme et du pCriplasme. Un zymogramme des glucanases a dkmontre, qu'8 l'exception de l'endoglucanase 2, les CBPs majeures assocites ? I la membrane ne posskdaient pas d'activitt endoglucanase. Des anticorps dirigCs contre la CBP de 180 kDa reagissaient fortement de faqon croisCe avec de nombreuses protkines de l'enveloppe cellulaire de haut et bas masse molCculaire, incluant la cellobiosidase induite par le chlore. Le traitement de la CBP de 180 kDa et des protkines de l'enveloppe cellulaire avec le periodate a rCsult6 en une perte presque totale de la liaison de l'anticorps, ce qui suggbre que ces protkines possbdent un Cpitope commun constituC d'hydrates de carbone. Le marquage immunologique B l'or colloidal des cellules complbtes en utilisant des anticorps spCcifiques pour la CBP de 180 kDa a dCmontrC que cette CBP ou des protCines relikes qui possbdent un Cpitope responsable d'une rCaction croisCe Ctaient situCes B la surface bactkrienne. Ces Cpitopes Ctaient distribuks uniformCment sur les bactkries non likes 8 la ce...

WbpO is associated with B-band lipopolysaccharide biosynthesis in Pseudomonas aeruginosa serotype O6. This protein is thought to catalyze the enzymatic conversion of UDP-N-acetyl-D-galactosamine (UDP-GalNAc) to UDP-N-acetyl-D-galactosaminuronic acid (UDP-GalNAcA). WbpO was overexpressed with a C-terminal hexahistidine tag. The soluble form of expressed WbpO (WbpO Sol ) exhibited a secondary structure with 29.2% ␣-helix and 20.1% ␤-strand. However, no enzymatic activity could be detected using either high performance anion exchange chromatography or capillary electrophoresis-mass spectrometry analysis. An insoluble form of expressed WbpO was purified in the presence of guanidine hydrochloride by immobilized metal ion affinity chromatography. After refolding, this preparation of WbpO (designated as WbpO Rf ) exhibited stable secondary structure at pH 7.5 to 8.2, and it was enzymatically active. Capillary electrophoresis-mass spectrometry and tandem mass spectrometry analysis showed that WbpO Rf catalyzed the conversion of UDP-GalNAc to UDP-GalNAcA. 26 and 22% of the substrate could be converted to UDP-GalNAcA in the presence of NAD ؉ and NADP ؉ as the cofactors, respectively. The K m values of WbpO Rf for UDP-GalNAc, NAD ؉ , and NADP ؉ were 7.79, 0.65, and 0.44 mM, respectively. WbpO Rf can also catalyze the conversion of UDP-GlcNAc to UDP-GlcNAcA. In conclusion, this is the first report of the overexpression, purification, and biochemical characterization of an NAD ؉ /NADP ؉ -dependent UDP-GalNAc dehydrogenase. Our results also complete the biosynthetic pathway for GalNAcA that is part of the O-antigen of P. aeruginosa serotype O6 lipopolysaccharide.

Lactate dehydrogenases which convert lactate to pyruvate are found in almost every organism and comprise a group of highly divergent proteins in amino acid sequence, catalytic properties, and substrate specificity. While the L-lactate dehydrogenases are among the most studied enzymes, very little is known about the structure and function of D-lactate dehydrogenases (D-LDHs) which include two discrete classes of enzymes that are classified based on their ability to transfer electrons and/or protons to NAD in NAD-dependent lactate dehydrogenases (nLDHs), and FAD in NAD-independent lactate dehydrogenases (iLDHs). In this study, we used a combination of structural and phylogenomic approaches to reveal the likely evolutionary events in the history of the recently described FAD binding oxidoreductase/transferase type 4 family that led to the evolution of D-iLDHs (commonly referred as DLD). Our phylogenetic reconstructions reveal that DLD genes from eukaryotes form a paraphyletic group with respect to D-2-hydroxyglutarate dehydrogenase (D2HGDH). All phylogenetic reconstructions recovered two divergent yeast DLD phylogroups. While the first group (DLD1) showed close phylogenetic relationships with the animal and plant DLDs, the second yeast group (DLD2) revealed strong phylogenetic and structural similarities to the plant and animal D2HGDH group. Our data strongly suggest that the functional assignment of the yeast DLD2 group should be carefully revisited. The present study demonstrates that structural phylogenomic approach can be used to resolve important evolutionary events in functionally diverse superfamilies and to provide reliable functional predictions to poorly characterized genes.