Characterization of terminal NeuNAcalpha2-3Galbeta1-4GlcNAc sequence in lipooligosaccharides of Neisseria meningitidis

Group B and C Neisseria meningitidis are the major cause of meningococcal disease in the United States and in Europe. N . meningitidis lipooligosaccharide (LOS), a major surface antigen, can be divided into 12 immunotypes of which L1 through L8 were found among Group B and C organisms. Groups B and C but not Group A may sialylate their LOSs with N-acetylneuraminic acid (NeuNAc) at the nonreducing end because they synthesize CMP-NeuNAc. Using sialic acid-galactose binding lectins as probes in an ELISA format, six of the eight LOS immunotypes (L2, L3, L4, L5, L7, and L8) in Groups B and C bound specifically to Maackia amurensis leukoagglutinin (MAL), which recognizes NeuNAcalpha2- 3Galbeta1-4GlcNAc/Glc sequence, but not to Sambucus nigra agglutinin, which binds NeuNAcalpha2-6Gal sequence. The combination of SDS-PAGE and MAL-blot analyses revealed that these six LOSs contained only the NeuNAcalpha2-3Galbeta1-4GlcNAc trisaccharide sequence in their 4.1 kDa LOS components, which have a common terminal lacto-N-neotetraose (LNnT, Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc) structure when nonsialylated as shown by previous studies. The LOS-lectin binding was abolished when the LOSs were treated with Newcastle disease viral neuraminidase which cleaves alpha2-->3 linked sialic acid. Methylation analysis of a representative LOS (L2) confirmed that NeuNAc is 2-->3 linked to Gal. Thus, these LOSs structurally mimic certain glycolipids, i.e., paragloboside (LNnT-ceramide) and sialylparagloboside and some glycoproteins in having LNnT and N-acetyllactosamine sequences, respectively, with or without alpha2-->3 linked NeuNAc. The molecular mimicry of the LOSs may play a role in the pathogenesis of N.meningitidis by assisting the organism to evade host immune defenses in man.      

The three transglycosylation reactions catalyzed by cyclodextrin glycosyltransferase from Bacillus circulans (strain 251) proceed via different kinetic mechanisms.

Cyclodextrin glycosyltransferase (CGTase) catalyzes three transglycosylation reactions via a double displacement mechanism involving a covalent enzyme-intermediate complex (substituted-enzyme intermediate). Characterization of the three transglycosylation reactions, however, revealed that they differ in their kinetic mechanisms. Disproportionation (cleavage of an alpha-glycosidic bond of a linear malto-oligosaccharide and transfer of one part to an acceptor substrate) proceeds according to a ping-pong mechanism. Cyclization (cleavage of an alpha-glycosidic bond in amylose or starch and subsequent formation of a cyclodextrin) is a single-substrate reaction with an affinity for the high molecular mass substrate used, which was too high to allow elucidation of the kinetic mechanism. Michaelis-Menten kinetics, however, have been observed using shorter amylose chains. Coupling (cleavage of an alpha-glycosidic bond in a cyclodextrin ring and transfer of the resulting linear malto-oligosaccharide to an acceptor substrate) proceeds according to a random ternary complex mechanism. In view of the different kinetic mechanisms observed for the various reactions, which can be related to differences in substrate binding, it should be possible to mutagenize CGTase in such a manner that a single reaction is affected most strongly. Construction of CGTase mutants that synthesize linear oligosaccharides instead of cyclodextrins thus appears feasible. Furthermore, the rate of interconversion of linear and circular conformations of oligosaccharides in the cyclization and coupling reactions was found to determine the reaction rate. In the cyclization reaction this conversion rate, together with initial binding of the high molecular mass substrate, may determine the product specificity of the enzyme. These new insights will allow rational design of CGTase mutant enzymes synthesizing cyclodextrins of specific sizes.     

Degradation of halogenated aliphatic compounds by Xanthobacter autotrophicus GJ10

A bacterium that is able to utilize a number of halogenated short-chain hydrocarbons and halogenated carboxylic acids as sole carbon source for growth was identified as a strain of Xanthobacter autotrophicus. The organism constitutively produces two different dehalogenases. One enzyme is specific for halogenated alkanes, whereas the other, which is more heat stable and has a higher pH optimum, is specific for halogenated carboxylic acids. Haloalkanes were hydrolyzed in cell extracts to produce alcohols and halide ions, and a route for the metabolism of 1,2-dichlorethane is proposed. Both dehalogenases show a broad substrate specificity, allowing the degradation of bromine- and chlorine-substituted organic compounds. The results show that X. autotrophicus may play a role in the degradation of organochlorine compounds and that hydrolytic dehalogenases may be involved in the microbial metabolism of short-chain halogenated hydrocarbons in microorganisms.     

Export,purification, and activities of affinity tagged <Emphasis Type="Italic">Lactobacillus reuteri</Emphasis> levansucrase produced by <Emphasis Type="Italic">Bacillus megaterium</Emphasis>

Fructosyltransferases, like the Lactobacillus reteri levansucrase, are important for the production of new fructosyloligosaccharides. Various His₆- and Strep-tagged variants of this enzyme were recombinantly produced and exported into the growth medium using the Gram-positive bacterium Bacillus megaterium. Nutrient-rich growth medium significantly enhanced levansucrase production and export. The B. megaterium signal peptide of the extracellular esterase LipA mediated better levansucrase export compared to the one of the penicillin amidase Pac. The combination of protein export via the LipA signal peptide with the coexpression of the signal peptidase gene sipM further increased the levansucrase secretion. Fused affinity tags allowed the efficient one-step purification of the recombinant proteins from the growth medium. However, fused peptide tags led to slightly decreased secretion of tested fusion proteins. After upscaling 2 to 3 mg affinity tagged levansucrase per liter culture medium was produced and exported. Up to 1 mg of His₆-tagged and 0.7 mg of Strep-tagged levansucrase per liter were recovered by affinity chromatography. Finally, the purified levansucrase was shown to synthesize new fructosyloligosaccharides from the novel donor substrates d-Gal-Fru, d-Xyl-Fru, d-Man-Fru, and d-Fuc-Fru. R. Biedendieck and R. Beine contributed equally to this work.     

Linkages among RFLP,RAPD, isozyme,disease-resistance,and morphological markers in narrow and wide crosses of cucumber

An algorithm of automatic classification is proposed and applied to a large collection of perennial ryegrass wild populations from France. This method is based on an ascendant hierarchical clustering using the Euclidian distance from the principal components extracted from the variance-covariance matrix between 28 agronomic traits. A contiguity constraint is imposed: only those pairs of populations which are defined as contiguous are grouped together into a cluster. The definition of contiguity is based on a geostatistical parameter: the range of the variogramme, i.e. the largest distance above which the variance between pairs of population no longer increases. This method yields clusters that are generally more compact than those obtained without constraint. In most cases the contours of these clusters fit well with known ecogeographic regions, namely, for macroclimatic homogeneous conditions. This suggests that selective factors exert a major influence in the genetic differentiation of ryegrass populations for quantitatively inherited adaptive traits. It is proposed that such a method could provide useful genetic and ecogeographic bases for sampling a core collection in widespread wild species such as forage grasses.Institut National de la Recherche Agrononique     

Structural features in the KshA terminal oxygenase protein that determine substrate preference of 3-ketosteroid 9α-hydroxylase enzymes

Rieske nonheme monooxygenase 3-ketosteroid 9α-hydroxylase (KSH) enzymes play a central role in bacterial steroid catabolism. KSH is a two-component iron-sulfur-containing enzyme, with KshA representing the terminal oxygenase component and KshB the reductase component. We previously reported that the KshA1 and KshA5 homologues of Rhodococcus rhodochrous DSM43269 have clearly different substrate preferences. KshA protein sequence alignments and three-dimensional crystal structure information for KshA(H37Rv) of Mycobacterium tuberculosis H37Rv served to identify a variable region of 58 amino acids organized in a β sheet that is part of the so-called helix-grip fold of the predicted KshA substrate binding pocket. Exchange of the β sheets between KshA1 and KshA5 resulted in active chimeric enzymes with substrate preferences clearly resembling those of the donor enzymes. Exchange of smaller parts of the KshA1 and KshA5 β-sheet regions revealed that a highly variable loop region located at the entrance of the active site strongly contributes to KSH substrate preference. This loop region may be subject to conformational changes, thereby affecting binding of different substrates in the active site. This study provides novel insights into KshA structure-function relationships and shows that KSH monooxygenase enzymes are amenable to protein engineering for the development of biocatalysts with improved substrate specificities.     

NepA is a structural cell wall protein involved in maintenance of spore dormancy in Streptomyces coelicolor

Streptomycetes have a complex morphogenetic programme culminating in the formation of aerial hyphae that develop into chains of spores. After spore dispersal, environmental signals trigger dormant spores to germinate to establish a new colony. We here compared whole genome expression of a wild-type colony of Streptomyces coelicolor forming aerial hyphae and spores with that of the chp null mutant that forms few aerial structures. This revealed that expression of 244 genes was significantly altered, among which genes known to be involved in development. One of the genes that was no longer expressed in the Δ chpABCDEFGH mutant was nepA , which was previously shown to be expressed in a compartment connecting the substrate mycelium with the sporulating parts of the aerial mycelium. We here show that expression is also detected in developing spore chains, where NepA is secreted to end up as a highly insoluble protein in the cell wall. Germination of spores of a nepA deletion mutant was faster and more synchronous, resulting in colonies with an accelerated morphogenetic programme. Crucially, spores of the Δ nepA mutant also germinated in water, unlike those of the wild-type strain. Taken together, NepA is the first bacterial structural cell wall protein that is important for maintenance of spore dormancy under unfavourable environmental conditions.     

Multilocus sequence types of Finnish bovine <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> isolates and their attribution to human infections

Background  

Campylobacter jejuni is the most common bacterial cause of human gastroenteritis worldwide. Due to the sporadic nature of infection, sources often remain unknown. Multilocus sequence typing (MLST) has been successfully applied to population genetics of Campylobacter jejuni and mathematical modelling can be applied to the sequence data. Here, we analysed the population structure of a total of 250 Finnish C. jejuni isolates from bovines, poultry meat and humans collected in 2003 using a combination of Bayesian clustering (BAPS software) and phylogenetic analysis.     

Differential Metabolism of Exopolysaccharides from Probiotic Lactobacilli by the Human Gut Symbiont Bacteroides thetaiotaomicron

Probiotic microorganisms are ingested as food or supplements and impart positive health benefits to consumers. Previous studies have indicated that probiotics transiently reside in the gastrointestinal tract and, in addition to modulating commensal species diversity, increase the expression of genes for carbohydrate metabolism in resident commensal bacterial species. In this study, it is demonstrated that the human gut commensal species Bacteroides thetaiotaomicron efficiently metabolizes fructan exopolysaccharide (EPS) synthesized by probiotic Lactobacillus reuteri strain 121 while only partially degrading reuteran and isomalto/malto-polysaccharide (IMMP) α-glucan EPS polymers. B. thetaiotaomicron metabolized these EPS molecules via the activation of enzymes and transport systems encoded by dedicated polysaccharide utilization loci specific for β-fructans and α-glucans. Reduced metabolism of reuteran and IMMP α-glucan EPS molecules may be due to reduced substrate binding by components of the starch utilization system (sus). This study reveals that microbial EPS substrates activate genes for carbohydrate metabolism in B. thetaiotaomicron and suggests that microbially derived carbohydrates provide a carbohydrate-rich reservoir for B. thetaiotaomicron nutrient acquisition in the gastrointestinal tract.