Homologous and Heterologous Overexpression in Clostridium acetobutylicum and Characterization of Purified Clostridial and Algal Fe-Only Hydrogenases with High Specific Activities

Clostridium acetobutylicum ATCC 824 was selected for the homologous overexpression of its Fe-only hydrogenase and for the heterologous expressions of the Chlamydomonas reinhardtii and Scenedesmus obliquus HydA1 Fe-only hydrogenases. The three Strep tag II-tagged Fe-only hydrogenases were isolated with high specific activities by two-step column chromatography. The purified algal hydrogenases evolve hydrogen with rates of around 700 μmol H₂ min⁻¹ mg⁻¹, while HydA from C. acetobutylicum (HydA_Ca) shows the highest activity (5,522 μmol H₂ min⁻¹ mg⁻¹) in the direction of hydrogen uptake. Further, kinetic parameters and substrate specificity were reported. An electron paramagnetic resonance (EPR) analysis of the thionin-oxidized HydA_Ca protein indicates a characteristic rhombic EPR signal that is typical for the oxidized H cluster of Fe-only hydrogenases.     

Karyotype variation inAllium commutatum (Liliaceae s. lato)

Chromosome numbers (2×, 3×, 4×), B-chromosomes, and variation among marker chromosomes inAllium commutatum Guss. was studied. These different chromosomal types exhibit distinct distribution patterns and a centre of variation has been located in the central Aegean area. A common origin ofA. commutatum, the Aegean endemicA. bourgeaui Rech. fil.,A. ampeloprasum L. and the cultivatedA. porrum L. is discussed.     

Structure elucidation of the core regions from Citrobacter O4 and O36 lipopolysaccharides by chemical and enzymatic methods, gas chromatography/mass spectrometry, and NMR spectroscopy at 500 MHz

Novel enterobacterial core oligosaccharides were isolated from Citrobacter O4 and O36 lipopolysaccharides, and their structures were determined by methylation analysis, Smith degradation and enzymatic degradations, gas chromatography/mass spectrometry, and two-dimensional phase-sensitive correlated, relayed coherence transfer, double-quantum, triple-quantum-filtered, and nuclear Overhauser effect (NOE) 1H NMR spectroscopy at 500 MHz. In the formulas, all hexose residues are D-hexopyranoses, and heptoses are L-glycero-D-manno-heptopyranoses; the alternative locations of the side-chain heptose and pyrophosphorylethanolamine (PPEtN) residues are marked by dashed lines; dOclA stands for 3-deoxy-D-manno-octulosonic acid. (formula; see text) Along with these complete cores, incomplete ones, lacking the hexosamine trisaccharides, occur in the lipopolysaccharides of both types. Qualitative NOE data were in good agreement with the minimum energy conformation of the above O36 oligosaccharide, calculated with the aid of the SUGAR program [Sundin, A., Carter, R. E., & Liljefors, T. (1988) J. Mol. Graphics (in press)].     

Interaction between phosphofructokinase and aldolase from Saccharomyces cerevisiae studied by aqueous two-phase partitioning

Phosphofructokinase (EC 2.7.1.11) and aldolase (EC 4.1.2.13) have been highly purified from Saccharomyces cerevisiae by improved protocols. Partitioning of the enzymes in aqueous polymer two-phase systems was used to detect complex formation. The partition of each enzyme was found to be affected by the presence of the other enzyme. AMP affected the partition of the individual enzymes as well as the mixture of the two. The activities of the respective enzymes were stimulated in the putative complex in an AMP-dependent manner. Two strictly conserved residues belonging to an acidic surface loop of class II aldolases, are a potential site for electrostatic interaction with the positively charged regions close to the active site in phosphofructokinase.     

Novel structural insights into rotavirus recognition of ganglioside glycan receptors

Rotaviruses ubiquitously infect children under the age of 5, being responsible for more than half a million diarrhoeal deaths each year worldwide. Host cell oligosaccharides containing sialic acid(s) are critical for attachment by rotaviruses. However, to date, no detailed three-dimensional atomic model showing the exact rotavirus interactions with these glycoconjugate receptors has been reported. Here, we present the first crystallographic structures of the rotavirus carbohydrate-recognizing protein VP8^? in complex with ganglioside G_M3 glycans. In combination with assessment of the inhibition of rotavirus infectivity by N-acetyl and N-glycolyl forms of this ganglioside, our results reveal key details of rotavirus-ganglioside G_M3 glycan recognition. In addition, they show a direct correlation between the carbohydrate specificities exhibited by VP8^? from porcine and by monkey rotaviruses and the respective infectious virus particles. These novel results also indicate the potential binding interactions of rotavirus VP8^? with other sialic acid-containing gangliosides.     

Effect of pH and the bacteriocin carnocin 54 on growth and cell morphology of two Leuconostoc strains

Leuconostoc carnosum LA54A produces carnocin 54, a bacteriocin inhibitory to Listeria and closely related lactic acid bacteria. The effects of the pH of cell-free LA54 culture supernatants on the antibacterial activity of carnocin 54 was assessed using Leuc. mesenteroides DSM 20343 and TA10C as indicator strains. Carnocin 54 showed greatest activity against both strains at pH 4.5. At pH 6.5, activity was reduced, especially against Leuc. mesenteroides TA10C. Scanning electron microscopy showed irregular and rough surfaces on bacteriocin-treated cells at both pH values.