Inactivation of the streptokinase gene prevents Streptococcus equisimilis H46A from acquiring cell-associated plasmin activity in the presence of plasminogen

Abstract The effects of some physico-chemical parameters on production of extracellular α-L-arabinofuranosidase by Aspergillus nidulans were examined. Highest levels of α-L-arabinofuranosidase were generated with cultures grown on 1% (w/v) purified beet pulp arabinan at 30°C and at an initial pH of 7.0. The enzyme was shown to be very sensitive to the action of proteases. Zymogram overlay of a protein profile obtained by SDS-PAGE revealed the occurrence of a band ( M _r 36 000) exhibiting α-L-arabinofuranosidase activity. The isoelectric pH of the enzyme lay near 4.3. Temperature and pH optima for the activity of crude α-L-arabinofuranosidase preparations were 55°C and 5.5, respectively. Enzyme activity was greatly reduced by thiol reagents such as Hg²⁺ and p -hydroxymercuribenzoate and showed a K _m value of 2.7 mM on p -nitrophenyl α-L-arabinofuranoside as substrate.     

Purification and properties of poly(3-hydroxyvaleric acid) depolymerase from Pseudomonas lemoignei

During growth on poly(3-hydroxyvaleric acid), P(3HV), or valerate Pseudomonas lemoignei secretes a P(3HV) depolymerase. This P(3HV) depolymerase was purified from the culture medium of valerate-grown cells by ammonium sulphate precipitation, chromatography on DEAe-sephacel and CM-Sepharose CL 6B. The relative molecular masses of the native as well as the sodium dodecyl sulphate (SDS)-treated enzyme were 53 000 or 54 000, respectively. In contrast to the poly(3-hydroxybutyric acid), P(3HB), depolymerase of Comamonas sp. and P(3HB) depolymerases A and B of P. lemoignei, which are specific for the hydrolysis of P(3HB), the purified P(3HV) depolymerase hydrolysed P(3HB), P(3HV) and co-polymers of 3-hydroxybutyric acid and 3-hydroxyvaleric acid at similar rates. Poly(hydroxyalkanoic acids), consisting of monomers with six and more carbon atoms or substrates characteristic for lipases such as Tween 80 or triolein were not hydrolysed. Maximum activities were measured in 50mm TRIS-HCl buffer, pH 8.0, at 55° C. The apparent K _m values of the purified P(3HV) depolymerase for P(3HB) and P(3HV) were 77 and 65 g polyester/ml, respectively. As the main product of enzymatic hydrolysis of P(3HV), 3-hydroxyvalerate was identified. The depolymerase was insensitive to p-hydroxymercuribenzoate but sensitive to dithioerythritol and phenylmethylsulphonyl fluoride, indicating the absence of active reduced sulphur groups and the presence of essential disulphide bonds and serine residues. Correspondence to: D. Jendrossek     

Evidence for the Existence of Differential O-Glycosylated α5-Subunits of the γ-Aminobutyric AcidA Receptor in the Rat Brain

Abstract: Polyclonal antibodies were raised to synthetic peptides having amino acid sequences corresponding with the N- or C-terminal part of the γ-aminobutyric acid_A (GABA_A) receptor α₅-subunit. These anti-peptide α₅(2–10) or anti-peptide α₅(427–433) antibodies reacted specifically with GABA_A receptors purified from the brains of 5–10-day-old rats in an enzyme-linked immunosorbent assay and were able to dose-dependently immunoprecipitate up to 6.3 or 13.1% of the GABA_A receptors present in the incubation, respectively. In immunoblots, each of these antibodies reacted with the same two protein bands with apparent molecular mass of 53 or 57 kDa. After exhaustive treatment of purified GABA_A receptors with N -Glycanase, each of these antibodies identified two proteins with apparent molecular masses of 46 and 48 kDa. Additional treatment of GABA_A receptors with neuraminidase and O -Glycanase resulted in an apparently single protein with molecular mass of 47 kDa, which again was identified by both the anti-peptide α₅(2–10) and the anti-peptide α₅(427–433) antibody. These results indicate the existence of at least two different α₅-sub-units of the GABA_A receptor that differ in their carbohydrate content. In contrast to other α- or β-subunits of GABA_A receptors so far investigated, at least one of these two α₅-subunits contains O-linked carbohydrates.     

A novel type of meso-diaminopimelic acid-based peptidoglycan and novel poly (erythritol phosphate) teichoic acids in cell walls of two coryneform isolates from the surface flora of French cooked cheeses

The primary structure of the peptidoglycan and the teichoic acids of two coryneform isolates from the surface flora of French cooked cheeses, CNRZ 925 and CNRZ 926, have been determined. In the peptidoglycan, meso-diaminopimelic acid was localized in position three of the peptide subunit. It contained an d-glutamyl-d-aspartyl interpeptide bridge, connecting meso-diaminopimelic acid and d-alanine residues of adjacent peptide subunits. The -carboxyl group of d-glutamic acid in position two of peptide subunits was substituted with glycine amide. The teichoic acid pattern and composition differed between the strains: both contained an erythritol teichoic acid and strain CNRZ 925 also contained an N-acetylglucosaminylphosphate polymer. The erythritol teichoic acids differed in terms of the quality and quantity of substituents, but they both had N,N-diacetyl-2,3-diamino-2,3-dideoxyglucuronic acid in common.Abbreviations DNP dinitrophenyl - Ery erythritol - Gal galactose - GlcN glucosamine - GlcNAc N-acetylglucosamine - GlcUANAc₂ N,N-diacetyl-2,3-diamino-2,3-dideoxyglucuronic acid - Hex UANAc₂ N,N-diacetyl-2,3-diamino-2,3-dideoxyhexuronic - acid m-Dpm, meso-diaminopimelic acid - Mur muramic acid - MurNAc N-acetylmuramic acid     

2-KETO-SUGAR ACIDS IN GREEN FLAGELLATES: A CHEMICAL MARKER FOR PRASINOPHYCEAN SCALES1,2

The chemical composition of cell walls (thecae) of three taxa of scaly green flagellates (Prasinophyceae) was investigated. The theca of Tetraselmis striata, Tetraselmis tetrathele, and Scherffelia dubia consists mainly of carbohydrate (80% of dry weight), with proteins (5%), calcium (4%), and sulfate (6%) as minor components. The principal sugars (60% of dry weight) are the 2-keto-sugar acids 3-deoxy-manno-2-octulosonic acid (KDO), 3-deoxy-manno-5-O-methyl-2-octulosonic acid (5OMeKDO), and 3-deoxy-lyxo-2-heptulosaric acid (DHA). Arabinose, gulose, galactose, galacturonic acid, and in S. dubia, xylose and rhamnose were also found. Examination of scale preparations from Mantoniella squamata, Mesostigma viride, Pyramimonas amylifera, and Nephroselmis olivacea revealed that the 2-keto-sugar acids were always associated with the presence of typical prasinophycean scales on the cell surface. In contrast, 2-keto-sugar acids were not detected in the cell wall of Chlamydomonas reinhardtii nor in polymer preparations from the culture medium of Chlamydomonas reinhardtii, Dunaliella bioculata, Dunaliella primolecta, Asteromonas gracilis, Hafniomonas reticulate, Pedinomonas tuberculata, Monomastix sp., and Micromonas pusilla. We conclude that 2-keto-sugar acids are chemical markers for prasinophycean scales.     

CROSSING EXPERIMENTS,LIPID COMPOSITION,AND THE SPECIES CONCEPT IN ECTOCARPUS SILICULOSUS AND E. FASCICULATUS (PHAEOPHYCEAE,ECTOCARPALES)1

Sporophytes of Ectocarpus siliculosus (Dillwyn) Lyngbye and E. fasciculatus Harvey were collected in the vicinity of Roscoff Brittany, France. Gametophytes derived from meiospores were used for intra- and interspecific crosses. Intraspecific gamete combinations gave viable zygotes, which developed into fertile sporophytes. Interspecific crosses were unsuccessful. Gamete fusions did not occur between female gametes of E. fasciculatus and male gametes of E. siliculosus. Hybrid zygotes were formed in the reciprocal combination but died soon after germination. We conclude that the two species of Ectocarpus at Roscoff represent distinct taxonomic entities, which are separated by pre- and postzygotic compatibility barriers. These biological findings are confirmed by the differential occurrence of the chemotaxonomic marker betaine-lipid diacetylglycerylhydroxymethyltrimethyl-β-alanine, which is present in our cultures of E. fasciculatus but absent in E. siliculosus.     

Ti plasmid-encoded octopine and nopaline catabolism in Agrobacterium: specificities of the LysR-type regulators OccR and NocR, and protein-induced DNA bending

The occ and noc regions in octopine and nopaline Ti plasmids, respectively, are responsible for the catabolism of octopine and nopaline in Agrobacterium. The functions are activated in the presence of the opines by OccR and NocR, two related regulatory proteins, and the promoters contain common sequence motifs. We have investigated heterologous interactions between the regulators and the promoters. Previous experiments using all possible heterologous combinations of opines, regulators, and promoters in vivo had demonstrated that only the combination of nopalme, NocR, and the occ promoter led to limited promoter activation. We now show that OccR and NocR bind to the heterologous promoters in vitro and in vivo. The weak or non-existent promoter activation actually observed could be explained by the assumption that OccR and NocR use different activation mechanisms; we investigated protein-induced DNA bending because of reports that the two regulators differ in this respect. Analysis with a bending vector showed that both OccR and NocR induced a DNA bend that is relaxed in the presence of the respective opine. The data suggest that subtle differences in regulator/promoter interactions are responsible for the inactivity of the heterologous combinations. Investigations with a chimeric NocR/OccR protein indicated that it induced a DNA bend in both promoters. No opine-induced relaxation was detectable with the hybrid, and the inducible promoter was not activated. These findings suggest that bend relaxation may be an integral part of promoter activation.