Molecular cloning and functional expression of bacteriophage PK1E-encoded endoneuraminidase Endo NE

Homopolymeric α-2,8-linked sialic acid (PSA) has been found as a capsular component of sepsis- and meningitis-causing bacterial pathogens, and on eukaryotic cells as a post-translational modification of the neural cell adhesion molecule (NCAM). The polysaccharide is specifically recognized and degraded by a phage-encoded enzyme, the endo-N-acetylneuraminidase E (Endo NE). Endo NE therefore has become a valuable tool in the study of bacterial pathogenesis and eukaryotic morphogenesis. In this report we describe the molecular cloning of Endo NE and the expression of a functionally active recombinant enzyme. The cloned DNA sequence (2436 bp) encodes a polypeptide of 811 amino acids, which at the 5′ end contains a totally conserved neuraminidase motif. Expressed in Escherichia coli, the enzyme migrates as a single band of approximately 74 kDa in SDS-PAGE. A central domain of 669 amino acid residues is about 90% homologous to the recently cloned Endo NF. Both phage-induced lysis of bacteria and the catalysis of PSA degradation by the recombinant enzyme are efficiently inhibited by a polyclonal antiserum raised against the intact phage particle. The C-terminal region seems to be essential to enzymatic functions, as truncation of 32 amino acids outside the homology domain completely abolishes Endo NE activity. Our data also indicate that the 38 kDa protein, previously assumed to be a subunit of the Endo NE holoenzyme, is the product of a separate gene locus and is not necessary for in vitro depolymerase activity.     

A new anaerobic,sporing, acetate-oxidizing,sulfate-reducing bacterium,Desulfotomaculum (emend.) acetoxidans

Archives of Microbiology - A new strictly anaerobic, polarly flagellated, sporing, acetate-oxidizing, sulfate-reducing bacterium was isolated from anaerobic fresh or sea water mud samples. The...     

Physiology and biochemistry of streptomycetes. XIII. Biosynthesis of paromomycin by Streptomyces albus var. metamycinus nov. var. supplied with 14C-glucose, 14C-glucosamine, 14C-2-desoxystreptamine and 14C-ribose

Distribution of radioactivity in paromomycin ascertained after application of 14C-D-glucose, 14C-D-glucosamine, 14C-2-deoxystreptamine, respectively, 14C-D-ribose is taken as basis for a biosynthesis scheme: While ribose bound in the antibiotic originates from glucose by oxidation and following decarboxylation, glucosamine is formed via fructose-6-phosphate. Paromose I arises from glucosamine, but not the cyclohexan derivative 2-deoxystreptamine, whose biosynthesis pathway is directly branching off glucose.     

Indole-3-acetic acid levels after phytochrome-mediated changes in the stem elongation rate of dark- and light-grownPisum seedlings

The effect of red (R) and far-red (FR) light on stem elongation and indole-3-acetic acid (IAA) levels was examined in dwarf and tall Pisum sativum L. seedlings. Red light reduced the extension-growth rate of etiolated seedlings by 70–90% after 3 h, and this inhibition was reversible by FR. Inhibition occurred throughout the growing zone. After 3 h of R, the level of extractable IAA in whole stem sections from the growing zone of etiolated plants either increased or showed no change. By contrast, extractable IAA from epidermal peels consistently decreased 3 h after R treatments. Decreases of 40% were observed for epidermal peels from the top 1 cm of tall plants receiving 3 h R. Brief R treatments resulted in smaller decreases in epidermal IAA levels and these decreases were not as great when FR followed R. In lightgrown plants, end-of-day FR stimulated growth during the following dark period in a photoreversible manner. The uppermost 1 cm of expanding third internodes was most responsive to the FR. Extractable IAA from epidermal peels from the upper 1 cm of third internodes increased by 30% or more 5 h after FR. When R followed the FR the increases were smaller. Levels of IAA in whole stem sections did not change and were twofold greater than in dark-grown plants. In both dark- and light-grown tall plants, IAA levels were lower in epidermal peels than in whole stem segments. These results provide evidence that IAA is compartmentalized at the tissue level within the growing stem and that phytochrome regulation of stem elongation rates may be partly based on modulating the level of IAA within the epidermis.Abbreviations IAA indole-3-acetic acid - R red light - FR farred light We thank Yu-Xian Zhu for helping to develop methods for IAA analysis, James Reid for supplying the genetic lines of Pisum and Richard Cyr for the use of microscopy equipment. This work was supported by NSF grant DCB-8801880 and by Hatch funds from the College of Agriculture and Life Sciences at Cornell University. The gas chromatograph-mass spectrometer was funded by NSF grant DMB-8505974 and funds from the College of Agriculture and Life Sciences at Cornell University. A preliminary report of some of these experiments has appeared in Plant Growth Substances, 1991 (Behringer et al. 1992 b).     

Platelet membrane glycoproteins IIb and IIIa are substrates of purified pp60c-src protein tyrosine kinase.

Human platelet glycoproteins IIb and IIIa form the receptor for fibrinogen, von Willebrand factor and fibronectin. Isolated human glycoproteins IIb-IIIa are phosphorylated by purified pp60c-src protein tyrosine kinase. Analysis of the phosphorylated proteins on SDS-PAGE showed that under reducing conditions both phosphoproteins change their relative molecular masses from 135 to 120 kDa and from 97 to 105 kDa, which are characteristic properties of glycoproteins IIb-IIIa. Phosphorylated proteins could be immunoprecipitated with an antiserum against glycoproteins IIb-IIIa but not by control serum. Some kinetic properties of the glycoprotein phosphorylations are also investigated. How the glycoprotein IIb-IIIa complex acquires its receptor activity in stimulated platelets is unknown; however, phosphorylation could be an important mechanism.     

Histidine utilization by Alcaligenes eutrophus: Regulation of histidase formation under heterotrophic conditions of growth

Histidine supported good growth of Alcaligenes eutrophus strain H 16 as a nitrogen source, but only poor growth as a carbon and energy source. The facultative chemolithoautotrophic bacterium was also able to utilize urocanic acid, the first intermediate of histidine catabolism. The products of histidine degradation were ammonium, formate and glutamate. Three enzymes of the pathway, histidase, urocanase and formiminoglutamate hydrolase, were present in histidine-grown cells. Two types of spontaneous mutants, derived from the wild type, were characterized by an increased growth rate on histidine. One of these types was found to produce histidase constitutively and at a higher activity compared with the parental strain. The second type of mutant had apparently gained an improved histidine uptake system, which is supposed to be growth rate-limiting in the wild type. From the physiological studies the conclusion was drawn that the control of histidine-degrading enzymes is based on induction by urocanate and catabolite repression by carbon sources supporting fast growth, such as succinate or pyruvate. Ammonium was found not to affect catabolite repression, however, we obtained evidence that histidine uptake is subject to a nitrogen control.Abbreviation CTAB hexadecyltrimethylammonium bromide     

Biosynthesis of cyclopentenylglycine from α-ketopimelate in Idesia polycarpa callus cultures

The nonproteinogenic amino acid, cyclopentenylglycine, is found in certain Flacourtiaceae. This compound may be synthesized by two C₁-chain elongations of -ketoglutarate via -ketopimelate (C₅+2C₁) or by condensation of C₄ and C₃ units (C₄+C₃), a pathway not involving -ketopimelate. The following experimental design elucidated the biosynthetic pathway: Idesia polycarpa callus cultures were freshly established from leaf petioles; synthetic -[1,2-¹⁴C]ketopimelate was added to the medium and cultures were incubated for 3 weeks. After isolation and separation of free amino acids from the tissues, the radioactivity incorporated into cyclopentenylglycine was determined. The results establish -ketopimelate as a precursor for cyclopentenylglycine, thus providing evidence for the C₅+2C₁ biosynthetic path.     

Jagdgebiet und Ern?hrung der Rohrweihe (Circus aeruginosus) in Schleswig-Holstein

Zusammenfassung Die Ausdehnung des Jagdgebietes der Rohrweihe hängt neben dem Beuteangebot und der Struktur des Gebietes vom Stadium und der Größe der Brut ab. Die Jagdgebietsgröße, ermittelt in einem Mäusejahr an 5 Paaren zur Brutzeit, schwankte zwischen 300–900 ha.1973–1975 wurden an 36 Rohrweihenhorsten 1202 Beutereste gesammelt und ausgewertet. Jagdweise, Beutebehandlung sowie das Auswerten der Beuteaufsammlung werden beschrieben. 58 Wirbeltierarten wurden festgestellt. Der prozentuale Anteil von Säugern und Vögeln an der Beute war in den einzelnen Jahren recht unterschiedlich, während der Anteil der Vögel während zweier Jahre 81 bzw. 75% betrug, sank er in einem Mäusejahr auf ca. 20%. Reptilien, Amphibien und Fische sowie Aas und Vogeleier waren im Untersuchungsgebiet von untergeordneter Bedeutung.Die Frage wird diskutiert, ob Rohrweihen, abgesehen von ihrer angeborenen Vorliebe für Vogeleier, als Nahrungs-Spezialisten zu bezeichnen sind. Ferner werden Hinweise aufgezeigt, die auf große Effizienz beim Nahrungserwerb schließen lassen.

---

Hunting-area and food of the marsh-harrier (Cirus aeruginosus) in Schleswig-Holstein

Summary The hunting-area of five pairs of the Marsh-harrier during the breeding season has been investigated in a year with a graduation of field voles (Microtus arvalis). The area varied between 300 and 900 ha per pair. The size of the area was influenced by food availability, the structure of the countryside, clutch size and the growth of the nestlings.From 1973 to 1975, 1202 prey remnants and pellets were collected from 36 nests. The hunting and feeding behaviour of the birds are described. 58 species of vertebrates were identified. The percentage of mammals and birds in the prey was very different in each year. The proportion of birds during two years was 81% and 75% respectively. In a year with a small mammal gradation it decreased to 20%. Other vertebrates (reptiles, amphibians and fishes), carrion, and bird eggs were of minor importance in the study area.The question is discussed whether marsh-harriers tend to a genuine food specialisation, except for their innate preference for bird eggs. Furthermore hints are given concerning the great efficiency of predation.

     

The isolation of IS1 and IS2 DNA

Summary DNA of the IS-elements IS1 and IS2 was prepared by digestion of appropriate heteroduplex molecules with endonuclease S1, followed by sucrose gradient centrifugation or gel electrophoresis. The material obtained is homogeneous with regard to size. The length of IS1 DNA is 820±65 nucleotides, the length of IS2 DNA is 1.350±70 nucleotides. IS1 DNA is not cleaved by the restriction endonucleases Eco R1, Hind II or Hind III. IS2 DNA is cleaved once by each of the two latter enzymes. The buoyant density determined by equilibrium centrifugation of Hg-complexes in Cs₂SO₄ corresponds to a GC content of approximately 50%. Labelling with polynucleotide kinase indicates that both IS DNA's have a guanosyl residue at both of their 5-termini.