Culture conditions for stimulating cholera toxin production by Vibrio cholerae O1 El Tor

A method that stimulates cholera toxin (CT) production by Vibrio cholerae O1 biotype El Tor (El Tor vibrios) to the level of several micrograms per ml in the culture fluid was established. Such a large amount of CT was obtained by the following method: El Tor vibrios were cultured in AKI medium (1.5% Bacto peptone, 0.4% yeast extract-Difco, 0.5% NaCl, 0.3% NaHCO3) at 37 C for 4 hr in a stationary test tube and then for 16 hr in a shaken flask, with inoculum sizes of 10(5) to 10(7)/ml. With this method, 35 strains out of 60 examined produced 2 to 16 micrograms/ml of CT as determined by the reversed passive latex agglutination test (RPLA). Thirty-three randomly selected strains out of the 60 produced reasonable amounts of rabbit skin vascular permeability factor, reflecting the amount of CT titrated with RPLA.     

Sialylation of glycoprotein oligosaccharides with N-acetyl-, N-glycolyl-, and N-O-diacetylneuraminic acids

Four common sialic acids (Sia), NeuAc, N-glycolyl-neuraminic acid (NeuGc), 4-O-acetyl-N-acetylneuraminic acid (4-O-Ac-NeuAc), and 9-O-Ac-NeuAc were examined for activation to their corresponding CMP-sialic acid conjugates and subsequently for their transfer to glycoprotein oligosaccharides by purified mammalian sialyltransferases. CMP-sialic acid synthetases from calf brain and from bovine and equine submaxillary glands were found to convert NeuAc, NeuGc, and 9-O-Ac-NeuAc to their corresponding CMP-sailic acids. In contrast, no conversion of 4-O-Ac-NeuAc to CMP-4-O-Ac-NeuAc was observed for any of the three synthetases examined. A new procedure for the preparation of CMP-9-O-Ac-NeuAc, CMP-NeuGc, and CMP-NeuAc in high yield and purity was developed, using the calf brain CMP-sialic acid synthetase. Each of these derivatives was tested as donor substrates for six mammalian sialyltransferases purified from porcine, rat, and bovine tissues, including a bovine GalNAc alpha 2,6 sialyltransferase whose purification is described in this report. The sialyltransferases examined represent those which form the Sia alpha 2,6Gal beta 1,4-GlcNAc-, Sia alpha 2,3Gal beta 1,3(4)GlcNAc-, Sia alpha 2,3Gal beta 1,3-GalNAc- and Sia alpha 2,6GalNAc- sequences found on N-linked and O-linked oligosaccharides of glycoproteins. CMP-NeuAc and CMP-NeuGc were equally good donor substrates for all six sialyltransferases. However, transfer of 9-O-Ac-NeuAc from CMP-9-O-Ac-NeuAc varied from only 10% to nearly 70% that of the transfer of NeuAc from CMP-NeuAc. Results are viewed to define the relative roles of direct transfer of these sialic acids and modification of glycosidically bound NeuAc in glycoproteins.     

Acetyl-coenzyme A:polysialic acid O-acetyltransferase from K1-positive Escherichia coli. The enzyme responsible for the O-acetyl plus phenotype and for O-acetyl form variation

The capsular polysaccharide of Escherichia coli K1 is a linear polymer of N-acetylneuraminic acid in alpha-2,8 linkage. Certain substrains of E. coli K1 (designated OAc+) modify the polysaccharide by O-acetylation of the sialic acids. We demonstrate here an acetyl-coenzyme A: polysialosyl O-acetyltransferase activity that is found only in E. coli K1 OAc+ substrains. When form variation between the O-acetyl-positive and -negative states occurred in strain D698:K1, the fluctuations were accompanied by appropriate changes in the expression of enzyme activity. Thus, expression of this enzyme can account for the OAc+ phenotype and for the form variation between OAc+ and OAc-. The enzyme was solubilized in nonionic detergent and freed of endogenous acceptor activity by DEAE-cellulose chromatography, and its general properties were determined. Analysis of the reaction product showed a highly preferential acetylation reaction that was confined to polysialosyl units of greater than 14 residues. Acetyl groups were shown to be transferred to both the 7- and the 9-positions of the sialic acid residues. The partially purified enzyme was stable even after prolonged incubation at 57 degrees C. In contrast, any further purification resulted in loss of activity, even at 4 degrees C. Treatment of the stable enzyme with a polysialic acid-specific endoneuraminidase caused a similar loss of enzyme stability. This effect of the endoneuraminidase could be protected against by the addition of exogenous polysialic acid. This indicates that the partially purified enzyme contains traces of endogenous polysialic acid substrate that are required for the stability of the enzyme. Finally, the enzyme can O-acetylate the polysialic acid chains on the eucaryotic protein neural cell adhesion molecule, suggesting that enzymatic recognition of the substrate requires only the polysialic acid sequence.     

Wildlife detector dogs and camera traps: a comparison of techniques for detecting feral cats

A major challenge in controlling overabundant wildlife is monitoring their populations, particularly as they decline to very low density. Camera traps and wildlife detector dogs are increasingly being used for this purpose. We compared the cost-effectiveness of these two approaches for detecting feral cats (Felis catus) on two pastoral properties in Hawke's Bay, North Island, New Zealand. One property was subject to intensive pest removal, while the other had no recent history of pest control. Camera traps and wildlife detector dogs detected cats at similar rates at both sites. The operating costs of each method were also comparable. We identify a number of advantages and disadvantages of each technique, and suggest priorities for further research.     

Targeted disruption of genes for gp138, a cell-fusion-related protein in Dictyostelium discoideum, revealed the existence of a third gene

The sexual cycle of the cellular slime mold, Dictyostelium discoideum , offers a suitable experimental system to analyze sexual cell interactions. We have been analyzing molecular mechanisms involved in sexual cell fusion using complementary heterothallic strains in D. discoideum and have identified several cell surface proteins involved in the process. One of them, gp138 is present in strains of both mating types and considered to be responsible for membrane fusion itself. Two genes with high mutual homology, GP 138 A and GP 138 B , have been identified so far as encoding this protein. Expression of antisense RNA for GP 138 B has been shown to suppress sexual cell fusion, confirming the critical importance of these genes in sexual cell fusion. However, neither the functional relationship of the two gp138 genes nor the possibility of the existence of more genes that encode gp138 has been determined yet. In the present study, GP 138 A and GP 138 B were disrupted by homologous recombination in an effort to clarify these points. Analysis of the double knock-out mutants suggested the presence of a third gene for gp138.     

Characterization of Vibrio cholerae O139 Synonym Bengal Isolated from Patients with Cholera-Like Disease in Bangladesh

Vibrio cholerae O139 (synonym Bengal), a novel serovar of V. cholerae, is the causative agent of large outbreaks of cholera-like illness currently sweeping India and Bangladesh. Eight randomly selected V. cholerae O139 isolates were studied for their biological properties, which were compared with those of V. cholerae O1 and other V. cholerae non-O1. The V. cholerae O139 isolates were characterized by the production of large amount of cholera toxin, hemagglutination, weak hemolytic properties, resistance to polymyxin B, lysogeny with, and production of, kappa type phage (4/8 isolates only), and resistance to both classical and El Tor-specific phages. Thus, V. cholerae O139 isolates had an overall similarity with V. cholerae O1 El Tor.     

Detection of Legionella spp. in cooling tower water by the polymerase chain reaction method.

The presence of Legionella spp. in cooling tower water was investigated by using the polymerase chain reaction. Total Legionella spp. detection was performed with 20-mer 5S rRNA complementary DNA sequence primers, and specific Legionella pneumophila detection was performed with 20-mer and then 21-mer macrophage infectivity potentiator gene sequence primers. Of 27 cooling tower water samples, 25 were positive for Legionella spp., and 14 of these contained L. pneumophila.     

Purification and some properties of the citrate synthase from a marine Pseudomonas.

Citrate synthase (citrate-oxaloacetate lyase (CoA acetylating), EC 4.1.3.7) has been purified to electrophoretic homogeneity from a marine Pseudomonas. The enzyme was made up of identical subunits, with a molecular wieght of about 53 000, as determined by sodium dodecyl sulphate - polyacrylamide gel electrophoresis. The native enzyme (citrate synthase II, CS II) could be dissociated by dialysis against 20 mM phosphate (Pi), pH 7; the enzyme thus obtained (citrate synthase I, CS I) was still active, but presented different molecular weight and kinetic and regulatory properties. CS II was activated by adenosine monophosphate (AMP), Pi, and KCl, and inhibited by reduced nicotinamide adenine dinucleotide (NADH), being apparently insensitive to adenosine triphosphate (ATP) and adenosine diphosphate (ADP). The inhibition by NADH was completely counteracted by 0.1 mM AMP, but not by 50 mM Pi or 0.1 M KCl. The activation by KCl and Pi, or by KCl and AMP was nearly additive, whereas that by AMP and Pi was not. The activators acted essentially by increasing Vmax, although they also caused a decrease in the Km values. CS I was inhibited by ATP, ADP, AMP, and KCl, and was insensitive to NADH. CS I could be reassociated after elimination of Pi by dialysis, regaining the higher molecular weight and the activation by AMP characteristic of CS II.