Pseudomonas putida Mutants Defective in the Metabolism of the Products of meta Fission of Catechol and Its Methyl Analogues

A selection procedure is described which was used to isolate mutants of Pseudomonas putida strain U in the following enzymes of the meta-fission pathway of phenol and cresols: hydrolase, tautomerase, and decarboxylase. Strains deficient in the hydrolase are unable to use either o- or m-cresol as a sole carbon source and were shown to accumulate 2-hydroxy-6-keto-2,4-heptadienoate when incubated in the presence of o- or m-cresol. When 2-hydroxymuconic semialdehyde (plus nicotinamide adenine dinucleotide, oxidized form) was metabolized by phenol-induced extracts of tautomerase-deficient strains, the enol tautomer of 4-oxalocrotonate accumulated and was then converted slowly to the keto tautomer by a nonenzymatic reaction. Phenol-induced extracts of decarboxylase-deficient strains accumulated the keto tautomer of 4-oxalocrotonate from 2-hydroxymuconic semialdehyde (plus nicotinamide adenine dinucleotide, oxidized form). Strains with an inactive decarboxylase are unable to completely metabolize either phenol or p-cresol. Tautomerase-defective strains are unable to grow with p-cresol as the sole carbon source and grow only very slowly on phenol.     

Pyridoxal phosphate-sensitized photoinactivation of glutamate decarboxylase from Clostridium perfringens

1. l-Glutamate decarboxylase (EC 4.1.1.15) from Clostridium perfringens was inactivated by exposure to visible light at pH6.2. 2. Inactivation does not occur at pH4.6 or in the absence of bound pyridoxal phosphate. 3. On prolonged photo-oxidation six histidine residues per molecule of enzyme were destroyed. 4. The loss of six cysteine residues per molecule occurred both in irradiated samples and in controls oxygenated in the dark. 5. This dark-oxidation of cysteine residues is apparently required before the photo-oxidation process. 6. The absorbance, fluorescence and circular-dichroism properties of the enzyme as well as its elution volume during Sephadex gel-filtration were unaffected by prolonged irradiation. 7. However, an apparently homogeneous product of photo-oxidation could be separated from the control enzyme by ion-exchange chromatography. 8. The K(m) for l-glutamate was unchanged in an irradiated sample retaining 22% of control activity. 9. These data and the catalytic role of imidazole residues at the active sites of amino acid decarboxylases are discussed.     

Morphogenesis of Newcastle Disease Virus in Chorioallantoic Membrane

Chick embryo chorioallantoic membrane, infected with the Blacksburg strain of Newcastle disease virus, was examined with an electron microscope to investigate the sequence of viral-induced host cell alterations. These were evident mostly in the endodermal epithelial cells lining the allantoic sac and were divided arbitrarily into three stages. Stage 1 was characterized by commencement of cell hypertrophy and hyperplasia and presence of fewer cytoplasmic inclusion bodies normally found in the cells; in stage 2, juxtanuclear nucleocapsid-glycogen aggregates appeared, and there were increased numbers of microvilli; stage 3 was characterized by increased cytoplasmic density and evidence of viral assembly and release. The morphological features of viral assembly and the virion are also described.