- Volume 75, Issue 1, 1973

SUMMARY: A single-step procedure was developed which enabled the phenol oxidase of Aspergillus nidulans to be recovered in a state of high purity. Ion-exchange chromatography was used to separate the phenol oxidase from an inhibitory protein. Kinetic analysis of the enzyme revealed that it was a tyrosinase (EC. 1.10.3.1) and had a variable ratio of o-hydroxylase to o-diphenol oxidase activity. The enzyme had a relatively low affinity for oxygen (Km of 125 μM) but, compared to other phenol oxidases, a high affinity for its phenolic substrate (Km DOPA of 250 μM). The Aspergillus tyrosinase was heterogeneous and appeared to exist in a monomer (mol. wt 1.3 × 105)-tetramer (5.2 × 105) equilibrium. The protein inhibitor was of similar size to the tyrosinase monomer and caused non-competitive inhibition of the enzyme. Both of the tyrosinase activities and the inhibitor had a substantial component of ribonucleic acid. The RNA was not removed from the enzymes or the inhibitor during purification; non-specific complexes may be formed between these proteins and RNA or true ribonucleoproteins may exist. A model is proposed for the physiological function of tyrosinase in A. nidulans under conditions where secondary metabolism does not occur.

SUMMARY: The mycelial and blastospore forms of Candida albicans have been grown under conditions in which the only environmental variable was temperature. The activity of phosphoglucose isomerase, phosphofructokinase and the first enzyme of the hexose-monophosphate pathway and the pathways for chitin and mannan synthesis has been determined in extracts at different times in the cell cycle. Phosphofructokinase has been partially purified from both forms and the effect of adenosine phosphates on its activity determined. In this yeast, concentrations of glucose-6-phosphate, fructose-6-phosphate, ATP, ADP and AMP differed in the two growth forms and at different times in the growth cycle. L-Glutamine-D-fructose-6-phosphate aminotransferase activity at concentrations of fructose-6-phosphate found in the cell was appreciably greater in mycelium than in blastospores. The metabolism of [14C]glucose through the hexose monophosphate pathway was low after 4 and 18 h growth, and considerably increased at 10 h. The results support the concepts of a requirement for NADPH for cell division and suggest that control over the synthesis of chitin and mannan may, in part, be provided through control of the activity of phosphofructokinase by adenosine phosphates.

SUMMARY: A recent report that the major soluble carbohydrates of Aspergillus clavatus include ribitol and sorbose is wrong. The pentitol is arabitol and no sorbose could be detected, demonstrating that A. clavatus is not unlike many other moulds. Previous work on the metabolism of acyclic polyols in A. clavatus is re-evaluated.

SUMMARY: Growth of Methylococcus capsulatus on methane was inhibited by methanol (0.1%, v/v, and above), ethanol, n-propanol and n-butanol (0.01%, v/v, and above), but was unaffected by galactose, glucose, fructose, maltose, sucrose (at 0.1 M) or lactose (0.05 M). About one organism in 7 million grew well on solid medium using methanol vapour as a sole source of carbon and energy, but [14C]methanol was readily metabolized and assimilated by cultures growing on methane. Labelling patterns from [14C]methane and [14C]methanol were similar, indicating their assimilation by a common pathway. Dissimilarities between the labelling patterns obtained with 14CH4 and 14C-labelled formaldehyde, formate and carbonate indicated that the ribose phosphate cycle of formaldehyde assimilation may not account for all the carbon assimilated by M. capsulatus: significant incorporation of formate, carbon dioxide and possibly of intermediates of methane oxidation more reduced than formaldehyde may occur. 14C-Labelled ethanol and acetate showed restricted incorporation into lipid, leucine, glutamate, proline and arginine, indicating that M. capsulatus can produce acetyl coenzyme A from both compounds and introduce it into an incomplete biosynthetic tricarboxylic acid cycle. Methylococcus capsulatus was unable to assimilate more than trace amounts of [14C]glucose or sucrose.

SUMMARY: Scanning electron microscopy of intact fixed trophozoites of Toxoplasma gondii showed typical crescent-shaped organisms with clear polar structures. Examination of living organisms exposed to mouse peritoneal macrophages and L-cells and then fixed suggested that phagocytosis is one method of entry of the parasite into both phagocytic cells and those usually considered non-phagocytic. Antibody and accessory factor collapsed the organisms; irregular vacuoles appeared on their surfaces and cytoplasmic contents were extruded.

SUMMARY: In cytochrome α-deficient mutants of Bacillus subtilis, which grow to a reduced extinction of 600 nm and cannot sporulate, exceptionally large membranous structures (mesosomes) were observed with the electron microscope. They appeared not only at the sites of septation but also attached to unfinished, unseparated cross-walls of cell division and prespore septa. The parental strain grew and sporulated normally and did not show any unusual membranous structures.

SUMMARY: An analysis, by means of varying gene dosage at the tetraploid level, of the kinetic relationships which link the activity of individual enzymes to the functioning of the entire arginine biosynthetic pathway is presented. Tetraploid gene dosage series were prepared for each of the following gene-enzyme pairs: argB coding for acetylglutamate kinase, argF for ornithine transcarbamylase, argH for argininosuccinase and cpaI coding for one of the two polypeptidic constituents of the arginine-pathway-specific carbamoylphosphate synthetase.

In each of these series, the growth rates remain constant whatever gene dose is present. When the dose of the argB+ and cpaI + alleles is decreased, the specific enzyme activity per wild-type allele increases, as a result of derepression. Consequently, the enzymes which are coded by these genes and located at the two entries of the arginine pathway are rate-limiting for the pathway.

In contrast, a decrease of the argF + or argH + alleles does not result in derepression, implying that the corresponding enzymes function far below their maximal capacities. Thus the activity of these enzymes is adapted to the metabolite flux imposed by the rate-limiting enzymes.

The interpretation of these observations with respect to the dominance relations is discussed. The wild-type alleles of argF and argH are dominant because of over-production of the corresponding gene products. For the argB and cpaI genes, dominance results from a release of repression.

SUMMARY: Ten independent lipoamide dehydrogenase mutants (lpd) of Escherichia coli were isolated by selecting strains which required supplements of acetate plus succinate for best growth on glucose. They would not grow on unsupplemented medium (except anaerobically) nor would they grow with single supplements of acetate or lipoate, but they responded slowly to lysine plus methionine or succinate. Bacteria-free extracts of the mutants had between 1 and 10% of parental lipoamide dehydrogenase activity and no activity for the pyruvate and α-ketoglutarate dehydrogenase complexes could be detected. Evidence that the mutants contained the dehydrogenase (E1) and transacylase (E2) components of the complexes and were deficient only in the lipoamide dehydrogenase (E3) components was obtained from studies with mixtures containing lpd mutant extracts and either extracts of other mutants having defined lesions or purified lipoamide dehydrogenases, e.g. overall pyruvate dehydrogenase complex could be reconstituted with extracts of aceE and F mutants and the α-ketoglutarate complex was similarly reconstituted with sucA and B extracts. Furthermore, both complexes could be restored by adding extract of an aceE, sucA double-amber mutant (which lacks both types of E1 and E2 component but has 30% of parental lipoamide dehydrogenase activity) or with purified bacterial and mammalian lipoamide dehydrogenases. The bacterial enzymes were several times more efficient than the mammalian enzyme for restoring pyruvate dehydrogenase complex activity.

Genetic studies indicated that the wild-type phenotype could be restored by single reversion or transduction events and they confirmed that the mutants are deficient only in lipoamide dehydrogenase. The mutant phenotype was introduced into a recipient strain by cotransduction with leu +. This indicates that there is a lipoamide dehydrogenase gene in the leu region of the Escherichia coli linkage map and strongly supports the view that the E3 components of both α-ketoacid dehydrogenase complexes are specified by a single lipoamide dehydrogenase gene (lpd).

SUMMARY: In phototrophic Euglena gracilis, chlorophyll content/organism is modulated both by varied amounts of chlorophyll/chloroplast and by number of chloroplasts/ organism. In unlimited growth, Euglena never has less than 7 to 10 chloroplasts on the average, each containing 0.35 to 1.0 pg chlorophyll, depending on conditions and age of culture. Chloroplasts rarely contain more than 1.1 pg chlorophyll, and organismic levels greater than 10 to 12 pg are accomplished by an increased number of chloroplasts (to 60 in this study). Chloroplast replication is tightly coupled to that of the organism in log phase growth, but not in the lag and early stationary periods of growth.

SUMMARY: Washed Fusobacterium polymorphum did not initiate growth under semi-anaerobic conditions unless reducing compounds were added to the medium. In the presence of glutathione or cysteine, however, cultures incubated under air achieved a turbidity twice that of anaerobic cultures. The increased growth occurred in a diauxic manner and was inhibited by several compounds that block oxidative phosphorylation or other oxidative reactions. Growth was not affected by inclusion of fermentative by-products in the medium. Flushing of the atmosphere with oxygen at the beginning of the second growth phase enhanced the rate of the increased growth. Fusobacterium polymorphum was capable of oxidizing glutathione under all growth conditions tested. The diauxie was apparently mediated by the oxidation of GSH by F. polymorphum.

SUMMARY: Tissues of cowpea and pea underwent cellular browning following infection by tobacco necrosis virus and pea early browning virus respectively. In both species antifungal isoflavanoids accumulated in the brown tissues. The phytoalexins, phaseollidine, kievitone and phaseollin were isolated from virus-infected cowpea; pisatin from virus-infected pea. These results agree with previous indications that the destruction of host cells promotes the formation and accumulation of antifungal compounds within the tissues.

SUMMARY: Oxygen-limited chemostat cultures of nitrogen-fixing Azotobacter chroococcum showed an inverse relation between biomass and dilution rate, accounted for largely by increased polysaccharide and polyhydroxybutyrate content. Abrupt increase in pO2 led to immediate increase in CO2 output followed later by increase in biomass and transition to N2 limitation; viability on N-free and NH4-containing media remained at 80 to 100% during O2 stress. P-limited populations showed no respiratory reponse to O2 stress, viability dropped rapidly on N-free medium though the populations were 100% viable on NH4 medium. These findings support the view that respiration in these bacteria has, in part, a protective function for nitrogenase.

SUMMARY: Wild-type strains of Pseudomonas putida, P. cepacia and P. acidovorans grew in minimal salts media with acetamide or phenylacetamide as the carbon or nitrogen source. Wild-type strains of P. aeruginosa utilized acetamide but not phenylacetamide. Pseudomonas putida A90 and mutants isolated from P. putida A87 and from P. cepacia 716 grew on phenylacetamide but not on acetamide. These activities were due to two types of amidase: (i) aliphatic amidases, which hydrolyse acetamide, produced by P. aeruginosa and strains of P. putida, P. cepacia and P. acidovorans; and (ii) phenylacetamidases, which hydrolyse phenylacetamide, produced by some strains of P. putida, P. cepacia and P. acidovorans.

SUMMARY: A mathematical expression for oxygen-induced death of dehydrated bacteria is reported. Data in the literature for Serratia marcescens 8UK were analysed and the expression describes viability changes over a range of 104, oxygen concentration effects over a range of 400 and the effect of time up to 3 h. The expression should apply to the oxygen-induced death of other dehydrated bacteria.