- Volume 81, Issue 1, 1974

SUMMARY

The pyruvate carboxylase of Verticillium albo-atrum had a pH optimum of 7·8 and a specific requirement for ATP. At the optimum pH, magnesium ions were required for maximum activity, while at pH 6·8 manganese was more effective than magnesium. Potassium was stimulatory while sodium was ineffective. Avidin and p-chloromercuribenzoate strongly inhibited the enzyme while biotin and dithio- threitol, respectively, reversed the effect of the inhibitors. Aspartate and oxalacetate were inhibitory while acetyl-CoA and CoA reversed the inhibition by aspartate. These cofactors were ineffective in the absence of aspartate. None of the tested metabolic intermediates was stimulatory to pyruvate carboxylase activity while NADP+ and 2,3-diphosphoglycerate were the most effective inhibitors (75 %) at a concentration of 6·7 mm. Levels of pyruvate carboxylase in cells grown on glucose, acetate, malate, xylose, glycerol or aspartate differed only slightly. The data indicated that the physiological role of pyruvate carboxylase in V. albo-atrum is the anaplerotic biosynthesis of C4 Krebs-cycle intermediates from pyruvate.

SUMMARY: Partially purified phosphoenolpyruvate carboxykinase from Verticillium albo- atrum had a pH optimum of 6·2 and required manganese for maximum activity, having lesser activity with iron or cobalt. Sodium and potassium ions were slightly stimulatory. Adenosine-5′-diphosphate increased activity and inosine-5′- diphosphate supported low activity, but other nucleotides were ineffective. Inhibition of the enzyme by p-chloromercuribenzoate was partially reversed by dithiothreitol. Avidin had no effect on enzyme activity. Oxalacetate slightly stimulated the enzyme and NADP+ strongly inhibited, but aspartate and acetyl- CoA showed no effect. Low levels of phosphoenolpyruvate carboxykinase were present in cells grown on glucose, xylose, or glycerol. Aspartate, pyruvate, and acetate as carbon sources resulted in higher levels of activity and malate gave the highest. The data indicate that the enzyme functions physiologically in the gluconeogenic conversion of oxalacetate to phosphoenolpyruvate.

The pathway of glucose metabolism in Sporocytophaga myxococcoides was studied by a radiorespirometric technique and assays of enzyme activity in cell-free extracts. The primary catabolic pathways in the organism were examined by measurement of relative rates of 14CO2-production from different carbon atoms of labelled glucose, pyruvic acid and acetic acid. These substrates appeared to be degraded solely by enzymes of the Embden-Meyerhof-Parnas pathway in conjunction with the tricarboxylic acid cycle. The results were confirmed by studies of enzyme activity, which showed a lack of two enzymes, glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate dehydrogenase, EC. 1.1.1.49) and 6-phospho-gluconate dehydrogenase [6-phospho-D-gluconate: NADP oxidoreductase (decarboxylating), EC. 1.1.1.44], in the pentose pathway, which indicated a biosynthetic function of the non-oxidative part of this pathway.

SUMMARY: Strains of Selenomonas ruminantium, Anaerovibrio lipolytica and Veillonella alcalescens contained cytochrome b. Peaks corresponding to cytochromes a and a carbon monoxide-binding pigment were also observed. By means of dualwavelength experiments with crude membrane fractions it was established that cytochrome b functioned in anaerobic electron transport to fumarate. In V. alcalescens and one strain of S. ruminantium which reduced nitrate, anaerobic electron transport to nitrate was found. Glycerol i-phosphate and NADH were active as hydrogen donors for cytochrome b reduction in glycerol-grown A. lipolytica, lactate and pyruvate were active in lactate-grown V. alcalescens, and NADH was active in lactose-grown S. ruminantium. Oxidative phosphorylation associated with these electron transfer systems might explain the high molar growth yields previously found for these micro-organisms. Fermentation products were measured in supernatant fluids of cultures grown in the presence and absence of nitrate. Nitrate did not influence the fermentation of lactose to lactate by S. ruminantium, and inhibited propionate formation by V. alcalescens.

SUMMARY: α-l-Arabinofuranosidase (AF) was detected in apple fruitlets experimentally infected by Sclerotinia fructigena. In extracts of such fruitlets, three AF isoenzymes were separated by preparative isoelectric focusing. When the fungus was grown in shake culture with different carbon sources, AF was detected in each culture filtrate and mycelial homogenate. Although fungus growth and total AF varied with the carbon source, the AF isoenzyme pattern was similar in each instance to that obtained when grown on sodium polypectate.

Each of the partially purified AF isoenzymes behaved differently in substrate specificity and inhibitor studies; however, each showed a specificity for α- l-arabino-furanosides. The two extracellular AF isoenzymes released monomeric arabinose when incubated with araban or apple cell walls. External AF III (pI 6·5) was more active on a substrate of apple cell wall material than external AF I (pI 3·0). The latter form of the enzyme was less susceptible to inhibition by either oxidized or unoxidized apple juice. Two isolates of Sclerotinia fructigena with low growth rate in vivo secreted no AF III in vitro.

The NADP-specific glutamate dehydrogenase (NADP-GDH) activity of Aspergillus nidulans was rapidly lost from cultures starved for a carbon source. This loss of NADP-GDH was blocked by protein synthesis inhibitors. Glutamate repressed NADP-GDH but did not cause rapid loss of activity. Since NADP-GDH is involved in the participation of ammonium in the regulation of nitrogen metabolism, the loss of NADP-GDH activity accompanying carbon starvation may be important in the interaction between carbon and nitrogen metabolism. Increased NAD specific glutamate dehydrogenase activity (NAD-GDH) was observed when mycelium was transferred to medium lacking glucose. The increase in NAD-GDH activity was greatest when glutamate was present. Protein synthesis inhibitors did not prevent this increase in activity.

Two mutants, amdT102 and amdT19, which are altered in regulation of nitrogen metabolism, are similar to the wild-type strain with regard to regulation of NADP-GDH and NAD-GDH.

The wall structure of the fission yeast Schizosaccharomyces pombe, examined by enzymic techniques, consists of a galactomannan, an α-glucan and β-glucan. The structures of the α-glucan and galactomannan were investigated by methylation. The wall structure is discussed in relation to the taxonomic position of the genus Schizosaccharomyces.

SUMMARY: Phages T3 and T7 were freeze-dried, freeze-thawed or aerosolized. Freezedrying and aerosolization caused dehydration and resulted in a loss of viability due mainly to one factor, loss of tails. However, the extent of loss of viability and tails was critically dependent upon the manner of rehydration, slow rehydration being best for the preservation of the head-tail complex and viability. Slow freezing (at about 1 °C/min), but fairly rapid thawing (at about 200 °C/min), resulted in marked loss of viability of T7 but only slight loss for T3, through causes other than loss of tails or lysis. Collection of aerosols of T3 and T7 by impinger resulted in much lower viabilities than were obtained by subsonic impaction (e.g. about 10 and 80 % respectively).

The results indicate that the dehydration induced by freeze-drying and aerosolization did not cause loss of viability, but that loss of viability occurred during rehydration. Dehydration of T3 and T7 did not appear to occur during slow freezing (at about 1 °/min). Mechanisms different from those operating through shear stresses induced by freeze-drying and aerosolization caused loss of viability when T3 and T7 were slowly frozen at about 1 °/min.

SUMMARY: Of 86 strains of Salmonella cholerae-suis isolated in the U.K., 83 possessed the O antigen 617 and three the O antigen 627. All the O 617 strains were latently infected with the same phage which was active on the O 627 strains; during lyso-genization this phage converted O 627 strains to O 617. None of 900 strains of Escherichia coli was latently infected with this phage; nor were they susceptible to it.

Two O 627 strains converted to O 617 by lysogenization were significantly more virulent for chicks than were their O 627 parent strains; lysogenization of one of these O 627 strains by two different non-converting phages was not accompanied by an increase in virulence. One of the converted strains tested in mice was more virulent for these animals than was the O 627 strain from which it was derived.

The O 617 organisms were found at higher concentrations in the blood and liver of chickens into which they had been injected intravenously than were organisms of the O 627 strain from which they were derived. No difference was detected between the survival rates of the two kinds of organisms in normal chicken serum.

SUMMARY: Growth of Mucor hiemalis, Geotrichum candidum, Aspergillus nidulans, Neurospora crassa and Penicillium chrysogenum was studied by time lapse photography. The total hyphal length of the mycelium of each species increased at an exponential rate; in M. hiemalis exponential growth continued until the mycelium had a total hyphal length in excess of 10 mm. After spore germination there was an initial phase of discontinuous tip production followed by a phase of ‘ continuous ’ tip production. The hyphal length and number of tips possessed by a mycelium increased exponentially at approximately the same specific growth rate.

The amplitude of the oscillations in the length of the hyphal growth unit of a mycelium decreased progressively during mycelial growth and eventually the growth unit attained a more or less constant value. The results support the hypothesis that mycelial growth involves the duplication of a ‘ growth unit ’ which consists of a tip and a certain mean length of hypha.

A method was developed for the fractionation of Fusarium culmorum homogenates in order to obtain purified cytoplasmic membranes. The fractions obtained were characterized by studying their ultrastructure and enzymic properties. Since ATPase is an enzyme present in the cytoplasmic membrane, we studied the levels of this activity in our fractions as an indication of the presence of cytoplasmic membrane fragments. Since this activity is also present in mitochondria, we determined the levels of fumarase, a mitochondrial enzyme, and took the ratio of the two activities as an index of the purity of the membrane preparations.

The sediment obtained by centrifuging the cell-free extract at 40000 g was fractionated in a discontinuous sucrose gradient. This led to three types of fractions. The most dense had a high ATPase/fumarase ratio and its ultrastructure showed that it contained membrane fragments having a triple-layered structure.

We concluded that this fraction was rich in cytoplasmic membrane fragments; it was clearly distinguishable from the intermediate and less dense fractions. These latter fractions had a much lower ATPase/fumarase ratio and, judging by their ultrastructure, they were respectively a mitochondrial fraction and a fraction consisting of vesicles probably related to the endoplasmic reticulum. Analysis of the cytoplasmic membrane-rich fraction showed that it consisted of protein, lipid and 30 % carbohydrate

The distribution of Bacillus subtilis in a pine-forest soil has been investigated by using the fluorescent-antibody technique and dilution plating. In the acid, mineral horizon of the soil, the organism occurred mainly in the vegetative condition, while in the alkaline, mineral horizon it was mostly present as spores. Within these soil horizons, the bulk of the vegetative bacteria was found on organic matter particles, but of those found on mineral particle surfaces, almost all were on cryptocrystalline quartz particles with iron oxide inclusions.

SUMMARY

A cell-free preparation of the blue-green alga, Anabaena variabilis, that incorporates 14C-labelled amino acids into protein has been prepared and characterized.

The activation of amino acids to amino acid transfer RNAs was characterized, and assembly of these into peptides has been described with respect to cofactor requirements and antibiotic sensitivity. The properties of these systems and the effect of antibiotics on them are similar to those of bacteria. Both natural and synthetic messenger RNA were effective in peptide formation. The kinetics of incorporation of [14C]uracil into RNA has been examined and the stability of labelled RNA from A. variabilis measured by radioactivity loss and by its role in directing peptide synthesis. The half-life of messenger RNA from this organism is approximately 12 min, and as such is comparable to that of bacteria when based upon the mean generation time of the respective organisms.

SUMMARY

Of more than 100 R factors transferred from naturally occurring Providence strains, approximately 60 % belonged to the A-C compatibility complex. Plasmids of groups F1 J, N, P and X, and the prototype of a new group G were also transferred. The Providence R factor set differs from those of Proteus rettgeri and P. morganii most strikingly in the abundance of plasmids of the A-C group and the infrequency of N group plasmids.

The immunological properties of ten lipoamide dehydrogenase mutants of Escherichia coli were investigated with antiserum raised against purified lipoamide dehydrogenase. Seven mutants were CRM+ (cross-reacting material present) as they contained lipoamide dehydrogenase proteins exhibiting either complete or partial immunological identity with the wild-type protein. This indicates that at least seven of the mutations affect the lipoamide dehydrogenase structural gene (lpd). The remaining three mutants (CRM-) contained no detectable cross-reacting protein. None of the lpd mutations were sensitive to any of three different amber-suppressors. Genetic analysis by P1-transduction showed that all the lpd mutant sites were clustered very near the distal gene (aceF) of the ace region which specifies the dehydrogenase (aceE) and transacetylase (aceF) components of the pyruvate dehydrogenase multienzyme complex. Calculations based on the recombination frequency between an aceF mutant and the nearest lpd mutant site support the conclusion that apart from the possible presence of a regulatory element, the aceF and lpd genes are contiguous.