- Volume 80, Issue 1, 1974

SUMMARY: Rubratoxin A or B can be assayed by overnight incubation at 25 °C of a standard inoculum of Tetrahymena pyriformis (Ehrenburg) strain w in nutrient medium containing differing toxin concentrations. Growth is estimated by measuring the extinction of the organism suspension after inactivation with Hayem’s fluid. The values obtained are expressed as a percentage of a normal control. Dose/res-ponse curves of percentage inactivation against log concentration of rubratoxin had linear ranges of 2·6 to 23 μg/ml for rubratoxin A and 8·5 to 30 μg/ml for rubratoxin B. The concentrations of toxin required to produce 50 % inhibition of growth in standard suspensions of T. pyriformis strain w were 8·75 μg/ml and 20·5 μg/ml respectively.

The octadecenoic acid growth-requirement for Acholeplasma (Mycoplasma) laidlawii a can be effectively replaced by cis-9,10-methylenehexadecanoic acid but not by m-9,10-hexadecenoic acid. Once incorporated into the membrane polar lipids, this acid is also elongated to cis-11,12-methyleneoctadecanoic acid. This is the first instance of the elongation of a cyclopropane fatty acid by a microorganism. cis-9,10-Methylenehexadecanoic acid, like cis-9,10-octadecenoic acid, alters the osmotic fragility of this mycoplasma, but while a correlation exists between fatty acid concentration and yield of organism, there is no apparent relationship between fatty acid concentration, maximal yield and greatest membrane osmotic stability for this organism. The fatty acid composition of the membrane polar lipids of A. laidlawii a grown with cis-11,12-octadecenoic and cis-9,10-methylenehexadecanoic acids shows each acid to be incorporated to a similar extent. A cell-free system from A. laidlawii a synthesized less cw-11,12- methyleneoctadecanoic and tetradecanoic acids but more octadecanoic acid when compared with the fatty acid composition of the intact organism.

SUMMARY: The entire sexual apparatus (oospore-oogonium-antheridium, or ooa) of Phytophthora megasperma var. sojae was isolated in large quantities free of mycelium. The cells were fractured in a Teflon tissue homogenizer and the wall fraction was washed with a cytoplasmic dialysate to prevent agglutination and a tendency to adhere to glassware. The wall preparations were a mixture of oospore and oogonium walls (oow) and probably unrecognizable antheridial wall fragments. The walls constituted 47% of the dry weight of the ooa. Insoluble glucans comprised almost 80 % of the wall; the main component was non-cellulosic β-glucan(s) linked mainly through the 3-position. Less than 10% of the oow was cellulose. In addition to glucose, there were small amounts of mannose and glucosamine in the wall polymers. Proteins comprised about 12% of the oow; the most abundant amino acids were arginine and glycine. Hydroxyproline, a component of oomycetous hyphal walls, was also present. Readily extracted lipids varied from 2·8 to 5·9%, while bound lipids comprised 5·4 to 5·9% of the wall. Most of the readily extractable lipids of the ooa were found in the walls whereas bound lipids were mainly in the cytoplasmic fraction. Sterols (0·025%) were detected in the readily extracted lipid fraction. Phosphorus amounted to 0·55 % of the oow. There was substantial similarity in gross chemical composition between oow and hyphal walls of Phytophthora spp.

SUMMARY: Experiments were carried out to establish whether glutamine synthesis in yeast is subject to control through the operation of cumulative feedback inhibition on the biosynthetic enzyme glutamine synthetase.

15N isotope was used to measure the in vivo rate of amide synthesis in a,culture growing on ammonia. The steady-state rate measured corresponded quite closely with a value calculated from a knowledge of the enzyme content of the yeast, and indicated that under these conditions yeast did not possess a large excess of biosynthetic capacity. In another experiment yeast was subjected to a brief period of nitrogen deprivation and rates of amide synthesis were determined immediately after addition of ammonia to the culture. This established that no significant increase in the rate of amide synthesis per unit of enzyme accompanied a reduction in the cell concentration of glutamine and other amino acids, and that the small increase in rate observed was caused by an increase in enzyme amount.

Measurements of the rate of glutamine synthesis in yeast with very high levels of enzyme confirmed that the enzyme content was of prime importance in determining the rate of synthesis in the organism. Thus when ammonia was added to yeast growing on glutamate, the rates of amide synthesis were initially nearly sixfold higher than ones observed on ammonia; this enhancement of rate almost exactly paralleled the increase in amount of enzyme in the yeast. Although the cell concentration of glutamine had greatly increased at this time and the concentration of some of its derivatives was unchanged, virtually no restraint operated to limit the synthetic activity of the enzyme in the organism. The rapid decline in the rate of glutamine synthesis which ensued occurred in response to a depletion of enzyme substrates as well as to enzyme inactivation, and the relative importance of these two factors in the control of glutamine synthesis was quantitatively assessed.

SUMMARY: In Candida utilis, the amount of glutamine synthetase is finely attuned to the availability of ammonia in the culture medium, and any change in ammonia availability results in a compensatory adjustment to enzyme level. In yeast growing with different sources of nitrogen there was a good inverse correlation between the rate of enzyme synthesis and the size of the cell pool of glutamine: glutamine and not ammonia appeared to act as a co-repressor of the formation of glutamine synthetase.

Sudden changes in ammonia availability or carbon supply could lead to the inactivation of glutamine synthetase. This was shown to be associated, under some conditions, with a marked increase in the pool of glutamine and it is possible that it was brought about by sudden changes in the relative cell concentrations of glutamine and glutamate. However, the process is clearly more complex than this and catabolism of carbon substrates is also involved.

The specific activity of the NAD-specific (deaminating) glutamate dehydrogenase also varied with the availability of ammonia, but neither it nor glutamine synthetase was subject to parallel regulation.

* All communications should be addressed to A.P.S.

In Candida utilis a sudden increase in ammonia supply or a decrease in glucose supply can result in a rapid and extensive reduction in glutamine synthetase activity, a fall that is much faster than can be accounted for by the immediate repression of enzyme synthesis followed by the dilution of existing enzyme by the other cell proteins produced during continued growth. This decrease in enzyme activity is not caused by the build-up within the cell of an inhibitor of the enzyme, by the rapid turnover in vivo of the enzyme, or by the conversion of the enzyme from a more active to a less active form, but by the specific inactivation of the enzyme. Inactivation appears to be largely irreversible, and studies using inhibitors of protein synthesis indicate that the reappearance of the enzyme after removal of ammonia is dependent on de novo protein synthesis.

Glutamine synthetase activity can be estimated by using either the transferase assay or the synthetase assay: the ratio of these activities is virtually constant in preparations from yeast growing under a wide range of conditions and during inactivation and reappearance of the enzyme.

SUMMARY: Fifteen isolates of Brevibacterium linens were tested by polyacrylamide gel electrophoresis. Protein patterns and zymograms of 18 intracellular enzyme activities (α-naphthyl acetate and β-naphthyl butyrate esterases, tributyrinase, alcohol-, lactate-, malate-, succinate-, glucose-6-phosphate-, 6-phosphogluconate-, isocitrate-, alanine- and glutamate-dehydrogenases, tetrazolium oxidase, catalase, peroxidase, arylamidase, proteinase and amylase) and four extracellular enzyme activities (α-naphthyl acetate esterase, arylamidase, proteinase and amylase) were examined. Enzyme activities were demonstrated by either specific staining reactions or by the subsequent diffusion of the proteins into a substrate layer. Striking variations of the enzyme patterns occurred among the strains. The variation, however, depended largely on the activity under consideration. Eight different enzymes were selected to calculate a matrix of similarity. The results indicated a separation of the strains into two major subdivisions or biotypes. Only one strain (bl107) differed markedly from the others.

SUMMARY: The lipid composition of the psychrophilic bacterium Micrococcus cryophilus (ATCC 15174) has been examined. The chloroform-methanol extractable (i.e. ‘free’) lipid represents nearly 10% of the bacterial mass and is composed of 85% polar lipid and 15% neutral lipid. The polar lipid consists of 46% phosphatidyl ethanolamine, 43% phosphatidyl glycerol and 9% cardiolipin, plus a fourth unidentified minor component which cannot be detected in all extracts. The neutral lipid is characterized by the presence of a mixture of simple waxes, which constitute 86% of this fraction, together with smaller amounts of free fatty acids, alcohols and a ubiquinone, probably Q8. Simple waxes have not been found previously in such large amounts in true bacteria. The implications of the results of the lipid analysis are discussed with regard to the taxonomic position of M. cryophilus.

SUMMARY: Escherichia coli possesses permeases that specifically transport exogenous peptides with a free or β-N-methylated terminal amino group, but not with a terminal acyl group. To determine whether steric hindrance or loss of positive charge might be responsible for inactivity of acyl peptides we prepared and tested a series of β-N -alkyl peptides, several with alkyl substituents larger than the inactivating acyl groups. β-N-dimethyl, -ethyl, -propyl, -butyl, -isopropyl and -isobutyl peptides were prepared by NaBH4 treatment of aldehyde or ketone-peptide complexes. The positively charged monoalkyl derivatives of triglycine were all nutritionally active, all competed with unsubstituted peptides, and all failed to be absorbed by a mutant that lacked the oligopeptide permease. Dimethyltriglycine was nutritionally and competitively inactive. The results negate the explanation of steric hindrance and suggest a requirement for an N-terminal β-amino hydrogen for peptide transport.

SUMMARY: Starvation of Peptoraccus prvotii caused extensive loss of RNA which produced a large flux of adenine nucleotides. The adenylate energy charge remained essentially constant the period of rapid decline of RNA but then fell when the residual RNA was being degraded more slowly. Loss of viability became more rapid when the adenquate energy charge fell below 0·4 to 0·5.

SUMMARY: 14C-labelled retinol, β-C18-ketone* and 4-hydroxy-β-C18-ketone fed to mixed plus and minus Blakeslea trispora cultures were efficiently incorporated into trisporic acids. This allowed the biosynthetic route from β-carotene to these hormones to be set out, leaving some uncertainties as to the sequence of later steps in the process.

Strains of Pseudomonas aeruginosa (P. pyocyanea) were examined for pyocin production by means of a standard pyocin-typing technique and also a modified method which depends on the ability of some pyocins to pass through a cellulose acetate membrane. The results suggested that strains could produce (1) pyocins incapable of spontaneous passage through the membrane, (2) pyocins capable of passage through the membrane, (3) both kinds of pyocin simultaneously. Electron microscopy revealed that pyocins in the first circumstance included contractile pyocins and those in the second included flexuous, rod-like particles. P. aeruginosa 430 produced both contractile and flexuous pyocins simultaneously. The pyocins could be separated by absorption with sensitive bacteria or by gel filtration. The two pyocins differed serologically and in their range of inhibitory activity against strains of P. aeruginosa.

SUMMARY: The germination rate of Clostridium bifermentans spores and the manner in which it is influenced by pH, sodium ion concentration and temperature differ according to the organic germinant used. Only l-alanine initiated germination when present alone. The germination rate with optimal l-alanine was increased by l-phenylalanine, l-arginine, l-histidine or l-lysine but not by other common amino acids or l-lactate. With suboptimal concentrations of l-alanine, addition of l-lactate, l-arginine or l-phenylalanine increased the germination rate more than an equal concentration of l-alanine. The mechanisms whereby l-phenylalanine and l-arginine stimulate germination appear to be different from those involved when l-alanine or l-lactate are the germinants.

SUMMARY: The sensitivity of Saccharomyces cerevisiae to sphaeroplast formation has been examined during the transition from the exponential phase of growth to the stationary phase. Exponential-phase yeasts are sensitive to sphaeroplast formation while stationary yeasts are resistant. During the transition period, there is a rapid increase in resistance to sphaeroplast formation. This increase can be inhibited by treatment with either cycloheximide or 5-fluorouracil. It is suggested that the resistance to sphaeroplast formation characteristic of stationary-phase yeasts is the result of a specific modification of the yeasts during the transition period which is dependent on both RNA and protein synthesis.

SUMMARY: Mutant Mir M7 of Klebsiella pneumoniae exhibits a pH-dependent morphological interconversion; growing cells are rod-shaped at pH 5·5 but round at pH 7. Round cells cannot divide or make DNA, RNA and proteins at pH 5·5. At this pH, cell division and DNA synthesis are resumed either when substances such as Mg2+, Na+, sucrose or spermine are added to the growth medium or immediately after the appearance of a normal rod morphology. Round cells, but not rods or the wild type, are lysed by low concentrations of detergents and are killed by Mdl-amino acids. Their growth rate is not improved by vigorous aeration and they make spheroplasts at an incubation temperature of 8 °C. This behaviour is the opposite of that of rods and the original strain. The transition from rods to round cells at pH 7 does not occur when cell division is inhibited by penicillin. Round cells must divide several times in an acid medium to give regular and uniform rods.

The possibility of a different state of the membrane in round and rod-shaped cells and a dependence of cell division, DNA synthesis, and morphology on the state of the membrane are discussed.

SUMMARY: A special technique permitted observation of sexual reactions between mycelia of Mucor mucedo without diffusion contact via the substrate. Morphogenetic phenomena (zygophore formation) appeared to be induced by volatile substances in a sex-specific way. Zygophores, induced via the air, showed chemotropic activity. The volatile stimuli were partly purified by thin-layer chromatography and appeared to have the same RF values as precursors of trisporic acids. Trisporic acids are currently assumed to be responsible for the induction of zygophore formation via the substrate.