- Volume 92, Issue 1, 1976

Summary: Rabbit antisera against l-asparaginase preparations from Escherichia coli, Erwinia carotovora, Citrobacter sp. and Chromobacterium violaceum showed on immunoelectrophoresis that only the enzymes from E. coli and Citrobacter are immunologically related. Purified preparations had to be used to determine the immunological cross-reactions. Immunoelectrophoresis at different pH values yielded the zero mobility points of the enzymes. The activity of the Er. carotovora preparation was enhanced up to fourfold by homologous antiserum but not by normal sera. Heterologous antisera also enhanced, but only at a higher concentration. Less enhancement was observed for the other enzymes with antisera as well as with bovine serum albumin. Inhibition was not observed.

Summary: Rifampicin at a concentration of 10 μg/ml completely inhibited protein synthesis in exponential-phase cultures of Bacillus amyloliquefaciens. At this same concentration the drug was shown to have no effect on the stability of mRNA, determined as the functional and hybridizable material, when compared with hybridizable mRNA in an uninhibited system. In each case, the half-life of the mRNA had a value in the range 5 ± 1 min, at 30 °C.

Summary: The ultrastructural characteristics of Neurospora cells during dedifferentiation and redifferentiation of conidiospores into vegetative cells have been determined. This germination process occurs between 2 and 5 h after inoculation; by 3·5 h, approximately 50 % of the cells have germinated. The cells enter the exponential phase of dry-weight gain between 4 and 5 h after inoculation. Several unusual structures are observed in Neurospora cells during germination. Whorled structures are frequently seen in the cytoplasm during germination, and occasionally at other times. They appear to be derived from the cytoplasmic membrane. Whorled structures of different appearance were observed in the mitochondria between 2 and 4 h after inoculation. Their number was related to the level of metabolizable carbohydrate, and was higher in 15 % glucose- than in 2 % sucrose-supplemented medium, and very low in medium containing 15 % mannitol, or 2 % sucrose + 13 % 2-deoxyglucose, or no added carbohydrate. The mitochondrial inclusions were osmiophilic and could be removed by treatment with 90 % aqueous acetone in the cold, indicating that they were composed at least in part of lipid. The strong dependence of the number of mitochondrial inclusins on time and on carbohydrate supplementation, suggests that there is a physiological basis for these structures and that they reflect changes occurring in the mitochondria at times significant to cellular differentiation.

Summary: Changes in the capacity of mitochondria to carry out oxidative phosphorylation and in the rate of synthesis and incorporation of phospholipids into mitochondria were measured during the germination of conidiospores of Neurospora crassa. The competence of isolated mitochondria to carry out coupled respiration was very low during the first 3 h growth, but it increased rapidly, reaching maximal levels at 5 to 6 h growth. Changes in mitochondrial function were the same in cells grown in 2 % sucrose- or 15 % glucose-supplemented medium. The rate of synthesis of mitochondrial phospholipids was very low during the first 2 h growth and increased to maximal levels between 3 and 5 h. The rate of synthesis of mitochondrial phospholipids was approximately three times higher in cells grown in 15 % glucose than in those grown in 2 % sucrose. The maximal rate of synthesis of mitochondrial phospholipids occurred during spore germination and preceded attainment of full competence for oxidative phosphorylation. The lipid-rich condition of the mitochondria resulting from the high rate of synthesis of phospholipids in glucose-grown cells is postulated to be related to the whorled inclusions observed in thin sections of Neurospora cells.

Summary: The effect of environmental conditions on the morphology of the blue-green alga Chlorogloea fritschii is described. Availability of reduced carbon substrate, light and nitrogen all caused alteration in cell type, as did increase in temperature. The two major cell types were irregular clumps of cells (aseriate), and filaments; in photoautotrophic conditions the former predominated during exponential growth at 34 °C. The presence of sucrose imposed aseriate morphology in both phototrophic and heterotrophic cultures. The development of differentiated cells (heterocysts) following deprivation of nitrate and the interrelationships between different cell types are described.

Summary: The nets produced by protoplasts of Saccharomyces cerevisiae in liquid culture media consisted of microfibrils about 20 nm wide, forming flat, fairly straight bundles of variable width and length, up to about 500 nm wide and 4 μm long. Ends of microfibrils were seldom found. They were not attacked by chitinase or dilute acids, but the net structure disappeared in 3% (w/v) NaOH, leaving about 60% dry wt of the nets as partly microfibrillar clusters. The X-ray powder pattern from the nets, in contrast to that from normal walls, exhibited a set of well-defined rings which identified two micro-crystalline constituents: chitin and unbranched chains of β(I → 3)-linked D-glucose residues. These latter were the alkali-soluble fraction. The X-ray diagram of the glucan, corresponding to that of paramylon, indicated an in vivo crystal modification. Up to 15% dry wt was chitin which was formed de novo by the protoplasts.

A fine net structure of microfibrils about 7·5 to 10 nm thick with meshes about 20 to 60 nm wide was demonstrated in normal walls, forming the entire inner layer and consisting mainly of yeast glucan. This glucan and chitin were only slightly crystalline in these walls. The features of the glucan and chitin of the protoplast nets indicate that enzymes active in normal wall formation were differentially removed or inactivated by the liquid medium.

SUMMARY: In Podospora anserina, protoplasmic incompatibility due to interactions between non-allelic genes was suppressed by the effect of mutations in two modifier genes, mod-1 and mod-2. It is shown that mod-1 and mod-2 are involved in the production of three specific proteins, a phenoloxidase and two previously identified proteases ( Bégueret & Bernet, 1973a ) which are associated with the phenomenon of protoplasmic disintegration. These enzymes, whose messengers are normally latent during vegetative growth, appear at this stage of the life cycle only as a consequence of incompatible gene interactions.

The mod-1 and mod-2 genes and each of the five incompatibility loci involved in non-allelic incompatibility systems also participate in the formation of the protoperithecia. This pleiotropic effect suggests that protoplasmic incompatibility is a deviation in the normal physiological processes of protoperithecial formation.

Summary: Two mutants are described in which the synthesis of tryptophanase is unusually insensitive to catabolite repression. Neither mutation is linked by transduction to the tryptophanase structural gene, neither mutation renders the synthesis of β-galactosidase insensitive to catabolite repression, and the mutations do not permit tryptophanase to be synthesized in strains deficient in adenyl cyclase. During growth in glucose-minimal medium the mutants maintained a similar intracellular concentration of cyclic AMP to their wild-type parent; but since in the wild type the concentration of cyclic AMP was the same in glycerol-minimal medium as in glucose-minimal medium, it is doubtful whether catabolite repression is mediated by measurable changes in the concentration of this nucleotide.

Summary: From a strain lacking adenyl cyclase and the catabolite-sensitive gene activator protein, two mutants were isolated that can synthesize tryptophanase. Each mutation is extremely closely linked to the tryptophanase structural gene. The mutations differ from one another in the rate of synthesis of tryptophanase that they permit in the genetic background in which they were isolated; they differ from one another and also from the wild type in the maximum rate of synthesis of tryptophanase that they permit in a genetic background with intact adenyl cyclase and catabolite-sensitive gene activator protein. Both mutations appear to lie in the tryptophanase promoter.

Summary: A new temperature-sensitive mutant of E. coli, defective in cell division, was isolated after selection for tolerance to colicin E2. The mutant strain, ash124, growing in either minimal or complex medium, commences filament formation immediately upon shift to high temperature. High densities of bacteria or the presence of 0·44 m-sucrose prevents filament formation at 42 °C and division continues. Filament formation in the mutant is reversible and upon return to 29 °C the multinucleate filaments divide up into normal-sized bacteria by a series of rapid but sequential divisions. In the presence of chloramphenicol at 29 °C, 25% of these division sites are still expressed. A genetic locus designated ftsH, apparently controlling both temperature sensitivity and filament formation, was provisionally mapped at minute 80 on the E. coli k12 map.

Summary: Purified gamma haemolysin of Staphylococcus aureus was characterized in relation to the alpha, beta and delta haemolysins. The sedimentation coefficient of the gamma lysin was 2·65, somewhat higher than the s 20,w values of 1·4 for freshly purified alpha lysin and 1.8 for the beta lysin. The molecular weight of gamma lysin determined by gel filtration was 45000 daltons. The pI of gamma lysin was 6·0, while that of the alpha, beta and delta lysins ranged from 8·5 to 9·6.

The amino acid analysis of gamma lysin was characterized by low levels of methionine and histidine. Methionine was, however, the N-terminus, which suggested that all of the amino acid might be involved in the N-terminal group.

The gamma lysin was immunologically distinct from the alpha, beta and delta lysins by quantitative precipitin tests; in Ouchterlony agar gel diffusion tests, single lines of precipitation were observed which showed no evidence of cross-reactions amongst the four haemolysins.

Gamma, beta and delta lysins had no effect in mice when injected at increasing doses ranging from 0 to 100 μg. The alpha lysin killed mice, the LD50 dose being 0·68± 0·12 μg, or 27 to 34 μg/kg mouse tissue. Gamma lysin was, however, lethal for guinea pigs when 50 μg quantities were injected intracardially. Gamma lysin also lysed human leucocytes and destroyed C-6 (human lymphoblast) cells.

Some nitrogen and phosphorus was released from human erythrocyte membranes treated with gamma lysin, when compared to untreated cells, and the rate of this release was linear over a 3 h period. Gamma lysin had no detectable effect on phospholipids extracted from erythrocytes or on lipid-free membrane protein. However, the haemolytic reaction was inhibited by erythrocyte membranes when these were added to lysin-red cell suspensions. Furthermore, human red cell phospholipids competitively inhibited haemolysis. Haemolysis was also inhibited by EDTA and activity could be restored by dialysis. The haemolytic reaction required sodium ions.

SUMMARY: Several type C and D strains of Clostridium botulinum, which had been converted to the toxigenic state by phages, were serially transferred through cooked meat medium with and without specific anti-phage serum. Most of the converted strains lost their toxigenicity even during transfer without antiserum, and the non-toxigenic variants that appeared were resistant to lysis and conversion by the original phage. However, in some combinations of phage and host bacteria toxigenicity was stable after ten transfers, though it showed a transient decrease, and the non-toxigenic variants that arose remained sensitive to lysis and conversion. When converted strains were transferred in medium containing anti-phage serum, toxigenicity was lost more rapidly than in the absence of serum and the non-toxigenic variants that appeared remained sensitive to lysis and conversion by the parent phage.

Filtrates of the supernatants of culture fluids of strains transferred without anti-phage serum converted non-toxigenic strains to toxigenicity at varying rates. However, a non-converting phage was demonstrated in one of these filtrates. This phage was identical to the original converting phage in its morphology, host range and antigenicity. Indicator strains treated with this phage acquired resistance to lysis by the parent phage.

The results suggest that re-infection and conversion to toxigenicity occurred in combinations showing stable toxigenicity after ten transfers. However, in those combinations that lost toxigenicity, re-infection with a non-converting mutant of the original phage may have occurred with the result that non-toxigenic variants became resistant to the converting phage. This appears to be one of the causes of the loss of toxigenicity which is common in some type C and D strains of C. botulinum.

SUMMARY: Addition of calcium ions increased 2- to 3-fold the growth of Saccharomyces carlsbergensis 21 in a minimal glucose-containing medium. The minimal concentration enhancing growth was 25 to 50 μg/ml CaCl2. Other divalent and trivalent cations tested, except for strontium ions, did not duplicate the calcium effect. Actively growing and dividing cells took up 45Ca2+, while resting yeast cells did not. The radiocalcium taken up was incorporated into newly synthesized structural material, presumably into the membrane protein.

Summary: The medium redox potential (E h) influenced the folate-limited growth of Lactobacillus casei; the growth response was maximal at an E h of +120 mV (pH 6·35). At raised E h serum folate would support less growth than pteroyl-glutamic acid, and the response to N 5-methyl tetrahydrofolic acid was intermediate between them. Pteroylglutamic acid was not destroyed during 24 h incubation at 37 °C in medium with E h values between +40 and +440 mV. Destruction of N 5-methyl tetrahydrofolic acid occurred within 24 h when the medium E h was greater than +125 mV. Folate was taken up rapidly by L. casei with an E h optimum at + 270 mV.