- Volume 76, Issue 2, 1973

Summary: 3-Deoxy-3-fluoro-D-glucose (3FG) was converted to 3FG-6-phosphate by the phosphoenolpyruvate-dependent phosphotransferase system in frozen and thawed Escherichia coli. Up to 0.03g 3FG was taken up/g bacterial dry wt. Uptake of 3FG was not lethal, though 3FG at 0.1 to 10 mM completely prevented or severely inhibited utilization of lactose, fructose, glycerol, succinate, acetate and pyruvate. It prevented lactose utilization by inhibition of the synthesis and activity of both β-galactosidase and galactoside permease. 3FG-resistant mutants were isolated which were deficient in the Enzyme II of the phosphoenolpyruvate-dependent phosphotransferase system specific for glucose and for 3FG. Our findings support the view that the ‘glucose effect’ may depend upon glucose itself or a glucose derivative, rather than upon catabolic products.

Summary: Axenic cultures of Puccinia graminis f. sp. tritici cannot reduce sulphate for sulphur-amino-acid synthesis. Although [35S]sulphide is incorporated into these amino acids, the fungus still has a nutritional requirement for a sulphur amino acid. Most of the incorporated sulphide was found in the culture filtrate in the form of cysteine, s-methylcysteine, glutathione and cysteinylglycine. There were appreciable amounts of labelled methionine in mycelial protein but very little of this amino acid appeared in the culture filtrate. The results indicate that the nutritional requirements for sulphur amino acids by the rust fungus is caused by loss of cysteine and some of its derivatives into the medium.

Summary: Pseudomonas AMI, which can grow on C1 compounds, also grows well on pyruvate and lactate. Extracts of the pyruvate-grown organism catalysed the synthesis of only small amounts of phosphoeno/pyruvate directly from pyruvate. Experiments tracing the incorporation of radioactivity into bacterial constituents from pyruvate or lactate, each labelled in the C-1 or C-3 position, indicated that these substrates are mainly assimilated after removal of the C-1 atom. A mutant, C5, of Pseudomonas AMI was isolated by a procedure intended to select organisms lacking phosphoeflo/pyruvate carboxylase. Although this enzyme was present in mutant C5, growth on lactate or pyruvate was impaired and there was no growth on C1 compounds or ethanol. Good growth on each of these substrates was restored by a supplement of glyoxylate or glycollate.

A scheme is proposed for the assimilation via malate synthase and phosphoenol-pyruvate carboxykinase of the C2 fragment produced from pyruvate and lactate.

Summary: The extracellular lipolytic activity produced by Anaerovibrio lipolytica strain 5s is associated with large particles composed of protein, lipid and nucleic acid. These particles have a membranous appearance when viewed by electron microscope and in the ultracentrifuge they have a sedimentation coefficient (S 20, W) of 13.1 S. Numerous ‘blebs’ are associated with the walls of Anaerovibrio lipolytica during the phase of logarithmic growth when lipase production is maximal. The lipolytic enzyme is released from the bacteria without visible bacterial lysis.

Summary: Protoplast release from young mycelium of Schizophyllum commune by a lytic enzyme preparation from Trichoderma viride was accompanied by degradation of the three wall polymers, s-glucan, r-glucan and chitin. Part of the s-glucan was resistant. The resistance of s-glucan to enzymatic degradation increased with culture age and concomitantly the yield of protoplasts was reduced. Isolated s-glucan was also partly resistant to degradation, probably due to its crystallinity. In living cells s-glucan protected chitin and possibly r-glucan against degradation by external enzymes. s-glucanase, r-glucanase, chitinase and exo-laminarinase were purified from the Trichoderma enzyme mixture. Addition of only s-glucanase and chitinase was essential for protoplast release; r-glucan was degraded endogenously.

Summary: The effects of the self-inhibitor, N,N-dimethylguanosine, on the metabolism of spores of Dictyostelium discoideum were investigated. It is concluded that since both self-inhibited and activated spores had similar initial Q O2 values, the site of action of the self-inhibitor was not on respiratory metabolism. Incorporation of [U14C]glucose revealed that organic acids and amino acids accumulated in self-inhibited spores and that protein synthesis was inhibited. Succinic acid accumulated only when the activated self-inhibited spores became anaerobic so its accumulation was not directly related to self-inhibition. Phenylalanine, methionine and tryptophan were absent in dormant spores but appeared after heat-activation and accumulated in self-inhibited spores. The accumulation of amino acids was related to the inability of self-inhibited spores to synthesize protein. In vivo analysis showed that protein synthesis began early in spore germination and that the self-inhibitor immediately arrested this process when added to activated spores and slowed it when added to germinating spores. No effect of self-inhibitor on RNA synthesis during germination was detected. Thus, we conclude that the self-inhibitor, a methylated base of guanine, regulated spore germination by preventing the synthesis of protein necessary for subsequent development.

Summary: The advancing edge of the active swarm of Bacillus licheniformis is surrounded with a fringe of non-flagellate, septate filaments. The mucoid portion of the colony is composed of short, flagellate bacilli. The two phases resemble Rough and Smooth forms occurring in the same colony.

Summary: Reciprocal crosses and repeated backcrosses have shown that the distance of growth before the first mycelium of a population exhibits ‘senescence’ and the rate at which the remaining mycelia exhibit ‘senescence’ are cytoplasmically inherited in several races of Podospora anserina. A nuclear gene (the plus mating type allele of race S) was found to influence the timing of ‘senescence’ in interracial hybrids carrying the cytoplasmic determinants of race A. Known nuclear genes segregated normally in the interracial crosses employed in the work.

Summary: Phage 34 transduces some R factors to Proteus mirabilis strain 13. These trans-ductants can transmit the R factor by conjugation. Transduction frequencies vary from 1×10−5/plaque-forming unit (p.f.u.) adsorbed (higher than chromosomal marker transduction) to 5×10−8/p.f.u. adsorbed. Transduced drug resistance markers of other R factors were detected only in the presence of resident fi − factors in recipients and recombination between the R factors concerned could account for the fact that these transductants were able to transmit hybrid R factors by conjugation. Transduction of portions of R factors, including an fi + factor occurred and some transductants were unable to transmit transduced markers by cell-to-cell contact despite the presence of resident fi − R factors. In transductants which were conjugally infectious the transduced R factor did not appear to be associated with the genome of the phage. Incompatibility had no demonstrable effect on transduction rates. The presence of an fi − resident factor reduced transduction rates when these were measurable in the absence of the resident. In two cases which involved N group resident plasmids this reduction was possibly due to restriction and host modification of transduced R factor DNA. Because of the natural resistance of the P. rettgeri host only R factors which bore the marker for kanamycin resistance could be selected in transductions with phage 7.R49. Only a T group factor was transduced. The transduction frequency was similar to that of a chromosomal marker but transductants were non-infectious.

Summary: Two complementary frameshift mutations in the trpA gene of Escherichia coli have been isolated following recombination of the pseudowild double mutant with the wild-type strain. One of these mutations recombines with a third, distal frameshift mutation to give a slow-growing Trp+ strain. Consideration of the relevant RNA codon sequences suggests that the slow-growing recombinant should contain a unique tryptophan codon in the altered reading phase between the frameshift mutations. This prediction has been verified by analysis of the purified tryptophan synthetase A protein from the double mutant. The preferential synthesis of A protein normally associated with prolonged tryptophan starvation is eliminated in the strain which has a tryptophan residue in its A protein.

Summary: Escherichia coli uvr502 bacteria have a higher spontaneous mutability and are 4.6 times more u.v.-sensitive than isogenic wild-type bacteria. Bacteria deficient in dimer excision (uvrA6) are 15 times more u.v.-sensitive than isogenic uvrA + bacteria. Double mutant uvrA6 uvr502 bacteria are only 1.5 times more u.v.-sensitive than uvrA6 bacteria and double mutant uvr502recA56 bacteria were as u.v.-sensitive as recA56 bacteria, suggesting that there exists a uvrA +- and recA +- dependent repair pathway which is blocked by the uvr502 mutation. We suggest that the uvr502 mutation affects primarily the excision repair pathway. Strict additivity of the uvr502 and recB21 mutations on u.v. sensitivity suggests that recB +-mediated repair acts in part of repair pathway different from excision repair. U.v.-induced DNA degradation in the uvr502 bacteria is only slightly greater than that in uvr + bacteria and the uvr502 mutation does not affect the ဘrecklessမ character of u.v.-induced DNA breakdown in the recA56 bacteria. The avr502-uvrA6 and uvr 502 rec A56 or uvr502recB21 double mutants have spontaneous mutation frequencies similar to that of the single uvr502 strain.

Summary: The sensitivities of lon strains of Escherichia coli to the filament inducing agents nalidixic acid, gentian violet, crystal violet and penicillin were examined. The strains used included those in which the lon gene is suppressed by sul, exr and rec as well as strains in which the Lon phenotype is expressed. The sensitivity of the bacteria to agents which act on DNA was reduced when lon was suppressed, but the extent of the decrease in sensitivity was dependent on the mechanism by which suppression was effected. Sensitivity to penicillin, on the other hand, was reduced only in the sul strain and by a mechanism which appears to be independent of the direct suppression of lon. A model to account for these results indicates possible sites at which the lon, sul, exrA and recA genes are expressed.

Summary: Two types of subunit, with molecular weights estimated to be 28000 and 8000, were demonstrated in the cholera exo-enterotoxin by sodium dodecylsulphate polyacrylamide electrophoresis. The light (L) component but not the heavy (H) was demonstrable in the antigenically identical natural toxoid. A spilt of the H component by reduction and alkylation suggested it to be a disulphide-bridged dimer.

Gel filtration at acid pH permitted separation of the H and L subunits in isolated form. Fractions with isolated subunits were non-toxic (skin test), in contrast to fractions with intermediate filtration rate containing both subunits. Fractions with a L:H ratio of about 3.5 were even more toxic than the intact toxin (L:H ratio about 2). Addition of the isolated subunits to toxic fractions generally caused inhibition and only exceptionally potentiation of toxicity.

The data suggest that the cholera toxin might consist of an aggregate of seven L and one H subunits while the natural toxoid seems composed of only the aggregated L chains.

Summary: The conditions for induction of thymine deficiency by sulphonamides and their effect on viability were investigated in Escherichia coli. In minimal medium, sulphonamides led to a slight decrease in the viable count over a prolonged length of time. This decrease was more pronounced in Casamino acid supplemented medium. Concentrations of as low as 1 μg/ml of sulphamethoxazole or some other sulphonamides killed up to 99.9% of bacteria within 4 h after manifestation of inhibition in media supplemented by Casamino acids and inosine. No strict relation was found between sulphonamide concentration and the number of surviving bacteria. In all experiments sulphonamides exerted their effects after a time lag of 3 to 4 h. Addition of thymidine prevented death. Effects similar to those in media containing Casamino acids and inosine were demonstrated in growth medium containing human blood. The possible role of this kind of action of sulphonamides in chemotherapy is discussed.

Summary: Strains of Penicillium chrysogenum derived from a virus-infected culture have been investigated for reduction in virus titre. These strains were obtained following treatment with either heat (74°C) or specific antimetabolites. Virus titres were measured with a combination of immunological and radiochemical assays. One strain was cured of the mycophage and exhibited an increased growth rate and stability. Other strains, which show an apparent reduction in intracellular virus titre, actually released increased levels of virus from cells. Lytic plaques developed when infected strains were grown on an unbuffered, high lactose-containing medium, and virus titres could be correlated with plaque formation. All strains, regardless of virus titre, retained the ability to synthesize penicillin.

Summary: Growth of Aspergillus nidulans in a thin layer of liquid minimal medium was exponential. The dry-weight doubling time at 37°C was 10 h when the inoculum density was 1.3×105 viable spores/ml. Compared with shake-flask cultures, growth was equally fast, but exponential growth ceased sooner. Aerial conidiation occurred, however, and the method was simple and permitted the collection of extracellular products in the absence of cell breakage.

Summary: The growth of a mycelium of Podospora anserina ceases after a period of time characteristic for a given race. As the mycelium grows out from the germinated spore it passes through two physiological states; a ‘non-senescent’ state where the growth potential of the various regions of the growing mycelium remains constant and a ‘senescent’ state where the growth rate remains constant but the growth potential of the mycelium progressively decreases.

The ‘senescent’ state is apparently due to the presence of a variant cytoplasmic determinant which is discontinuously distributed among the cells of the mycelium and whose concentration increases exponentially during growth. The rate of transformation from the ‘non-senescent’ to ‘senescent’ state is directly proportional to the number of hyphae per mycelium. The median length of growth can be greatly extended by short periods of growth in the presence of cycloheximide or cyanide, but not by a variety of other drugs which inhibit mitochondrial functions.