- Volume 99, Issue 1, 1977

Summary: The stimulatory action of zinc on aflatoxin production by Aspergillus parasiticus nrrl3240 has been investigated by studying the levels of tricarboxylic acid (TCA) cycle intermediates and related enzymes in the fungal mycelium. During the stationary phase of growth, the levels of α-keto acids declined in zinc-sufficient cultures compared with those in zinc-deficient cultures. TCA cycle enzymes did not show any significant changes due to zinc availability. In zinc-deficient cultures, enzymes of the TCA cycle had maximum activity on the fourth day, after which their activity declined. In zinc-sufficient cultures, some enzymes showed maximum activity on the fourth day, others on the second day.

Summary: Chitin synthase (EC 2.4.1.16) from membrane preparations of Aspergillus nidulans was characterized and the optimum conditions for enzyme activity were determined. A reaction velocity-substrate concentration plot was sigmoidal, but was hyperbolic in the presence of N-acetylglucosamine when the K m for UDP-N-acetylglucosamine was 3·1 mmol 1−1. Chitin synthase activity could be increased sixfold by digestion of enzyme preparations with trypsin for short periods. The trypsin-activated enzyme had altered kinetic and storage properties.

SUMMARY: The development of resistance to amphotericin methyl ester, measured in terms of the amount of drug required to induce a standard rate of release of K+ from suspensions of washed organisms, has been followed in Candida albicans in starved cultures under controlled conditions of aeration, stirring and temperature. Resistance develops at a rate which increases with the rate of aeration, limited by the onset of damage due to turbulence. Resistance decreases rapidly if gassing with N2 is substituted for aeration, but sensitivity does not reach that of exponentially growing cells. Resumption of aeration is followed by a slow recovery of resistance.

The addition of inhibitors of protein synthesis (trichodermin, verrucarin) or uncoupling agents (2,4-dinitrophenol, sodium azide) at the beginning of starvation results in an increased rate of development of resistance. Adding inhibitors at a later stage, when resistance has developed after 72 h aeration, does xnot affect the decrease in resistance produced by gassing with N2 but the presence of trichodermin or verrucarin delays the recovery of resistance on resumption of aeration.

SUMMARY: Pseudomonas aeruginosa paci grows poorly on l-lysine as sole source of carbon but mutant derivatives which grow rapidly were readily isolated. Studies with one such mutant, P. aeruginosa pac586, supported the existence of a route for l-lysine → catabolism which differs from those reported previously in other species of Pseudomonas. The postulated route, the cadaverine or decarboxylase pathway, is initiated by the decarboxylation of l-lysine and involves the following steps: l-lysine → cadaverine → i-piperideine → 5-aminovalerate → glutarate semialdehyde → glutarate. Evidence for this pathway is based on the characterization of the pathway reactions and the induction of the corresponding enzymes by growth on l-lysine. The first three enzymes were also induced by growth on cadaverine and to a lesser extent by 5-aminovalerate. No evidence was obtained for the presence of pathways involving l-lysine 2-monooxygenase or l-pipecolate dehydrogenase, but another potential route for l-lysine catabolism initiated by l-lysine 6-aminotrans-ferase was detected. Studies with mutants unable to grow on l-lysine supported the existence of more than one catabolic pathway for l-lysine in this organism and indicated that all routes converge on a pathway for glutarate catabolism which generates acetyl-CoA. Pipecolate catabolism also appeared to converge on the glutarate pathway in P. aeruginosa. The results suggested that the growth rate of the parental strain is limited by the rate of transport and/or decarboxylation of l-lysine. The cadaverine pathway was present, but not so highly induced, in the parental strain P. aeruginosa paci. Pseudomonas fluorescens contained enzymes of both the cadaverine (decarboxylase) and oxygenase pathways, strains of P. putida (biotypes a and b) contained enzymes of the oxygenase pathway but not the decarboxylase pathway and P. multivorans appeared deficient in both. All these species possessed l-lysine aminotransferase activity.

SUMMARY: A wall + membrane preparation from Micrococcus luteus was used to synthesize radioactively labelled peptidoglycan. The newly synthesized peptidoglycan either was cross-linked by transpeptidation to existing wall or remained associated with the membrane fraction but was not cross-linked. The average biosynthetic chain lengths, calculated from the ratio of free reducing groups of muramic acid to total muramic acid, were 66 disaccharide units for cross-linked and 26 disaccharide units for the uncross-linked material. The latter value was confirmed by the release of lactyl peptide side chains by β-elimination. Benzylpenicillin (1 µg ml−1) inhibited cross-linking but not overall synthesis of glycan whereas at concentrations above 10 µg ml−1 overall glycan synthesis was slightly inhibited. In the presence of 100 µ;g benzylpenicillin ml−1 the incorporation of disaccharide units to existing walls decreased to 25% of the control. This residual incorporation represented extension by transglycosylation of peptidoglycan already cross-linked to existing walls. Chains with an average length of between 30 and 45 disaccharide units were added during a 30 min incubation period. However, if incubation was continued for up to 120 min (in the presence of 100 µg benzylpenicillin ml−1) a considerable amount of the newly synthesized peptidoglycan was lost from the purified wall because autolytic enzymes were expressed in the wall + membrane preparation after the action of the antibiotic.

Summary: The rumen flagellate Piromonas communis is the zoospore of a phycomycete fungus inhabiting the rumen. Zoosporogenesis was stimulated by a dietary component (the inducer), and inhibited by compounds affecting membrane structure and function, but not by inhibitors of protein synthesis. The zoospores showed taxis towards the tissues surrounding the inflorescence of Lolium perenne L. in the rumen, invading principally the stomata and damaged tissues. The zoospores germinated on this substratum and the rhizoids of the developing vegetative stage penetrated the tissue, taking up 14C from labelled plant material, which was incorporated into the fungal cells. The conditions for maximum flagellate production (39 °C, pH 6·0 to 7·0, high concentration of CO2, absence of O2) resembled those found in the rumen. The organism was cultured in an undefined medium in vitro in the absence of other flagellates.

SUMMARY: The uhp gene, which specifies the uptake of hexose phosphates, and several other genes in the vicinity of minute 81 on the E. coli linkage map have been located by phage-mediated transductions. The order found is mtl-gpsA-pyrE-gltC-uhp-tna-dnaA. Alleles specifying the Uhp− and Uhp+ characters were separated from that specifying constitutivity of hexose phosphate uptake (Uhpc). Although cotransduction frequencies between gltC and uhp as high as 90%, and between uhp and tna as high as 80%, were observed, these frequencies were unusually strongly dependent on which marker was selected. This may be due to the proximity of the uhp region to the point of origin of chromosome replication.

SUMMARY: All seven mitotic chromosomes of the haploid cellular slime mould Dictyostelium discoideum have been identified and numbered using Giemsa banding techniques. Although size differences are not marked, the size distribution is one long, three medium length, and three shorter chromosomes. Each chromosome has a distinctive banding pattern. All seven chromosomes appear to have terminal or near terminal centromeres. Apparent connexions between chromosomes explain the controversy concerning chromosome number (five or seven) in D. discoideum. The mitotic index of exponential-phase axenic cultures of D. discoideum is 2%. By returning aggregating amoebae to growth medium the mitotic index could be reproducibly raised to approximately 6%.

SUMMARY: Conditions for obtaining colchicine-induced metaphase arrest of the chromosomes of the cellular slime mould Dictyostelium discoideum are described. Using this technique, which increases the mitotic index of exponentially growing amoebae from 1·5 to 11%, it has been demonstrated conclusively that D. discoideum has a haploid chromosome number of seven and that strain ax2 (atcc24397) is not highly aneuploid as has recently been suggested. Each of the seven chromosomes can be identified by a characteristic banding pattern seen after staining with Giemsa.

Summary: Non-flagellate Salmonella mutants representing complementation groups H1, H2, flaAI, flaAII, flaAIII, flaB, flaC, flaD, flaE, flaF, flaK, flaL, flaM, flaN, flaP, flaQ and flaR were examined for their sensitivity to the flagellotropic bacteriophage χ. H1, H2, flaL and flaR mutants were shown to be sensitive to χ by spot tests, although they were less sensitive than a parental flagellate strain; flaAIII mutants were also shown to be sensitive, although even less so, by their ability to propagate the phage at the spotted area. In liquid medium neither adsorption nor propagation of χ was observed on any of these sensitive non-flagellate mutants. Electron micrographs showed that χ particles were attached to the superhooks of flaR mutants, but the receptor sites of other sensitive non-flagellate mutants could not be recognized.

Summary: Liquid batch cultures of Fusarium culmorum showed a typical growth pattern coupled with a rapid fall in the pH of the medium followed by a rise in pH after the cessation of growth. Production of conidia was associated with this final pH rise. In glucose-limited continuous cultures without pH control, a low pH was maintained and the organism remained totally vegetative. Adjusting the pH to values above 5·0 for 6 h or more led to maximum production of conidia. The rate of development of conidia was independent of the growth rate but the rate of production of conidia was greatest at high growth rates. Glucose inhibited both production of conidia and the rate of development of conidia in phosphate-limited and nitrogen-limited cultures. The increased rates of conidium production at high growth rates were achieved not by the greater proliferation of apices, but by the development of a greater proportion of the apices into conidia.

Summary: The rate of carbon dioxide fixation by Fusarium culmorum during vegetative growth in glucose-limited continuous culture at pH 3·5 was 0·036 μmol CO2 fixed (mg hyphae)−1 h−1; during conidium production at pH 6·5 it reached a maximum value of 0·29 μmol CO2 fixed (mg hyphae)−1 h−1. During growth in phosphate-limited continuous cultures, containing sufficient glucose to suppress conidiation, the rate of CO2 fixation was 0·045 μmol CO2 fixed (mg hyphae)−1 h−1, and did not increase substantially following an increase in pH to 6·5. The internal hyphal pH remained at approximately 6·5 despite changes in the external pH from 3·5 to 6·5. Phosphoenolpyruvate carboxykinase (EC 4.1.1.32) and pyruvate carboxylase (EC 6.4.1.1) activities estimated in hyphal extracts gave a fixation rate approximately three times the rate of CO2 fixation in vivo in glucose-limited and phosphate-limited vegetative continuous cultures at pH 3·5. In conidiating cultures, phosphoenolpyruvate carboxykinase and pyruvate carboxylase activities approximately equalled the highest CO2 fixation rates in vivo. Fixed 14C was distributed amongst all the major cell components, with the highest percentage in the conidial protein fraction.

SUMMARY: Intracellular accumulation of zinc by Candida utilis nrrl-y-7634 was mediated by an energy- and temperature-dependent, highly specific process exhibiting saturation kinetics. In zinc-supplemented medium, uptake occurred only during the lag and late-exponential phases; this type of transport did not occur with zinc in bacteria nor with iron in either yeast or bacteria. Cells of C. utilis did not possess a zinc-efflux system; they could reduce their level of intracellular zinc only by dilution of the metal into daughter cells. Zinc-deficient organisms accumulated 12 times more zinc than did cells of the same culture age grown in zinc-supplemented medium. The varied, but experimentally reproducible levels of intracellular zinc that occurred in response to the physiological and environmental parameters had no detectable effects on respiration, rate of growth, total cell yield, or cell viability. Neither the mechanism underlying the cyclic accumulation of zinc nor the function of such behaviour are understood.

SUMMARY: Synchronous cultures obtained by selection at division were used to investigate the occurrence of cold-sensitive stages during the division cycle of Escherichia coli (Îindâ). There are two such stages within the 50 min cycle: one (early) at 10 to 20 min and the other (late) at 40 to 45 min. Similar results were obtained from calculations based both on the age frequency distribution of cells in exponential growth and on the size of the populations which accumulate as a result of a single change of temperature. Times of about 17 and 44 min were found for the early and the late stages, respectively. It is concluded that the two-step doubling of E. coli k12 cultures synchronized by a single cold shock is due to two cold-sensitive stages in the division cycle.

A micro-organism which grows on cyclohexane as sole carbon and energy source has been isolated from estuarine mud flats. It has been tentatively identified as a Nocardia. The organism, which is auxotrophic for biotin, grows on cyclohexane (supplied as a vapour) with a mean generation time of about 10 h and a Y SUB of 59·9 g dry wt (mol cyclohexane)−1. Growth on cyclohexane leads to the production of intracytoplasmic membrane structures which are not present after growth on succinate. Growth, respiration and enzyme studies are consistent with the degradation of cyclohexane via cyclohexanol, cyclohexanone, caprolactone and ε-hydroxycaproate. Extracts of organisms grown on cyclohexane contained cyclohexanol dehydrogenase, cyclohexanone monooxygenase and ε-caprolactone hydrolase; these enzymes are absent from, or at very low activity in, extracts of organisms grown on succinate.