- Volume 105, Issue 1, 1978

SUMMARY: Protoplasts were produced from Aspergillus nidulans mycelium using Trichoderma lytic enzyme. The influence of KC1 and MgSO4as stabilizer systems on the morphological variation of protoplasts produced during digestion and the pattern of release from hyphae were compared. The results suggest that protoplast release in the presence of KC1 followed a sequential fractionation of the hyphae with ‘early’ protoplasts originating from the tip regions and ‘late’ protoplasts from the distal regions. In MgSO4-stabilized systems the hyphae were disrupted in a less ordered fashion. Between 12 and 16% of the mycelial protein was recovered in protoplast form using the systems described.

The level of chitin synthase (EC 2.4.1.16) and the capacity for trypsin-activation of the enzyme in protoplast fractions was investigated. In KC1-stabilized systems, ‘early’ (1 h) fractions possessed higher specific activities than later fractions. Activatable enzyme was low in the early fraction but was present at high levels in later fractions. It is suggested that these observations are consistent with a model relating active and activatable enzyme to hyphal growth.

SUMMARY: Cells of Moniliella tomentosa were grown in the presence of several short-chain alcohols (C1 to C4). n-Propanol and n-butanol induced the appearance of a mitochondrial cyanide-insensitive respiration without affecting the normal cytochrome chain whereas methanol and ethanol had no effect. Preliminary experiments indicated that the C3 and C4 alcohols interfered with oxidative phosphorylation, which explains the low growth yield and the accumulation of ethanol in the growth medium.

Thirteen strains of bacteria able to grow on phenanthrene were isolated from soil; they included fluorescent and non-fluorescent pseudomonads, vibrios and unidentified bacteria. Two of the pseudomonads, like Aeromonas sp. s45p1, also grew on naphthalene. In all strains, growth on phenanthrene induced the enzyme responsible for the conversion of 1-hydroxy-2-naphthoate to 2-carboxybenzaldehyde, NAD-dependent 2-carboxybenzalde-hyde dehydrogenase and protocatechuate oxygenase, but not salicylate hydroxylase, catechol oxygenase or NAD(P)H-dependent 1-hydroxy-2-naphthoate hydroxylase. Growth on naphthalene induced salicylate hydroxylase and catechol oxygenase. It is suggested that the catabolism of phenanthrene occurs via protocatechuate in all these bacteria, and that the pathways for degradation of phenanthrene and naphthalene are separate.

Lipopolysaccharide (LPS) was isolated from two strains of Agrobacterium tumefaciens and analysed. It contained phosphate, hexose, hexosamine, fatty acids and 2-keto-3-deoxyoc-tonic acid. Of the compounds commonly found in LPS, heptose and galactosamine were absent. The fatty acid composition of the agrobacterial LPS is unique, since only 3-hydroxy-tetradecanoic acid and 3-hydroxyhexadecanoic acid were significant components. 3-Hydroxyhexadecanoic acid was linked via an amide bond to the carbohydrate backbone. Non-hydroxylated fatty acids were present only as impurities (< 3%).

An efficient technique for the enrichment of mutants of Mucor racemosus by differential freeze-killing is described. Ungerminated spores were resistant to freeze-killing. During germination susceptibility increased up to a maximum coinciding with the appearance of germ tubes. Under optimum conditions a thousandfold preferential survival of growth-limited spores over actively germinating spores was obtained. Using this differential killing, cultures treated with mutagen were enriched with respect to auxotrophic and temperature-sensitive mutants, with the desired mutants constituting a large fraction of the survivors of the freeze-thaw treatments. This technique may be applicable to mutant enrichment in other filamentous fungi.

Thirty-two clinical isolates of anthropophilic dermatophytes were examined for their capacity to produce antibiotics in liquid culture and on human stratum corneum in vitro. Antibiotics were detected and classified using agar diffusion bioassays and chromatographic analysis. Twenty-four isolates produced antibiotic substances in liquid culture filtrates; some strains produced more than one antibiotic. Only four isolates produced detectable levels of antibiotics when grown on stratum corneum unless an artificial sweat mixture was used as a nutrient supplement, when the number rose to 11. Representatives of all species studied produced benzyl penicillin-like substances. Some Trichophyton isolates also produced streptomycin-like antibiotics, a characteristic previously unrecorded for eukaryotic organisms. Other antibiotics, which apart from azalomycin F could not be properly classified, were produced by Epidermophyton floccosum. Antibiotic production occurred over the normal skin temperature range but sometimes the type of antibiotic produced and the frequency of detection appeared to be influenced by the incubation temperature.

Effective nitrogenase synthesis by nostocacean cyanobacteria (including one mutant strain unable to form heterocysts or fix nitrogen aerobically) was induced in the light in the absence of molecular oxygen. Anaerobiosis was maintained during induction by treating the organisms with dichloromethylurea which prevents photosynthetic oxygen production. Under these special conditions both synthesis and activity of nitrogenase were light-dependent, the required ATP being produced by cyclic photophosphorylation. Enzyme synthesis and activity were also dependent on the availability of an organic substrate that could serve both as a general source of carbon and as a source of reductant. The organic requirement could be fulfilled by the intracellular glycogen reserve or, in facultative hetero-trophs, by a utilizable sugar (e.g. glucose). Nitrogenase synthesized anaerobically was highly susceptible to inactivation by molecular oxygen in vivo: exposure of a suspension of anaerobically induced filaments to 20 % (v/v) O2 for 1 h caused total and irreversible destruction of the enzyme. Anaerobic nitrogenase synthesis was not accompanied by the differentiation of mature heterocysts, the morphogenetic process being arrested at an early (proheterocyst) stage. After the gratuitous anaerobic synthesis of nitrogenase, introduction of either N2, nitrate or ammonia to the illuminated, anaerobic suspension resulted in a rapid accumulation of cyanophycin granules in both vegetative cells and proheterocysts. Cyanophycin was randomly deposited in vegetative cells, but localized at the cell poles of the proheterocysts. The bearing of these findings on the role played by the heterocyst in nitrogen fixation is discussed.

The cell cycle of Tetrahymena pyriformis st was studied using synchronous cultures prepared by continuous-flow size selection. These organisms had cell cycle times (mean values 151 and 186 min in the first and second cycles respectively) similar to but not identical with the mean generation times of exponential cultures from which they were selected (mean value 165 min). The median volume of cells averaged over the duration of the cell cycle in synchronous cultures was similar to that found in exponential cultures. Total cell DNA, RNA and protein doubled during the cell cycle: there was one discrete S phase which occupied 40 to 50 % of the cell cycle time during the period between the end of the selection procedure and the first synchronous division. Total cell protein and RNA increased continuously.

The rate of glycylleucine uptake by Group N streptococci varied widely. One strain of Streptococcus cremoris did not transport the dipeptide or utilize tripeptides. In peptide-utilizing strains, amino acid, dipeptide and tripeptide transport were distinct, although dipeptides inhibited tripeptide utilization. Specificity determinants for peptide transport and utilization were similar to those reported in Gram-negative bacteria. Peptide utilization in S. lactis was not completely dependent on the transport of intact peptides.

SUMMARY: Spore germination was used as an assay to measure the sensitivities of selected fungi (Achlya bisexualis, Cladosporium sp., Trichophyton mentagrophytes and Microsporum gypseum) to cycloheximide and to determine their abilities to adapt to the drug. Two patterns of response were noted. The saprobes, A. bisexualis and Cladosporium sp., demonstrated acquired resistance. Spores from hyphae previously exposed to cycloheximide either germinated in the presence of concentrations of the drug that completely inhibited spores from unexposed hyphae (Achlya), or germinated with a shorter lag and to a greater extent in the presence of the antibiotic than did spores from unexposed hyphae (Cladosporium). Hyphae of Achlya adapted at concentrations of cycloheximide in which spores did not germinate and hyphae of Cladosporium adapted more rapidly than spores. Achlya adapted to only 12 μ m-cycloheximide whereas Cladosporium acquired resistance to 18 mm-cycloheximide. These fungi lost this acquired resistance after a single transfer to media lacking cycloheximide. The zoopathogens, T. mentagrophytes and M. gypseum, had a contrasting response, indicating constitutive resistance. Conidia from unexposed hyphae showed 90 to 100% germination on media containing up to 18 mm-cycloheximide; prior exposure to the drug did not affect their response.

Thirty-three collections of Agaricus from nature and several strains of cultivated Agaricus bisporus were compared by 16 criteria including type of sexuality, interfertility patterns, macroscopic and microscopic morphology, growth and cultural characteristics, production of extracellular enzymes, and isoenzyme patterns for one enzyme. All but three of the collections fell into six distinct groups as defined with respect to all criteria examined. Members within each group are clearly related, but relationships between groups could not be established. Sexuality and interfertility patterns are important criteria for distinguishing relatedness among the collections. The correlations observed provide a new and useful base of reference for the classification of members of the genus Agaricus. Several aspects of the basic biology of the genus have also been revealed.