- Volume 129, Issue 9, 1983

Most isolates of Nectria haematococca mating population (MP) VI detoxify pisatin, the major phytoalexin produced by pea. However, there is evidence of another pisatin tolerance mechanism that does not depend on pisatin degradation. To facilitate further studies on nondegradative tolerance, a bioassay was developed in which culture turbidity was used as a measure of mycelial growth. Under conditions of this assay, four N. haematococca MP VI isolates degraded 0·4 mm-pisatin within 15 h, but two other isolates required more than 24 h. A remaining group of four isolates did not degrade pisatin. These three groups were typified by the isolates 126–71. T-77 and 126–80, respectively. Regardless of their ability to degrade pisatin, however, all 10 isolates grew equally well after addition of 0·4 mm-pisatin, suggesting that detoxification may not be needed for pisatin tolerance during the short time interval of this assay. The existence of a nondegradative tolerance in isolate 126–71 was confirmed when additional experiments showed that growth could occur (i) before degradation had reduced pisatin below initially noninhibitory levels, and (ii) when pisatin degradation was inhibited by 2 % ethanol. When tested in growth medium, pretreatment with a noninhibitory concentration of pisatin for 3 h enhanced equally the ability of isolates 126–71 and 126–80 to tolerate pisatin. After cultures were pretreated with pisatin while temporarily suspended in buffer, isolates 126–71 and T-77 were stimulated to degrade pisatin, and they grew better than did isolate 126–80. Again, however, growth began before there was significant detoxification, especially for isolate T-77. Thus, these three isolates all appeared to have an inducible, nondegradative tolerance to pisatin. Nectria haematococca MP I isolate T-145 lacked an adaptive tolerance to pisatin and was more sensitive to pisatin than any of the MP VI isolates.

Isolates of Nectria haematococca mating population (MP) VI were found previously to possess an inducible tolerance to pisatin, a pea phytoalexin, regardless of their ability to detoxify this compound. Nondegradative tolerance to pisatin was further characterized by studying MP VI isolate 126-80, a strain that does not degrade pisatin. Tolerance was maximally induced in growth medium at 28 °C by a 3 h treatment with 0.1 mM-pisatin. Shorter time intervals or lower pisatin concentrations reduced the degree of induction. Adaptation to pisatin was inhibited if mycelium was temporarily suspended in buffer during the induction period. Enhanced tolerance in growth medium was lost completely within 4 h at 28 °C after pisatin was removed from the medium. However, incubation at 1 °C prevented both the induction and the loss of enhanced pisatin tolerance. These results suggest that the induction of tolerance requires an active metabolic response, and that the expression of tolerance is tightly controlled. Of six isoflavonoid derivatives tested that were structurally related to pisatin, only four were good inducers of pisatin tolerance. Cyanide and the polyene antibiotic amphotericin B also induced some pisatin tolerance. However, tolerance was not a general stress response, because three other antimicrobial compounds failed to induce tolerance. Besides inducing tolerance to itself, pisatin simultaneously induced nondegradative tolerance to the structurally related isoflavonoid phytoalexins medicarpin and phaseollin. A partial tolerance was also induced to the isoflavone biochanin A. Of the non-isoflavonoid toxicants tested, only amphotericin B was tolerated as a result of pisatin pretreatment. Tolerance to amphotericin B was expressed both as the ability to grow and as the decreased leakage of cellular constituents after treatment with normally inhibitory concentrations of amphotericin B. We suggest that the plasma membrane is modified during the induced adaptation to pisatin, and that such modifications could be involved in nondegradative tolerance to phytoalexins.

Natural-abundance 13C nuclear magnetic resonance spectroscopy was used to study the patterns of accumulation of osmotically active internal solutes in five different fungi. Four xerophilic fungi (Penicillium janczewskii, Eurotium chevalieri, Xeromyces bisporus and Wallemia sebi), and one non-xerophilic fungal species (P. digitatum) were grown at three different water activities (a w) on media containing sorbitol, glucose/fructose or NaCl as the controlling solute. Under all conditions studied, the major internal solutes detected in aqueous ethanol extracts of these fungi were simple polyhydric alcohols: glycerol, erythritol, arabitol and mannitol. The most important osmoregulatory solute accumulated by all species was glycerol. On the sorbitol and the glucose/fructose media, all five fungi were able to accumulate glycerol. However, when NaCl was used to control a w, only one species, W. sebi, was able to accumulate glycerol below 0.92 a w. Significant quantities of the controlling solutes were also present in the extracts.

When intact mycelia of P. janczewskii were examined by NMR, resonances of all the major internal solutes were clearly discernible, although they were not as well resolved as those from the fungal extracts. Relaxation measurements showed that the solutes were relatively mobile inside the cells.

Fractionation on sucrose density gradients of spheroplast lysates from Saccharomyces cerevisiae NCYC 366 yielded a fraction with a peak density of 1·05 g ml−1, intermediate between that of membranes and intracellular low-density vesicles. Electron microscopy showed the fraction to consist of membranes associated with intracellular vesicles. Evidence for the presence of plasma membranes in the association fraction was obtained by using 125I-labelled spheroplasts. The fraction was free from detectable amounts of cytochromes and DNA. Fractionation on sucrose density gradients of incubation mixtures containing isolated crude plasma-membranes and vesicles gave rise to a visible intermediate-density band, which electron microscopy showed to contain membranes associated with vesicles. Evidence for the presence of plasma membranes in the in vitro intermediate-density band came from incubating mixtures containing 125I-labelled crude plasma-membranes, and evidence for the presence of vesicles came from using 125I-labelled vesicles. Supplementing incubation mixtures with calcium chloride sharply increased the size of the intermediate-density band. Cycloheximide and methylbenzimidazol-2-yl-carbamate had no effect on its formation. Purified plasma-membranes failed to form an intermediate-density band when incubated with vesicles. Supplementing these incubation mixtures with calcium chloride did not produce an intermediate-density band, but caused extensive association of vesicles with plasma membranes that pelleted in gradients. Isolated vesicles were not osmotically active; their polyphosphate content was 1·61 μmol orthophosphate equivalent (mg vesicle protein)−1. They had diameters in the range 0·45-0·65 μm, as measured on electron micrographs of thin sections. The data reported provide further evidence for a role for intracellular low-density vesicles in envelope growth in S. cerevisiae.

Sixty-five strains of Clostridia of the butyricum group were studied by DNA-DNA hybridization, electrophoresis of cell proteins, gas-liquid chromatography, and fermentation of glycerol, inositol and ribose. The DNA-DNA hybridization results confirmed that strains of this group belong to two main species, Clostridium butyricum and C. beijerinckii. Five strains did not hybridize with the reference strains of these two species. Most of the strains could be identified by quantitative gas-liquid chromatographic analysis combined with fermentation patterns. The other strains could be identified by their protein electrophoretic patterns.

During studies on a desulphurizing, denitrifying effluent-treatment system, an organism which is able to grow aerobically and anaerobically on reduced sulphur compounds and hydrogen, while fixing carbon dioxide, was isolated. The new isolate is also capable of mixotrophic and heterotrophic growth on a wide range of substrates, and is therefore a facultatively aerobic, facultative autotroph. Comparisons with two similar species, Thiobacillus A2 and Paracoccus denitrificans, showed that the new isolate is significantly different from the other two, and merits separate classification. In view of its ability to oxidize reduced sulphur compounds, and because it is a chain-forming coccus rather than a rod, the new isolate has been given the generic name of Thiosphaera, and the species name pantotropha in recognition of its wide range of possible substrates.

Fifteen strains of Streptomyces, two of Streptoverticillium and one of Nocardia mediterranea were studied by the immunodiffusion technique and the results were compared with those of a previous phenetic study. Both the serological and phenetic analyses demonstrated that Nocardia mediterranea was different from strains of Streptomyces and Streptoverticillium. The latter two genera were phenetically distinguishable but demonstrated a comparatively close serological relationship. Those strains of Streptomyces and Streptoverticillium which shared a high number of precipitinogens also belonged to the same phenetic cluster, while strains which were less serologically related usually fell into different clusters. Thus there was good evidence of a general congruence between serological and phenetic similarity. The immunological results also supported the phenetic evidence in suggesting that many species in the genus Streptomyces should be combined. Similar evidence was obtained for the genus Streptoverticillium.