Aspergillus nidulans and Penicillium chrysogenum produce specific cellular siderophores in addition to the well-known siderophores of the culture medium. Since this was found previously in Neurospora crassa, it is probably generally true for filamentous ascomycetes. The cellular siderophore of A. nidulans is ferricrocin; that of P. chrysogenum is ferrichrome. A. nidulans also contains triacetylfusigen, a siderophore without apparent biological activity. Conidia of both species lose siderophores at high salt concentrations and become siderophore dependent. This has also been found in N. crassa, where lowering of the water activity has been shown to be the causal factor. We used an assay procedure based on this dependency to reexamine the extracellular siderophores of these species. During rapid mycelial growth, both A. nidulans and P. chrysogenum produced two highly active, unidentified siderophores which were later replaced by a less active or inactive product--coprogen in the case of P. chrysogenum and triacetylfusigen in the case of A. nidulans. N. crassa secreted coprogen only. Fungal siderophore metabolism is varied and complex.

The germination-essential substance (germination factor [GF]) that is lost from conidia of Neurospora crassa on exposure to solutions of low water activity has been isolated and identified as a group of iron-transport compounds, or siderochromes. The principal siderochrome of conidia is ferricrocin, a cyclic hexapeptide. A closely related substance, ferrichrome C, is tentatively identified as a minor constituent. The same substances are also present in extracts of mycelium along with small amounts of a third siderochrome, which has not been identified. The GF activity of culture filtrates is due to coprogen, the only siderochrome previously identified with N. crassa.

When the water activity (as.) of the medium is lowered by the addition of NaCl or nonelectrolytes, an inhibition of germination, growth rate, and total growth is observed in Neurospora crassa. Inhibition of conidial germination is separable from the other effects and is caused, in large measure, by the loss from conidia in media of low water activity of a substance that is essential for their germination. The substance is detectable in the medium and is also extractable from cultures. It is dialysable and thermostable, and it appears to be highly active. It is not detectable in complete medium.In the course of experiments designed to obtain basic information for a genetic study of water metabolism in Neurospora crassa, we have observed an unusual and, it appears, previously unknown cellular reaction to media of low water activity. with distilled water, and dried to constant weight. All growth data are averages of duplicate flasks.The water activity of Vogel's medium N with 2% sucrose is estimated to be 0.996, from Raoult's law. For complete medium, we estimate a,. to be about 0.994. The water activities were further lowered by addition of NaCl, sucrose, glucose, or glycerol. The amounts of solutes needed to bring a,.to the desired values were calculated from the tables given in Robinson and Stokes (2), Scott (3), and Ingram (4). In the calculations, additivity of the values of 1-a, of the medium and solute was assumed. RESULTS Effects of lowering a,. on germination and growth As has been found with other microorganisms (3) the effects of lowered a,. are threefold: (1) prolongation of the lag phase, including the time required for germination of conidia, (2) reduction of the growth rate, and (3) reduction of the total amount of growth. These responses are observed whether a. is lowered with NaCl or with nonelectrolytes (Fig. 1). The inhibitory effects produced by different solutes are qualitatively alike, but they show consistent quantitative differences; the order of increasing toxicity under the conditions of the experiment shown in Fig. 1 is glycerol < NaCl < glucose < sucrose.The foregoing conclusions hold for values of a,. less than about 0.98. At higher a., secondary effects, probably related to the metabolism of the solutes, may interfere. This is particularly noticeable with glucose which, at a,. ) 0.985, accelerates growth.In the remainder of this paper, we describe experiments performed in an attempt to overcome the inhibitory effects mentioned above.Additions of cations to the medium Previous results by Slayman and Tatum (5) with Neurospora, Brown and Gibbons (6) with halophilic bacteria, and LaHaye and Epstein (7) with higher plants suggested that additions of K+, Mg2+, or Ca2+ to the media might alleviate some of the effects of low a,.. The concentrations of these cations, and also of the trace metals in the media, were therefore increased. Doubling the K+ concentration (normally 36.8 mM) produced no, or a doubtful, effect on germination and growth (Table 1). Likewise, only a slight effect wa...

Neurospora crassa conidia incubating in buffer at low water activities (a,) release a germination-essential component as well as 260-nm absorbing and ninhydrin-positive materials, regardless of whether an electrolyte or non-electrolyte is used to reduce a,. Chloroform and antibiotics known to increase cell-membrane permeability have a similar effect. This suggests that membrane damage occurs in media of low a, and that an increase in permeability is responsible for the release of cellular components. The damage caused in media of low a, is nonlethal in most cases, and the conidia recover when transferred to nutrient medium. 261 on August 1, 2020 by guest

The highly active extracellular siderophores previously detected in young cultures of Aspergillus nidulans and Penicillium chrysogenum have been identified as the cyclic ester fusigen (fusarinine C), and its open-chain form, fusigen B (fusarinine B).