Copper And Differentiation

Studying the aspects of Streptomyces differentiation held more surprises. Extracting an agar plug with a brass cork borer from a plate with confluent growth of vegetative S. lividans mycelium resulted in a stimulation of development of aerial hyphae and of spores in the mycelium in the immediate surroundings where the plug was taken. This effect was not the result of mechanical stress because the stimulation of the development did not occur when a plastic device was employed to extract an agar plug. The source for the developmental induction was found to be the result of copper, one of the components of the alloy of which the cork borer was made (Kieser and Melton, unpublished). The effects of copper ions on reproductive growth was confirmed by Ueda et al. (50), who showed that a mutant of S. tanashiensis, deficient in both antibiotic production and sporulation, was restored in both aspects when extra copper salts were added to the medium. Although stimulation of the actinorhodin production in S. coelicolor by copper ions was also reported (50), this observation could not be reproduced by others (51). However, copper ions do seem to have an effect on (secondary) metabolism. The characteristic earthy odor of many streptomycetes and fungi is the result of geosmin (trans-1,10-dimethyl-trans-9-decanol) production. This volatile compound has a very low odor threshold. Dionigi et al. (52) observed that geosmin production is stimulated specifically by copper ions not only in S. tendae but also in the fungus, Penicillum expansum. The impact of copper ions on morphogenesis is not restricted to streptomycetes. The GRISEA mutant of the filamentous fungus Podospora anserina has a diminished production of aerial hyphae and a longer life-span than the wild type. This phenotype was reversed to wild-type characteristics upon the addition of extra copper salts to the medium (53). The protein encoded by the grisea gene turned out to be an ortholog of Macl, a copper-dependent transcription factor of Saccharomyces cerevisae (54). The wild-type grisea gene complements a macl mutant of S. cerevisae. The implication of these results is that GRISEA has a role in copper homeostasis in P. anserina similar to that of Macl in S. cerevisae. The observed phenotype for the grisea mutant shows a serious impact on morphogenesis, life-span control, and senescence, suggesting that disturbance of copper homeostasis does have a broad spectrum of effects and is better manifested in filamentous fungi than in yeast.

In S. lividans, an increase of the concentration copper in the growth media R2YE from 0.2 to 2 pM was sufficient to advance the switch from vegetative growth to the production of aerial hyphae by approx 24 h (Fig. 2). The abundance of aerial hyphae and spores formed, as judged from the "fluffy white appearance" and the intensity of the gray spore pigment, respectively, is higher in the presence of copper ions than eventually observed in control strains without extra copper added. The closely related strain S. coelicolor did not respond to the addition of extra copper. However, it should be noted that this strain is "quicker" when it comes to development than S. lividans. In the presence of 2 pM copper, salt differentiation in S. lividans is about as quick as that of S. coelicolor. The action of copper ions is unique and specific, no other metal ions stimulated the production of aerial hyphae and spores (50,51).

The importance of copper during the switch from vegetative growth to the reproductive growth was substantiated further by the effect of reduction of the bioavailable copper concentration by the addition of copper chelators. In media, equilibrium exists between Cu(I) and Cu(II). The reduced Cu(I) form is constantly oxidized by oxygen, whereas the oxidized species Cu(II) is reduced by reducing equivalents present in the growth medium. Upon binding of Cu(I) by the specific chelator

Fig. 2. Induction of development by copper. On the complex R2YE medium, S. lividans is slower in development than the closely related strain S. coelicolor. However, extra copper ions accelerate differentiation. This figure shows that immediately around a cotton filter disk that contains copper sulfate, aerial hyphae and spores develop faster than in the more distant regions of growth. Titration experiments have shown that raising the concentration of copper ions from 0.2 to 2 ^M is sufficient.

BCDA (bathocuproinedisulfonic acid), the equilibrium will shift toward the Cu(I) species and eventually all copper will be bound by the chelator.

Streptomyces coelicolor and S. lividans grown on medium containing BCDA (20 ^M) failed to produce aerial hyphae and spores (Fig. 3A). However, the presence of this copper chelator was not an impediment for vegetative growth or secondary metabolism. The BCDA effect could be reversed by the addition of extra copper ions (Fig. 3B). Remarkably, the developmental block that occurred after the addition of BCDA could still be relieved by the addition of extra copper salts after 1 wk. Therefore, the effect of BCDA is not the result of the toxicity of this chelator but rather the result of the diminished bioavailability of copper (51). The devastating effect of the reduction of the available copper ions by addition of BCDA on morphogenesis was observed only on rich yeast-extract-containing media such as R2YE. In contrast, neither this effect on differentiation nor the stimulation of differentiation by extra copper was present on minimal media containing mannitol as the carbon source. These observations support the idea that at least two pathways can be induced that lead into development.

None of the bld mutants could be stimulated to produce aerial hyphae by the addition of extra copper. The inability to restore development of the bld mutants by elevating the copper-ion concentration may indicate that we are dealing with a linear cascade and not two separate pathways. In the linear-cascade

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