Black fungal extremes - CBS

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Ce l l u l a r r e s p o n s e s t o d e s i c c at i o nA3000slow desiccation (SD) (SD)S500Aslow desiccation (SD)S µg/g dw2500200015001000500PCPE400300200100PC, PE µg/g dw0K D 24h0Bfast fast desiccation (FD) (FD)Bfast desiccation (FD)3000500S µg/g dw2500200015001000500400300200100PC, PE µg/g dw0KD 24hFig. 5. Neutral (storage) lipids in colonies o microcolonial strain A95 subjected toFigure Fig. 4. Membrane 4. Membrane lipids in lipids colonies in colonies of microcolonial of microcolonial strain A95 subjected strain A95 to slow subjected (A) to slow slow (A) and fast (B) desiccation. The time axis is not to scale - the distance between(A) and and fast (B) fast desiccation. (B) desiccation. The time The axis time is not axis to is scale, not to as scale, the distance as the between distance K between K and K D equals 8 wk of desiccation, while 24 h represents 24 hs after re-hydration.and D equals 8 8 wks weeks of desiccation, of desiccation, while 24 while h is 24 24 hs h after is 24 water hours was after added water to the was added K = to control, D = after 8 wk of desiccation; 24 h = re-hydrated colony after 24 h. TAGthe desiccated desiccated colonies. colonies. K = control K (stationary – control phase (stationary colony, before phase being colony, transferred before being = triacylglycerol; DAG = diacylglycerol, SE = sterol esters.transferred to a desiccator), D a = desiccator), after 8 wk of D desiccation; – after 824 weeks h = re-hydrated of desiccation; colony 2424h h later. – re-hydratedcolony 24 h later. S – sterols, PC – phosphatidylcholine, PE –S = sterols, PC = phosphatidylcholine, PE = phosphatidylethanolamine.phosphatidylethanolamine.were practically absent in SD colonies (Fig. 4A, 24 h), while FDcolonies showed a steady increase in both phosphatidylcholine andphosphatidylethanolamine levels (Fig. 4B).Desiccated cells contained high amounts of neutral lipids liketriacylglycerol (TAG) and diacylglycerol (DAG). Storage lipidsalso displayed significantly diverse dynamics between slow- andfast-dehydrated mycelia. Neutral lipids decreased during thedehydration when the process was slow (Fig. 5A), while a slightincrease occurred when desiccation was fast. TAG levels of SDcells were reduced by 50 % as compared to controls (Fig 5A),while FD cells contained 1,4-fold higher amounts of TAG (Fig.5B). After rehydration, the constant levels of neutral lipids liketriacylglycerols and sterol ethers suggest a stable metabolicstate that was not significantly influenced by desiccation stress(Fig. 5B). The proportion of rapidly and slowly oxidised lipids incell membranes and intracellular inclusions was checked in A95following the desiccation challenge. The proportion of unsaturatedfatty acids was always relatively high. In control cells, 81 % of allfatty acids were unsaturated. After 8 wk of anhydrobiosis the ratioof unsaturated fatty acids in SD and FD cells equalled respectively80 and 67 % of the total fatty acids. In desiccated cells after 24 h ofrehydration the values reached 86 % in SD cells, while rehydratedFD cells had equal proportions of saturated and unsaturated fattyacids (50:50 %).As shown by biochemical methods, intra-cellular structures infast desiccated cells were largely preserved and their biochemical0status (synthesis of carotenoids and phospholipids) supportedthese observations (Figs 4B, 5B, 6B). In contrast, SD colonies (Figs4A, 5A, 6A), were less viable although some cells in a colony werestill capable of re-growth. Decreased levels of triglycerols and sterolesters suggested lower levels of storage lipids in SD cells. Onlytrace amounts of phospholipids were found following rehydration,confirming the complete loss of membrane structure in SD colonies.Under TEM, these cells looked like vials filled with lipid droplets,confirming the decay of intracellular membrane structures.Pigment analysisAs expected (Gorbushina et al. 1993, Butler & Day 1997) melaninwas obvious under TEM (Figs 1, 2) and no further characterisationof this pigment group was carried out. Pigments found in A95absorbed radiation in UV- and visible-wavelengths. Both redcarotenoids and colourless UV-absorbing carotenoids as well asmycosporines were present. Five different carotenoid pigmentswere identified in A95: ß-carotene, ζ-carotene, phytoene, toruleneand torularhodin. Mycosporines included mycosporine-glutaminol,mycosporine-glutaminol-glucoside, mycosporine-glutamicol, andmycosporine-glutamicol-glucoside (Volkmann & Gorbushina 2006).Carotenoid contents also varied during desiccation / rehydrationstress (Fig. 6). Levels of phytoene, a colourless precursor ofcarotenoid synthesis were particularly sensitive to stresses, butthe amounts of red carotenoids decreased under SD (Fig. 6A) orremained unchanged in conditions of FD (Fig. 6B).www.studiesinmycology.org95
Go r b u s h i n a e t a l.Aphytoene, mkg/g mkg/g dw dry mBphytoene, mkg/g mkg/g dw dry m800070006000500040003000200010000800070006000500040003000200010000Fig. 6. Pigment composition in colonies of microcolonial strain A95 subjected to slowFigure (A) and fast 6. Pigment (B) desiccation. composition For further in colonies details see of Figs microcolonial 3 and 4. strain A95 subjected to slow(a) and fast (b) desiccation. For further details see Fig. 3 and 4.DISCUSSIONslow dessicstionslow desiccation (SD)PhytoeneRedK D 24hfast dessicationfast desiccation (FD)PhytoeneRedK D 24hCultivation, biochemical- and ultra-structural analyses have shownthat MCF strain A95 is capable of surviving desiccation / rehydrationstress and sudden changes in the availability of water. Desiccatedcells maintained their typical MCF spore-like ultra-structure(Sterflinger 1998, Gorbushina 2003, Gorbushina et al. 2003), andall colonies were able to re-grow after long-term desiccation, aswell as following the rehydration challenge. Large inocula (discs of5 mm) aided survival as parts of colonies always contained viablecells that gave rise to new colonies. Even when viable cells werefew, some survivor cells in the middle of a colony were protected bythe surrounding cells (Fig. 2) and were abundant enough to permitre-growth.The decrease in the proportion of unsaturated fatty acids inboth SD and FD colonies with and without dehydration indicatessignificant stress. Unsaturated fatty acids in membranes of A95remained high (50 to 80 %) in all cases. Rehydrated SD coloniesmanaged to keep the ratio of unsaturated fatty acids at the level ofunstressed (unchallenged) controls, while in FD cells this parameterdecreased to slightly more than 50 %. This value is, however, stillhigh and shows an intrinsic ability of A95 to adapt to desiccation /rehydration stress.Desiccated A95 cells contained high amounts of neutral lipidsthat served as a reserve for the synthesis of phospholipid-membranefollowing stress relief. Rapidly desiccated cells largely retained theirnormal physiological status. After 8 wk of matric stress and one day400350300250200150100500400350300250200150100500red red carotenoids, mkg/g dw dry mred red carotenoids, mkg/g dw dry mof rehydration, phospholipid synthesis was restored to levels thatpermitted correct functioning of membranes (Fig. 4B). TEM showedthat budding cells were indeed present after 24 h of rehydration,confirming that the colonies remained viable (Fig. 2).In contrast, slow desiccation (complete water loss over a periodof several days, which kept metabolism active for approx. 80 h)followed by rehydration, drastically reduced phospholipid contents(Fig. 4A), and resulted in complete degradation of membranesystems. This could have been caused by the exhaustion of internallipid resources as TAG levels of SD cells were reduced by 50 %compared to control values (Fig. 5A).Typical <strong>fungal</strong> carotenoids include molecules with 13conjugated double bonds that are important antioxidants (e.g.torulene and torularhodin), which help to stabilise membranesunder unfavourable conditions All carotenoids found in A95 havebeen previously observed in different asco- and basidiomycetousyeasts and thus are typical <strong>fungal</strong> pigments (Davoli & Weber 2002,Weber & Davoli 2002, Davoli et al. 2004). As A95 was grown inthe dark, the carotenoids described here belong to the naturalmetabolites of MCF. Constitutive levels of carotenoids in a majorityof MCF strains (data not shown) suggest a readiness to counteractstress. Fungal carotenoids are reported to occur predominantly incytoplasmic lipid bodies, the endoplasmatic reticulum, cell wallsand EPS layers (Rikkinen 1995). Carotenoids are abundant in lipidinclusions of fungi, but are also major components of the cell walland the cell membrane (Rikkinen 1995).Phytoene is generally accepted as a precursor of less-saturatedC 40carotenoids, which are synthesised from phytoene througha series of desaturation reactions (Simpson 1972). All types ofenvironmental stresses promote accumulation of this colourlessprecursor (Fig. 6). The amount of red carotenoids either remainedunchanged under conditions of fast desiccation or UV-radiationor was reduced when the cells were slowly desiccated. Fastdesiccation stimulated both phytoene and red carotenoid synthesis(Fig. 6B) while preventing degradation of protective pigments.During slow desiccation, colonies are forced to subsist withever lower levels of available water and as a result retain syntheticactivities until their internal resources (especially reserve lipids)are exhausted. In contrast, fast drying forces vegetative MCF toshift rapidly to a dormant state in which levels of reserve storagecompounds, protective carotenoid precursors and constitutiveantioxidants like melanins and mycosporines are maintained.In many rock surface environments, access to sufficient sourcesof energy, nutrients and water rarely coincides. For this reason,micro-organisms that dwell on and in rocks need to be able tomaintain biomass during extended periods of dormancy. We haveshown here that MCF are capable of fast recovery after prolongeddesiccation, proving that the cellular machinery remains in a state ofsuspended animation. This immediate revival from an anhydrobioticstate clearly demonstrates the ability of MCF to recover from waterdeficits that might be lethal to many prokaryotes.The most important findings of this study are: A the fact that MCFresponses to stresses are unspecific and thus can be employedagainst various environmental challenges (in this sense, toleranceto desiccation is perhaps part of a broader range of adaptationsto other stresses; Mattimore & Battista 1996), and B that rapid asopposed to slow desiccation best preserves the viability of MCF.This preference reflects an inherent capacity of MCF to respond todrastic changes in the environmental conditions typical of subaerialrock surfaces (Gorbushina 2003). Enhanced survival of MCF whensubjected to rapid changes in the environment provides strongexperimental support for the poikilotolerance hypothesis proposedfor rock inhabiting organisms (Gorbushina & Krumbein 2000).96
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Studies in Mycology 61 (2008)Black
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Studies in MycologyThe Studies in M
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PREFACEThe terms "black fungi" or "
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available online at www.studiesinmy
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Dr o u g h t m e e t s a c i dTable
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Dr o u g h t m e e t s a c i d2003/
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Dr o u g h t m e e t s a c i din Pe
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Dr o u g h t m e e t s a c i d99100
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Dr o u g h t m e e t s a c i dAnamo
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Dr o u g h t m e e t s a c i dFig.
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Dr o u g h t m e e t s a c i dFig.
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Dr o u g h t m e e t s a c i dconve
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Dr o u g h t m e e t s a c i dal. 2
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Za l a r e t a l.Table 1. List of a
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Za l a r e t a l.Table 1. (Continue
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Za l a r e t a l.Fig. 2. Consensus
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Za l a r e t a l.Fig. 4. Macromorph
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Za l a r e t a l.Fig. 5. Aureobasid
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Za l a r e t a l.Fig. 7. Aureobasid
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Za l a r e t a l.the type species o
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Za l a r e t a l.Vishniac HS, Onofr
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Yu r l o va e t a l.Table 1. Strain
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Page 52 and 53: Yu r l o va e t a l.RESULTSFourteen
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Page 56 and 57: Yu r l o va e t a l.AcetateMelanin(
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Page 68 and 69: Va u p o t i č e t a l.cerevisiae)
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Page 80 and 81: Pl e m e n i ta s e t a l.diversity
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Page 104 and 105: On o f r i e t a l.Table 1. Test pa
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Page 124 and 125: De n g e t a l.Table 1. Strains use
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Origin o f Ex o p h i a l a d e r m
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Sat o w e t a l.Table 1. Compositio
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Sat o w e t a l.Assimilation of min
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Sat o w e t a l.pollutants in biore
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s u b s t r at e s p e c i f i c i
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Bi o d i v e rs i t y o f t h e g e
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homogentisate 165, 171, 173Hortaea
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CBS Biodiversity SeriesNo. 6: Alter
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Studies in Mycology (ISSN 0166-0616
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Satow MM, Attili-Angelis D, Hoog GS
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Black fungal extremes - CBS

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