Indian Journal of BiotechnologyVol 2, July 2003, pp 293-301

Autolysis of Penicillium chrysogenum-A Holistic Approach

Istvan Pocsi", Tunde Pusztahelyi, Laszlo Saw and Tamas EmriDepartment of Microbiology and Biotechnology, Faculty of Science, University of Debrecen, Egyetem ter 1,

H-4010 Debrecen, Hungary

Received 20 January 2003; accepted 20 February 2003

Despite of its biotechnological significance, the autolysis of filamentous fungi is a poorly studied and understoodarea of fungal physiology. The autolysis of 13-lactam producing fungus, Penicillium chrysogenum shares some simi-larities with the apoptosis of higher eukaryotes. For example, the biosynthesis and processing of age-related hydro-lases were highly regulated in carbon-depleted cultures. The in vivo inhibition of autolytic chitinase activity hinderedconsiderably the disintegration of pelleted structures that are typical of the exponential growth phase. In the absenceof conidiation, round-ended "yeast-like" hyphal fragments were the dominant surviving morphological forms, whichwere characterised with decreasing total respiration, increasing cyanide-resistant respiration, intracellular accumu-lation of reactive oxygen species (ROS) and declining viability in the autolytic and post-autolytic phases of growth.The term "ageing" was used to describe these physiological changes, and the surviving fragments may undergo oxi-dative-stress induced programmed cell death. Although variations in oxygen tension and extracellular ROS concen-trations are key elements in the initiation of morphological changes, the genomic expression programmes of fungigoverning morphological transitions including autolysis are likely to be activated by different kinds of environmentalstress and signal transduction pathways. The glutathione (GSH) and ROS metabolisms of P. chrysogenum were in-fluenced by many extrinsic and intrinsic factors in each growth phase studied. As a consequence, no firm correlationwas found between the GSHIglutathione disulphide (GSSG) redox status, the intracellular ROS levels and the ob-served morphological and physiological characteristics of the cells.

Keywords: autolysis, apoptosis, ageing, fragmentation, vacuolation, chitinase, respiration

IntroductionThe autolysis of industrially important filamentous

fungi has been recently reviewed by White et al(2002). Fungal autolysis as a dynamic process of celldeath influences numerous biotechnological processesincluding secondary metabolite and heterologousprotein productions. Although the papers published inthis field are mainly either physiology or morphologyoriented, these approaches are aiming at the samephenomenon using different experimental tools. Bothintrinsic (differentiation, ageing and cell death pro-grammes of the micro-organism) and extrinsic(changes in the culture media, physical stress) effec-tors have significant, mostly inseparable impact onfungal autolysis observable in fermentation processes

*Author for correspondence:Tel: 36-52-512900, ext 2061; Fax: 36-52-533677E-mail: pocsi@tigris.klte.huAbbreviations: AOX: alternative oxidase; GOX: glucose oxidase;GPx: glutathione peroxidase; GR: glutathione reductase; GSH:glutathione; GSSG: glutathione disulphide; HexNAc-ase: N-acetyl-I3-D-hexosaminidase; ROS: reactive oxygen species; SOD:superoxide dismutase.

(White et al, 2002). Physiological differentiationalong the growing and stationary phase hyphae in-cluding progressing vacuolation in the older com-partments and weakening of cell wall due to the ac-tion of autolytic hydrolases influences the 'resistanceof mycelia to mechanical forces introduced by agita-tion (Paul et al, 1994; Harvey et al, 1998; Ji.isten et al,1998). On the contrary, the agitation intensity influ-ences considerably both the vacuolation (Ji.isten et al,1998) and hydrolase production (Harvey et al, 1998).Undoubtedly, a holistic approach is needed to get adeeper understanding of the underlying mechanism ofthe interdependent morphological and physiologicalevents leading to the disintegration of hyphae in sub-merged cultures in the absence of conidiation (Whiteet al, 2002).

In the last decade, the authors characterized theautolysis of P. chrysogenum NCAIM 00237, a high ~-lactam producing filamentous fungus, both physio-logically and morphologically. Because of their inter-est in the intrinsic cell death and ageing programmesof the microorganism, in the first place autolysis wastriggered by the depletion of carbon source in the ab-

294 INDIAN J BIOTECHNOL, JULY 2003

sence of penicillin side-chain precursors (Pusztahelyiet al, 1997a, b; Sami et al, 2001a, b; 2003). In thisreview, the findings are summarised and collated withthe propositions of the Dioxygen Theory of Cell Dif-ferentiation (Hansberg & Aguirre, 1990), the Free-radical Theory of Ageing (Harman, 1993) and the "Itis Better to Die than to be Wrong" Principle (orSamurai Law) of Biology (Skulachev, 2000, 2001).Possible signalling pathways in the activation of thegenomic programmes that may govern fungal autoly-sis have also been summarised and discussed.

Growth Phases of P. chrysogenum

The growth of the high B-Iactam producingP. chrysogenum NCAIM 00237 was characterised byfive phases including exponential (up to 34 hrs ofincubation), deceleration (34-40 hrs), stationary (40-50 hrs), autolytic (50-148 hrs) and cryptic or post-autolytic (from 148 hrs) phases of growth (Table 1).Autolytic hydro lases , including proteases (Pusztahelyiet al, 1997b; McIntyre et al, 2000), N-acetyl-B-D-hexosaminidase (HexNAc-ase') (P6csi et al, 1993;Pusztahelyi et al, 1997b; Sami et al, 2001 b) andzymogenic chitinases (Sami et al, 2001b),accumulated inside the cells in the deceleration andstationary phases of growth and were released into theculture media later, coinciding with progressingautolysis and fragmentation (Table 1). In autolyticand post-autolytic cultures, round-ended hyphalfragments typically consisting of two cells were thedominant surviving morphological forms. After theaddition of an extra dose of glucose, these "yeast-like" hyphal fragments germinated at both endssimultaneously and reverted to vegetative growth(Fig. 1).

Among the age-dependent hydrolytic enzymes,chitinases have important nutritional and morphoge-netic functions in fungi (Gooday et al, 1992; Gooday,1997). The authors used the pseudotrisaccharide allo-samidin, which is one of the most potent known in-hibitors of chitinases, to gain information about themorphogenetic functions of these enzymes in P.chrysogenum (P6csi et al, 2000; Sarni et al, 2001b).Allosamidin retarded significantly the autolysis andfragmentation of hyphae in ageing carbon-depletedcultures thus shedding light on the crucial rolechitinases play in the degradation of chitin microfi-brils and as a consequence, in the disruption of theinnermost, shape-determining and stress-bearing fab-ric of the fungal cell wall (P6csi et al, 2000; Sami et

al, 2001b) (Fig. 2). Hydrolases that attack the outerprotein and glucan cell wall layers (Pusztahelyi et al,1997b; Harvey et al, 1998; McNeil et al, 1998;McIntyre, 2000) may facilitate the action ofchitinases. Allosamidin also hindered the fragmenta-tion of the other major B-Iactam produing filamentousfungus, Acremonium chrysogenum (Sandor et al,1998) but did not influence the growth and antibioticproduction of either P. chrysogenum or A. chryso-genum (Sandor et al, 1998; P6csi et al, 2000). On theother hand, this chitinase inhibitor hindered signifi-cantly the out-growth of new hyphal tips from thetwo-celled "yeast-like" fragments after glucose sup-plementation (P6csi et al, 2000; Sami et al, 2001b)(Fig. 2), and the antifungal effect of allosamidin onautolysing P. chrysogenum mycelia was fungistatic(Sami et al, 2001b). The markedly different sensitiv-ity of growing and autolysing mycelia to allosamidinwas explained by the strict post-translational regula-tion (zymogen activation) and compartmentalisation(microsomes) of age-related chitinases (Sami et al,2001b). The inhibition of fungal autolysis and frag-mentation through blocking the action of chitinasesmay be exploitable in the bioprocessing industry(White et al, 2002) and in the treatment of humanmycoses caused by filamentous fungi (Cooper &Haycocks, 2000). The disruption of genes coding forfungal chitinases may also be beneficial but many ofthe chitinases are multifunctional (Takaya et al, 1998)and the absence of one chitinase can be compensatedfor by up-regulation of other chitinase gene(s)(Reichard et al, 2000).

As far as the free-radical metabolism is concerned,high intracellular peroxide and superoxide levels wererecorded in the exponential phase of growth, whenglucose and O2 were converted effectively to gluco-nate and H202 by GOX. In this period of time, highGR, GPx, catalase and cyanide-resistant mitochon-drial AOX activities provided the growing cells with asuitable antioxidative defence system (Table 1) (Samiet al, 2001a, 2003). Prior to autolysis, the utilisationof peptides as carbon sources (Pusztahelyi et al,1997a) did not give rise to ROS in high concentra-tions (Sarni et al, 2001a). The continuous accumula-tion of ROS in the autolytic and post-autolytic phasesof growth as well as the burst in the intracellular per-oxide concentrations after glucose supplementationwas attributed to disintegrating mitochondria (Trinci& Righelato, 1970; Sami et al, 2001a). Under theseconditions GR, GPx, SOD and AOX activities repre-

Table I-Morphologicall\Ild physiological characterization of the growth phases of P. chrysogenum NCAIM 00237 cultivated in a complex culture medium

Growth phase Morphology Hydrolase production Carbon source utilization Secondary Levels of glutathione and Specific activity ofand respiration" metabolite reactive oxygen species" antioxidant enzymes

production

Exponential hyphae,pellets no autolytic hydrolase glucose, gluconate; total none GSSG level is high; GPx and GR are high;(up to 34 hrs) GOX is high but decreasing GSHlGSSG ratio is low; catalase is increasing;

later; respiration and AOX peroxide and 020- levels are SOD is low

are high high'"00

Deceleration intracellular f3-lactarn, oxalate GPx, GR and catalaseo

hyphae, pellets; peptides; decreasing GOX; GSSG level is high; no ~(34-40 hrs) vacuolation is accumulation of respiration is decreasing; andNH3 change in GSH; GSHlGSSG are high; SOD is low ~

progressing protease, HexNAc-ase AOX is low productions start ratio is low; peroxide and ~and zymogenic 02

0• levels decrease ;l>

chi tinase starts C...,0r-

Stationary hyphae, pellets; intracellular peptides; decreasing GOX; f3-lactarn and GSSG is decreasing; no GPx and GR are slightly >-<en

(40-50 hrs) vacuolation is accumulation of respiration is decreasing; oxalate; NH3 change in GSH; GSHlGSSG decreased; catalase is •.....en

progressing protease, HexNAc-ase AOXislow production ratio is increasing; peroxide high; SOD is low 0and zymogenic continues and 02

0- levels are low

'"Ii"1:l

chitinase goes on ~-(jAutolytic disintegration of substantial extracellular peptides, proteins; GOX oxalate; GSH remains at constant GPx decreases and r::

r-(50-148 hrs) pellets and the chitinase, protease and goes down to zero; apparently no ~- level, GSSG tends to reaches a constant value -

appearance of HexNAc-ase respiration tends to lactam"; NH3 decrease with a plateau at 88 h; catalase ~hyphal fragments production; intracellular decrease and remain production is between 66 and 120 hrs; decreases and reaches a (j

hydrolase activities constant after 68 hrs; AOX increasing GSHlGSSG tends to constant value at 120 ~decrease increases increase with a plateau hrs; GR tends to ~

between 66 and 120 hrs; decrease and remainsQa

both peroxide and 020- constant after 115 hrs; ~levels increase steadily SOD increases up to 115

~hrs

Post-aur.o~tic round-ended chitinase and HexNAc- peptides, proteins; no oxalate, GSH - no significant GPx, GR and catalaseor crypuc hyphal fragments ase have reached GOX; respiration is low, apparently no 13- change; GSSG tends to are low, constant values;(148 hrs) consisting of plateaus; protease unchanged; AOX increases Iactam"; NH3 decrease further, . SOD is high, constant

mainly two cells increases further continuously level is high, GSHlGSSG ratio as well asconstant peroxide and 02

0- levels tend

to increase further

(Contd)

N\0V1

296 INDIAN J BIOTECHNOL, JULY 2003

sented the first line of antioxidative defence (Sami etal, 2001a, 2003) (Table 1).

Interestingly, the intracellular GSH concentrationswere insensitive to changes in the physiological con-ditions of the cells. GSH responded only to glucosesupplementation when the re-initiation of growth trig-gered the fast degradation of intracellular nitrogen andsulphur reserves, including GSH (Elskens et al, 1991;Mehdi & Penninckx, 1997; Ernri et al, 1998). TheGSHJGSSG ratios were only influenced by intracel-lular GSSG concentrations that decreased with in-creasing incubation times (Sami et al, 2001a)(Table 1).

Collation with the Dioxygen Avoidance Theory ofCell Differentiation

Transient hyperoxidant states characterised byredox imbalances, intracellular accumulation of ROSand the activation of antioxidant enzymes have beenhypothesised to trigger versatile cell differentiationprocesses in air-exposed Neurospora crassa mycelia,including the adhesion of hyphae, the emergence ofaerial hyphae and the formation of conidia (Hans berget al, 1993; Toledo et al, 1995). The intracellularaccumulation of ROS may also initiate sporegermination (Lledias et al, 1999), yeast ~ mycelialdimorphic conversions (Jiirgensen et al, 2001) andeven autolysis (Hansberg & Aguirre, 1990). Insubmerged cultures of P. chrysogenum, growinghyphae and the round-ended, "yeast-like" survivingfragments observable in autolysing and post-autolyticphases of growth were regarded as physiologicallystable, differentiated states of the fungus. Autolysismight represent a crucial part of the celldifferentiation process resulting in the "yeast-like"fragments in the absence of conidiation. Obviously,autolysis provided the surviving cells with substratesto facilitate their differentiation and maintain theircryptic growth under carbon starvation (Pusztahelyi etal, 1997a).

Authors' data did not support the hypothesis thatthe autolysis of P. chrysogenum cells in carbon-depleted cultures was preceded by an unstable hyper-oxidant state in the deceleration or stationary phasesof growth (Sarni et al, 2001a). Moreover, both theGSH and ROS metabolisms of P. chrysogenum wereinfluenced by many intrinsic and extrinsic factorsthroughout the incubation. As a consequence, no firmcorrelation was found between the GSHJGSSG redoxstatus, intracellular ROS levels and the observed mor-

POCSI et al: AUTOLYSIS OF PENICILLIUM CHRYSOGENUM

Autolysis 1between50-148 hrs ofincubation

Substantial autolytic hydrolase productionDecreasing respiration with increasingAOX proportionSignificant oxalate and NH3 productionIncreasing intracullular ROS levelsDecreasing antioxidative enzyme activitieswith the exception of SOD

1Hydrolase production ceases

Addition of a Respiration and AOX activity intensify temporarilysecond dose No oxalate production. likely NH3 consumptionof 51 mM Peroxide production is enhanced temporarilyglucose GPx and GR activities increase temporarily

GSH concentration and GSH/GSSG ratiodecrease

Fig. I-Sumrnarisation of the physiological and morphologicalchanges taking place in autolysing and glucose supplemented P.chrysogenum cultures. P. chrysogenum mycelium at 64 hrs ofincubation at the beginning of autolysis (A), and round-ended"yeast-like" fragments before (B) and after (C) the addition of anextra dose of glucose at 112 hrs incubation time are presentedaccording to Pusztahelyi et al (1997a). Typical dormant (a), ger-minating (b) and cryptically growing (c) fragments, as well as acalcium oxalate crystal (cr) are shown in part (B). Note the typicalfragments with two simultaneously outgrowing thin hyphae afterglucose supplementation (C). Bars in parts (A) and (C) are equalto 50 urn, while the bar in part (B) = 20 urn,

297

phological and physiological characteristics of thecells (Sarni et al, 2001a, 2003). For example, the per-oxide production was high in the exponential phase ofgrowth due to the action of GOX, and the intracellularROS concentrations dropped after all the starting glu-cose had been consumed by the otganism, i.e. justbefore the autolysis had started. But these favourablechanges in ROS levels were not reflected in the intra-cellular GSH concentrations and GSHlGSSG ratios atall (Table 1) (Sarni et al, 2001a, 2003).

On the other hand, the outgrowth of survivingfragments after glucose supplementation was pre-ceded by a certain hyperoxidant state. For example,peroxide accumulated inside the cells and antioxida-tive enzymes (GPx, GR) were activated transiently,but the intracellular superoxide levels even decreasedafter glucose re-addition and the specific catalase andSOD activities remained unchanged (Table 1) (Sarniet al, 2001a).

Undoubtedly, variations in oxygen tension and ex-tracellular ROS concentrations represent a cruciallyimportant kind of environmental stress that affectsprofoundly both the metabolism and the morphologyof aerobic filamentous fungi (Toledo et al, 1995; Hen-riksen et al, 1997; Priede et al, 1997; Ruijter et al,2002; Kreiner et al, 2003). Nevertheless, other typesof environmental stress, e.g. changes in the CO2 ten-sion, are also well-known inducers of morphologicalchanges in P. chrysogenum cultures (Ho & Smith,1986; Ju et al, 1991). Air-exposure (the formation ofan air/water interface) and red light are needed to in-duce the conidiation of Aspergillus nidulans but car-bon or nitrogen starvation stress that also affects GSHmetabolism (Penninckx, 2000) may substitute forthese factors in submerged cultures (Adams et al,1998). The genomic expression programme of fungigoverning the morphological transitions provoked byenvironmental stress seem therefore to be activated byseveral different signals, and not exclusively by theavailability of O2. More recently, transcriptome analy-ses using DNA rnicroarrays indicated that the envi-ronmental stress response of Saccharomyces cere-visiae was in fact stereotypical to each of the stressfulconditions tested but was controlled by differentregulatory systems according to the type of stress(Gasch et al, 2000).

Fungal Autolysis-Necrotic or Apoptotic CellDeath?

The existence of a highly coordinated cellular sui-cide programme has been demonstrated in S. cere-

298 INDIAN J BIOTECHNOL, JULY 2003

Autolysis andfragmentation incontrol cultures

Autolysis andfragmentation inallosamidin-treatedcultures

The chemical structure of allosamidin

Fig. 2-Retardation of fragmentation by 9.6 /lM aIlosarnidin in autolysing P. chrysogenum cultures. Late exponential growth phase (30hrs) pellets (A, bar = 100 urn) as well as autolysing (72 hrs control (B, bar = 200 urn) and allosarnidin-treated (C, bar = 200 urn) hyphalfragments are shown. The pseudotrisaccharide allosamidin inhibited age-related P. chrysogenum chitinases effectively (ICso = 1.4 /lM;Sami et at, 2001b).

visiae (Frohlich & Madeo, 2000; Laun et al, 2001;Madeo et al, 2002). In filamentous fungi, lovastatin(Roze & Linz, 1998) and viscosinamide (Thrane &Olsson, 1998) may induce apoptosis-like processes. Inaddition, the autolysis of P. chrysogenum is an active,energy-consuming process and hence, shares simi-larities with the apoptosis of higher eukaryotes(McIntyre et al, 1999). The biosynthesis and proc-essing of age-related hydrolases were highly regulatedin deceleration and stationary phase P. chrysogenum

cultures (Table 1) (Pocsi et al, 1993; Pusztahelyi et al,1997b, Sami et al, 2001b). These observations furtherstrengthen the view that fungal autolysis is clearlydistinct from the process of necrotic cell death(McIntyre et al, 1999).

In autolytic and post-autolytic phase cells, the ROSconcentrations increased and the cell vitality de-creased continuously although the specific SOD ac-tivity and the cyanide-resistant respiration (AOX ac-tivity) were elevated (Table 1) (Sami et al, 2001a,

PoeSI et al: AUTOLYSIS OF PENICILLIUM CHRYSOGENUM

2003). A decreasing total respiration rate (Sami et al,2003) together with mitochondrial disorganisation(Trinci & Righelato, 1970) may represent the onlymeasure of choice in autolysing cells to hinder thefree-radical dependent activation of cell death pro-grammes (mitoptosis) (Skulachev, 2000, 2001). Atlast, cells with seriously impaired mitochondrialfunction may commit suicide according to the Samu-rai Law of Biology (Skulachev, 2000, 2001).

Although ROS did not accumulate in decelerationand stationary phase P. chrysogenum cultures prior toautolysis and, hence, were unlikely to play any role inthe first initiation events of autolysis (Sami et al,2001a), an inherent physiological analogy betweenautolysis and apoptosis cannot be excluded. It isknown that the accumulation of ROS is not anobligatory prerequisite of the induction of apoptosis inmore complex eukaryotes (Coppola & Ghibelli,2000).

Unlike stationary phase hyphae, surviving "yeast-like" fragments of P. chrysogenam (Fig. 1) may un-dergo oxidative-stress induced apoptosis, which is apromising area for further cell death studies in fungi(Sami et al, 2001a, 2003).

Post-autolytic (Cryptic) Growth and AgeingIn post-autolytic P. chrysogenum cultures, numer-

ous physiological parameters reached a constant value(Table 1) (Sami et al, 2003) but this equilibrium be-tween cell death and cryptic growth was apparent be-cause cell vitality declined as a function of incubationtime (Sami et al, 2001b). The physiological changestaking place in these cultures (intracellular accumula-tion of ROS, increasing SOD activity) were verysimilar to those observed in ageing stationary culturesof S. cerevisiae (Jakubowski et al, 2000) and in yeastmother cell-specific ageing leading to apoptosis(Nestelbacher et al, 2000; Laun et al, 2001). The age-related increase in the specific AOX activity in cryp-tically growing P. chrysogenum (Sami et al, 2003) isreminiscent of the switch from cytochrome C oxidasemediated respiration to alternative respiration observ-able in senescent Podospora anserina cultures (Frese& Stahl, 1992). The increased AOX activity may bebeneficial to eliminate harmful free-radicals accumu-lating in cells with declining mitochondrial function(Dufour et 'ai, 2000; Karaffa et al, 2001; Silar et al,2001) and/or to improve the energy levesl of crypti-cally growing surviving fragments (Lorin et al, 2001).

The intracellular accumulation of ROS, declining

299

mitochondrial function and cell vitality as well as thedecreasing ability of the surviving fragments to revertto vegetative growth after glucose supplementation(Pusztahelyi et al, 1997a) are in good accordance withthe propositions of the Free-radical Theory of Ageing(Harman, 1993). Therefore, the authors have used theterm "ageing" to describe the changes characteristicof post-autolytic P. chrysogenum cultures (Sami et al,2001a).

More recent findings in yeast apoptosis research(Nestelbacher et al, 2000; Laun et al, 2001; Madeo etal, 2002) strengthen the view on a likely cross-communication between ageing and apoptosis at mo-lecular level, which is an intriguing possibility infilamentous fungi and mammalian systems too(Zhang & Herman, 2002).

Signalling Pathways in the Activation of AutolysisGenomic Programmes

The molecular background of the regulation offungal autolysis is poorly understood. There are someliterature data indicating that low molecular weightendogenous substances may trigger fragmentation(Natsume & Marumo, 1984) and vacuolation (Bottoneet al, 1998). Signals from the environment includingavailability of nutrients and extracellular enzymeproducts are likely to be received by membrane-bound receptors (Thevelein & de Winde, 1999) andforwarded by heterotrimeric G proteins likeFadAJSfaD in A. niduians (Rozen et al, 1999). Thefine tuning between growth, conidiation and autolysis,e.g. via the FlbA regulatory protein, is basically im-portant to support the development of conidiophores(Adams et al, 1998). The control of cell wall structureand cell integrity also relies on cAMP-dependent(Roze & Linz, 1998; Shimizu & Keller, 2001) andMAP kinase dependent (Igual et al, 1996; Lev et al,1999) regulatory pathways. At the level of transcrip-tional regulation, Yaplp, a b-Zip protein, seems tofulfil a major role in general mechanisms of cell lifeand death in the yeast S. cerevisiae (Dumond et al,2000).

AcknowledgementThe Hungarian Ministry of Education awarded a

Szechenyi Scholarship for Professor I P. T P was arecipient of a Hungarian Scientific Research Fundpostdoctoral fellowship. This project was supportedfinancially by the Office for Higher Education Pro-grammes (grant reference number 0092/2001) and by

300 INDIAN J BIOTECHNOL, JULY 2003

the Hungarian Scientific Research Fund (grant refer-ence numbers T 034315 and T 037473).

ReferencesAdams T H et al, 1998. Asexual sporulation in Aspergillus nidu-

lans. Microb Mol Bioi Rev, 62, 35-54.Bottone E J et al, 1998. Evidence of self-inhibition by filamentous

fungi accounts for unidirectional hyphal growth in colonies.Can J Microbiol, 44, 390-393.

Christensen L H et al, 1994. A robust liquid chromatographicmethod for measurement of medium components duringpenicillin fermentations. Anal Chim Acta, 296, 51-62.

Cooper C R & Haycocks N G, 2000. Penicillium marneffei: Aninsurgent species among the penicillia. J Eukaryot Microbiol,47,24-28.

Coppola S & Ghibelli L, 2000. GSH extrusion and the mitochon-drial pathway of apoptotic signalling. Biochem Soc Trans,28,56-61.

Dufour E et al, 2000. A causal link between respiration and se-nescence in Podospora anserina. Proc Natl Acad Sci USA,97,4138-4143.

Dumond H et al, 2000. A large-scale study of Yap lp-dependentgenes in normal aerobic and H20z-stress conditions: The roleof Yap1p in cell proliferation control in yeast. Mol Micro-bioi, 36, 830-845.

Elskens M T et al, 1991. Glutathione as an endogenous sulphursource in the yeast Saccharomyces cerevisiae. J Gen Micro-bioi, 137,637-644.

Emri T et al, 1997. Glutathione metabolism and protection againstoxidative stress caused by peroxides in Penicillium chryso-genum. Free Rad Bioi Med, 23, 809-814.

Emri T et al, 1998. Changes in the glutathione (GSH) metabolismof Penicillium chrysogenum grown on different nitrogen,sulphur and carbon sources. J Basic Microbiol, 38, 3-8.

Emri T et al, 1999. Analysis of the oxidative stress response ofPenicillium chrysogenum to menadione. Free Rad Res, 30,125-132.

Frese D & Stahl U, 1992. Oxidative stress and ageing in the fun-gus Podospora anserina. Mech Ageing Dev, 65, 277-288.

Frohlich K U & Madeo F, 2000. Apoptosis in yeast-A mono-cellular organism exhibits altruistic behaviour. FEBS Left,473,6-9.

Gasch A P et al, 2000. Genomic expression programs in the re-sponse of yeast cells to environmental changes. Mol BioiCell, 11,4241-4257.

Gooday G W et al, 1992. What are the roles of chitinases in thegrowing fungus? FEMS Microbiol Lett, 100,387-392.

Gooday G W, 1997. The many uses of chitinases in nature.Chitinase Chitosan Res, 3, 233-243.

Hansberg W & Aguirre J, 1990. Hyperoxidant states cause micro-bial cell differentiation by cell isolation from dioxygen. JTheor Bioi, 142,201-221.

Hansberg W et al, 1993. Reactive oxygen species associated withcell differentiation in Neurospora crassa. Free Rad BioiMed, 14,287-293.

Harman D, 1993. Free radical involvement in aging. Drugs Aging,3,60-80

Harvey L Met al, 1998. Autolysis in batch cultures of Penicilliumchrysogenum at varying agitation rates. Enzyme MicrobialTechnol, 22,446-458.

Henriksen C M et al, 1997. Influence of the dissolved oxygenconcentration on the penicillin biosynthetic pathway insteady-state cultures of Penicillium chrysogenum. BiotechnolProg, 13,776-782.

Ho C S & Smith M D, 1986. Morphological alterations of Peni-cillium chrysogenum caused by carbon dioxide. J Gen Mi-crobiol, 132, 3479-3484.

Igual J C et al, 1996. Coordinated regulation of gene expressionby the cell cycle transcription factor SWI4 and the proteinkinase C AMP kinase pathway for yeast cell integrity. EMBOJ, 15,5001-5013.

Jakubowski W et al, 2000. Oxidative stress during aging of sta-tionary cultures of the yeast Saccharomyces cerevisiae. FreeRad Bioi Med, 28, 659-664.

Ju L K et al, 1991. Effects of carbon dioxide on the rheologicalbehavior and oxygen transfer in submerged penicillin fer-mentations. Biotechnol Bioeng, 38, 1223-1232.

JUrgensen C W et al, 2001. Glutathione metabolism and dimor-phism in Aureobasidium pullulans. J Basic Microbiol, 41,131-137.

Ji.isten P et al, 1998. Dependence of Penicillium chrysogenumgrowth, morphology, vacuolation, and productivity in fed-batch fermentations on impeller type and agitation intensity.Biotechnol Bioeng, 59,762-775.

Karaffa L et al, 2001. Cyanide-resistant alternative respiration isstrictly correlated to intracellular peroxide levels in Acremo-nium chrysogenum. Free Rad Res, 34,405-416.

Kreiner M et al, 2003. Morphological and enzymatic responses ofa recombinant Aspergillus niger to oxidative stressors inchemostat cultures. J Biotechnol, 100,251-260.

Laun P et al, 2001. Aged mother cells of Saccharomyces cere-visiae show markers of oxidative stress and apoptosis. MolMicrobiol, 39,1166-1173.

Lev S et al, 1999. A mitogen-activated protein kinase of the cornleaf pathogen Cochliobolus heterostrophus is involved in co-nidiation, appressorium formation, and pathogenicity: Di-verse roles for mitogen-activated protein kinase homology infoliar pathogens. Proc Natl Acad Sci USA, 96, 13542-13547.

Lorin S et al, 2001. Overexpression of the alternative oxidaserestores senescence and fertility in a long-lived respiration-deficient mutant of Podospora anserina. Mol Microbiol, 42,1259-1267.

Madeo F et al, 2002. Apoptosis in yeast: A new model systemwith applications in cell biology and medicine. Curr Genet,41,208-216.

McIntyre M et al, 1999. Response of Penicillium chrysogenum tooxygen starvation in glucose- and nitrogen-limited chemostatcultures. Enzyme Microb Technol, 25,447-454.

McIntyre M et al, 2000. Role of proteases in autolysis of Penicil-lium chrysogenum chemostat cultures in response to nutrientdepletion. Appl Microbiol Biotechnol, 53, 235-242.

McNeil B et al, 1998. Measurement of autolysis in submergedbatch cultures of Penicillium chrysogenum. Biotechnol Bio-eng, 57,297-305.

Mehdi K & Penninckx M J, 1997. An important role of glutathi-one and y-glutamyltranspeptidase in the supply of growth re-quirements during nitrogen starvation of the yeast Saccha-romyces cerevisiae. Microbiology, 143, 1885-1889.

Natsume M & Marumo S, 1984. Arthrospore-inducing substancesfrom Acremonium chrysogenum which stimulate cephalospo-rin C production. Agric Bioi Chem, 48,567-569.

POCSI et al: AUTOLYSIS OF PENICILLIUM CHRYSOGENUM

Nestelbacher R et al, 2000. The influence of oxygen toxicity onyeast mother cell-specific aging. Exp Gerontal, 35,63-70.

Paul G C et al, 1994. Hyphal vacuolation and fragmentation inPenicillium chrysogenum. Biotechnol Bioeng, 44, 655-660.

Penninckx M J, 2000. A short review on the role of glutathione inthe response of yeasts to nutritional, environmental, and oxi-dative stresses. Enzyme Microb Technol, 26,737-742.

P6csi I et al, 1993. The formation of N-acetyl-~-D-hexosaminidase is repressed by glucose in Penicilliumchrysogenum. J Basic Microbiol, 33, 259-267.

P6csi I et al, 2000. Allosamidin inhibits the fragmentation andautolysis of P. chrysogenum. in Advances in Chitin Science,edited by M G Peter, A Domard & R A A Muzzarelli, Vol 4.University of Potsdam, Potsdam. Pp 558-564.

Priede M A et al, 1997. Control of the production of individualfusicoccins at different dissolved oxygen concentrations.World J Microbiol Biotechnol, 13, 665-670.

Pusztahelyi T et al, 1997a. Ageing of Penicillium chrysogenumcultures under carbon starvation. I: Morphological changesand secondary metabolite production. Biotechnol Appl Bio-chem, 25, 81-86.

Pusztahelyi T et al, 1997b. Ageing of Penicillium chrysogenumcultures under carbon starvation. II: Protease and N-acetyl-~-D-hexosaminidase production. Biotechnol Appl Biochem, 25,87-93.

Reichard U et al, 2000. Disruption of the gene which encodes aserodiagnostic antigen and chitinase of the human fungalpathogen Coccidioides immitis. Infect Immun, 68, 5830-5838.

Roze L V & Linz J E, 1998. Lovastatin triggers apoptosis-like celldeath process in the fungus Mucor racemosus. Fungal GenBioi, 25, 119-133.

Rozen S et al, 1999. The Aspergillus nidulans sfaD gene encodesa G protein ~ subunit that is required for normal growth andrepression of sporulation. EMBO J, 18,5592-5600.

Ruijter G J G et al, 2002. Production of organic acids by fungi. inThe Mycota, edited by K Esser & J W Bennett, Vol X,Springer- Verlag, Berlin. Pp 213-230.

Sami L et al, 2001a. Autolysis and ageing of Penicillium chryso-genum cultures under carbon starvation: Glutathione me-tabolism and formation of reactive oxygen species. MycolRes, 105, 1246-1250.

Sami L et al, 2001b. Autolysis and ageing of Penicillium chryso-genum cultures under carbon starvation: Chitinase production

301

and antifungal effect of allosamidin. J Gen Appl Microbiol,47,201-211.

Sami L, Karaffa L, Emri T & P6csi I, 2003. Autolysis and ageingof Penicillium chrysogenum under carbon starvation: Respi-ration and glucose oxidase production. Acta Microbiol Im-munol Hung, in press.

Sandor E et al, 1998. Allosamidin inhibits the fragmentation ofAcremonium chrysogenum but does not influence thecephalosporin-C production of the fungus. FEMS MicrobiolLett, 164,231-236.

Shimizu K & Keller N P, 2001. Genetic involvement of a cAMP-dependent protein kinase in a G protein signaling pathwayregulating morphological and chemical transitions in Asper-gillus nidulans. Genetics, 157,591-600.

Skulachev V P, 2000. Mitochondria in the programmed deathphenomena; a principle of biology: "It is better to die than tobe wrong". IUBMB Life, 49, 365-373.

Skulachev V P, 2001. The programmed death phenomena, ageing,and the Samurai law of biology. Exp Gerontol, 36, 995-1024.

Takaya N et al, 1998. Cloning and charaterization of a chitinase-encoding gene (chiA) from Aspergillus nidulans, disruptionof which decreases germination frequency and hyphalgrowth. Biosci Biotechnol Biochem, 62,60-65.

Thevelein J M & de Winde J H, 1999. Novel sensing mechanismand targets for the cAMP-protein kinase: A pathway in theyeast Saccharomyces cerevisiae. Mol Microbiol, 33, 904-918.

Thrane C & Olsson S, 1998. Do fungi die by controlled celldeath? Abstr 6'" Int Mycol Congr, Int Mycol Congr, Jerusa-lem. P 103.

Toledo I et al, 1995. Redox imbalance at the start of each mor-phogenetic step of Neurospora crassa conidiation. ArchBiochem Biophys, 319, 519-524.

Trinci A P J & Righelato R C, 1970. Changes in constituents andultrastructure of hyphal compartments during autolysis ofglucose-starved Penicillium chrysogenum. J Gen Microbiol,60,239-249.

White S et al, 2002. The autolysis of industrial filamentous fungi.Crit Rev Biotechnol, 22, 1-14.

Zhang Y & Herman B, 2002. Ageing and apoptosis. Mech AgeingDev, 123, 245-260.