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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 2003, p. 65006506 Vol. 69, No.

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0099-2240/03/$08.000 DOI: 10.1128/AEM.69.11.65006506.2003
Copyright 2003, American Society for Microbiology. All Rights Reserved.

Reactive Oxygen Species and Induction of Lignin Peroxidase in


Phanerochaete chrysosporium
Paula A. Belinky,1 Nufar Flikshtein,1,2 Sergey Lechenko,1 Shimon Gepstein,2
and Carlos G. Dosoretz3*
MIGAL-Galilee Technology Center, Kiryat Shmona 10200,1 and Faculty of Biology2 and Division of Environmental
Engineering, Infrastructure and Environmental Sciences, Faculty of Civil and Environmental Engineering,3
Technion-Israel Institute of Technology, Haifa 32000, Israel
Received 7 April 2003/Accepted 7 August 2003

We studied oxidative stress in lignin peroxidase (LIP)-producing cultures (cultures flushed with pure O2) of
Phanerochaete chrysosporium by comparing levels of reactive oxygen species (ROS), cumulative oxidative
damage, and antioxidant enzymes with those found in non-LIP-producing cultures (cultures grown with free
exchange of atmospheric air [control cultures]). A significant increase in the intracellular peroxide concen-
tration and the degree of oxidative damage to macromolecules, e.g., DNA, lipids, and proteins, was observed
when the fungus was exposed to pure O2 gas. The specific activities of manganese superoxide dismutase,
catalase, glutathione reductase, and glutathione peroxidase and the consumption of glutathione were all higher
in cultures exposed to pure O2 (oxygenated cultures) than in cultures grown with atmospheric air. Significantly
higher gene expression of the LIP-H2 isozyme occurred in the oxygenated cultures. A hydroxyl radical
scavenger, dimethyl sulfoxide (50 mM), added to the culture every 12 h, completely abolished LIP expression
at the mRNA and protein levels. This effect was confirmed by in situ generation of hydroxyl radicals via the
Fenton reaction, which significantly enhanced LIP expression. The level of intracellular cyclic AMP (cAMP)
was correlated with the starvation conditions regardless of the oxygenation regimen applied, and similar cAMP
levels were obtained at high O2 concentrations and in cultures grown with atmospheric air. These results
suggest that even though cAMP is a prerequisite for LIP expression, high levels of ROS, preferentially hydroxyl
radicals, are required to trigger LIP synthesis. Thus, the induction of LIP expression by O2 is at least partially
mediated by the intracellular ROS.

The white rot fungus Phanerochaete chrysosporium can de- cellular ultrastructure and chlamydospore development, prob-
grade and metabolize lignin and a broad range of recalcitrant ably in response to accumulating ROS. LIP activities were
organopollutants (14, 34). Lignin depolymerization is achieved similar in cultures of P. chrysosporium exposed to pure O2 gas
primarily by one-electron oxidation reactions catalyzed by ex- (oxygenated cultures) and in manganese-deficient cultures
tracellular oxidases and peroxidases in the presence of extra- grown with atmospheric air (36). No manganese-containing
cellular hydrogen peroxide (H2O2). Lignin peroxidase (LIP) is superoxide dismutase (MnSOD) activity was detected in the
an enzyme commonly associated with the extracellular degra- latter cultures, suggesting that both cultures are saturated by
dation of lignin by P. chrysosporium. It oxidizes phenols to the ROS needed to trigger LIP expression. The level of tran-
phenoxy radicals and nonphenolic aromatics to radical cations scription of manganese peroxidase in cultures of P. chrysospo-
(6, 21). Liquid cultures of P. chrysosporium must be starved rium was reported to correlate with the concentration of ex-
(nitrogen or carbon depletion) and exposed to a pure O2 at- ogenous hydrogen peroxide added in the absence or presence
mosphere to trigger LIP expression (4, 11, 26). Boominathan of Mn2 (28). The transcription of versatile peroxidase in
and Reddy (7) showed that transcription of genes encoding liquid cultures of Pleurotus eryngii was also found to correlate
some LIP enzymes is controlled by cyclic AMP (cAMP) levels. with the addition of exogenous hydrogen peroxide or hydroxyl
Increasing oxygen availability to liquid cultures of P. chryso- radicals (38).
sporium increases LIP levels (35, 36). The high O2 concentra- Protection against the deleterious effects of ROS has been
tion presumably leads to increased production of reactive ox- analyzed in depth in yeasts, including Saccharomyces cerevisiae
ygen species (ROS) relative to that during normal metabolism, (19), Schizosaccharomyces pombe (24), Candida albicans (20),
subjecting the fungus to a ROS-rich environment, in addition and Hansenula mrakii (42). The filamentous fungus Penicillium
to the toxic radical intermediates produced in the ligninolytic chrysogenum is also highly resistant to oxidative stress caused
phase. Zacchi et al. (4446) suggested that cultures of P. by high concentrations of peroxides and superoxide anions,
chrysosporium exposed to O2 to trigger LIP synthesis are sub- which are attributed to high levels of catalase and glutathione
jected to oxygen toxicity, which leads to disorganization of the peroxidase activity (12, 13). Catalase activity was examined in
different strains of P. chrysosporium under various growth con-
ditions (23, 29). In low-nitrogen cultures (i.e., excess glucose),
* Corresponding author. Mailing address: Division of Environmen- production of catalase preceded that of LIP by approximately
tal Engineering, Infrastructure and Environmental Sciences, Faculty of
Civil and Environmental Engineering, Technion-Israel Institute of
3 days, whereas in low-carbon cultures, catalase was produced
Technology, Haifa 32000, Israel. Phone: 972-4-8294962. Fax: 972-4- at an even higher level, but LIP activity was not detected
8228898. E-mail: carlosd@tx.technion.ac.il. during the incubation period (23). The specific activities of

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VOL. 69, 2003 ROS INDUCTION OF LIP GENE EXPRESSION 6501

both catalase and SOD decreased at the end of the primary tor of superoxide) was measured in the extracellular medium and in the cellular
growth phase before idiophase in liquid cultures of P. chryso- extract with a fluorescence spectrophotometer (F-2000; Hitachi, Tokyo, Japan).
To detect 2,7-dichlorofluorescein, the extinction and emission wavelengths
sporium (CMI 174727) (30). were 520 6 and 590 10 nm, respectively. To detect ethidium, the extinction
LIP production in liquid cultures of P. chrysosporium occurs and emission wavelengths were 501 and 521 nm, respectively.
only when the cultures are flushed with pure O2. The objective Cell extract preparations. Pellets were separated from extracellular fluid by
of this study was to determine the mode of action by which this filtration through 200-m-pore-size nylon mesh (Amiad Filtration Systems,
Amiad, Israel). Extracellular fluid was used for LIP activity measurements and
high O2 requirement triggers LIP synthesis. Our working hy-
the fractionation of heme proteins.
pothesis was that ROS induce LIP gene expression. The results Cell extracts were prepared differently for the four different aims.
of this work provide new insights in the understanding of the (i) Determination of oxidative damage of macromolecules, intracellular enzy-
signal transduction mechanisms triggering LIP synthesis in re- matic activities, and protein content. Samples were homogenized in the cold for
sponse to high oxygen levels in P. chrysosporium in particular 2 min in 50 mM phosphate buffer, pH 7, with an Ultra-Turrax T25 homogenizer
(IKA, Staufen, Germany). The homogenate was centrifuged at 20,000 g for 20
and in white rot fungi in general. min at 4C. PMSF (1 mM) was added to each sample at the time of homogeni-
zation.
(ii) Measurement of intracellular cAMP. EDTA (4 mM) in 2 ml of 5%
MATERIALS AND METHODS (vol/vol) perchloric acid was added to the samples before homogenization to
Reagents. Hematoxylin, 5,5-dithiobis-2-nitrobenzoic acid (DTNB), NADP prevent enzymatic degradation of cAMP. Homogenization and centrifugation
(NADPH), veratryl alcohol, phenylmethanesulfonyl fluoride (PMSF), reduced were performed as described above.
glutathione (GSH), oxidized glutathione (GSSG), glutathione reductase, 2,4- (iii) Measurement of GSH and GSSG content. Two milliliters of 5% (vol/vol)
dinitrophenylhydrazine (DNPH), p-nitrophenylphosphate, dimethyl sulfoxide perchloric acid was added to each sample before homogenization. The homog-
(DMSO), mannitol, MnSOD from Escherichia coli, calf thymus DNA, N-methyl- enates were centrifuged at 20,000 g for 15 min at 4C.
2-phenylindole, and bovine serum albumin were purchased from Sigma Chem- (iv) RNA and DNA isolation. Frozen biomass (frozen by treatment with liquid
ical Co. (St. Louis, Mo.). 1-Methyl-2-vinylpyridinium trifluoromethanesulfonate nitrogen) was ground with a mortar and pestle. The cell extract was separated
was purchased from Bioxytech (Oxis International Inc., Portland, Oreg.). Alde- from cell debris by centrifugation at 20,000 g for 20 min at 4C.
hyde reactive probe (ARP)-biotinylated alkoxyamine, and 2,7-dichlorodihy- Measurements of oxidative damage. (i) Oxidized lipids. The levels of oxidized
drofluorescein diacetate were from Molecular Probes (Eugene, Oreg.). Vec- lipids were detected with a lipid peroxidation assay kit (catalog no. 437634;
tastain ABC-alkaline phosphatase reagent was from Vector Laboratories Calbiochem, San Diego, Calif.) following the manufacturers instructions. This
(Burlingame, Calif.), and dihydroethidium was from Fluka (Buchs, Switzerland). kit detects a chromogen that absorbs 586-nm-wavelength light and is formed by
Fungal strain and culture conditions. The filamentous fungus P. chrysospo- the reaction between malondialdehyde (MDA), an end product of the peroxi-
rium Burds BKM-F-1767 (ATCC 24725) was used for this study. The fungus was dation of polyunsaturated fatty acids, and N-methyl-2-phenylindole.
maintained at 4C on 2% (wt/vol) malt extract agar slants and inoculated by the (ii) Oxidized proteins. The levels of oxidized proteins were monitored by
method of Tien and Kirk (41). The growth medium was prepared as previously measuring carbonyl accumulation, which serves as an early marker for metal-
described (35, 36) with initial concentrations of glucose, diammonium tartrate, catalyzed protein oxidation (40). Redox cycling cations, e.g., Fe2, can bind to
and MnSO4 H2O of 56, 2.4, and 0.225 mM, respectively. The fungus was grown cation-binding sites on proteins and, when combined with H2O2, transform side
in submerged liquid cultures (90 ml) at 175 rpm and at 37C in 250-ml flasks chain amine groups on some amino acids into carbonyl groups. Protein carbonyl
sealed with rubber stoppers, and the headspace was flushed twice a day with O2 content (PCC) in the cellular extracts was measured in an enzyme-linked immu-
for 2 min at a flow rate of 1 liter/min (oxygenated cultures). Oxygen gas was of nosorbent assay (ELISA), a modified version of the method described by Buss et
medical-grade purity. Cultures grown with free exchange of atmospheric air al. (8). The hydrazones obtained from derivatization of proteins with DNPH,
(flasks sealed with dense paper plugs) (aerated cultures) served as controls. were reacted with biotinylated anti-DNPH and quantified using Vectastain ABC-
Unless otherwise specified, the cultures were incubated for 5 days. For determi- alkaline phosphatase reagent instead of streptavidin-biotinylated horseradish
nation of LIP activity, ROS concentration, macromolecule damage, and antiox- peroxidase. A standard curve representing 0 to 4 nmol of PCC/mg of protein was
idant enzyme activities, the entire contents of flasks were taken after 24, 48, 72, prepared with oxidized bovine serum albumin (8).
96, and 120 h of culture. (iii) Oxidized DNA. The principal damage to DNA produced by ROS is the
To determine the effect of hydroxyl radical (OH ) on LIP synthesis, the fungus loss of a base and the production of abasic (AP) sites. Oxidative damage of DNA
was grown under the same conditions as described above, in oxygenated or molecules was measured as the number of AP sites/104 bp by an ELISA-like
aerated cultures with or without the addition of 50 mM DMSO every 12 h for assay (22). A biotinylated aldehyde-specific reagent (ARP-biotinylated alkoxy-
120 h (27). The effect of DMSO on cell viability was measured by inoculating amine) reacted specifically with the aldehyde group present in the open ring in
mycelial plugs onto agar plates containing the growth medium supplemented AP sites, and the amount of biotinylated AP sites was quantified using Vectastain
with various concentrations of DMSO and comparing the amount of radial ABC-alkaline phosphatase reagent. Calf thymus DNA containing 1 to 5 AP
growth. For determination of LIP expression (activity, transcription, and heme sites/104 bp, was used for the standard calibration curve. DNA was extracted by
protein analyses), the entire contents of flasks were taken after 96 and 120 h of a modified version of the rapid method of Raeder and Broda (33).
culture. GSH/GSSG ratio. The GSH/GSSG molar ratio was determined by using the
The direct effect of OH on LIP expression was studied through the in situ Bioxytech GSH:GSSG-412 assay kit (catalog no. 21040) according to the man-
generation of OH via the Fenton reaction with Fe(II)-EDTA (0.1 mM) and ufacturers instructions with slight modifications. This method employs DTNB,
H2O2 (0.5 mM). The Fenton reagents were added separately or together to which reacts with GSH to form 2-nitro-5-thiobenzoate anion (TNB2), a product
cultures grown for 96 h with free exchange of atmospheric air, and the cultures which can be detected spectrophotometrically with 412-nm-wavelength light with
were incubated for an additional hour. Cultures that were incubated without an extinction coefficient of 14,150 M1 cm1. The concentration of GSH was
Fenton reagents or OH scavengers served as controls. Cultures incubated with calculated from a calibration curve (0 to 3 M GSH). The assay was conducted
Fenton reagents in the presence of either 50 mM DMSO or 50 mM mannitol on two parallel sets of samples. One set of samples, preincubated with glutha-
served as negative controls. After the additional incubation, the sample flasks thione reductase, gave total glutathione (reduced and oxidized) as GSH. The
were harvested, and LIP transcription was then determined. second set, used for GSSG determination, was first treated with the thiol-scav-
ROS determination. Intracellular ROS concentrations were determined in enging reagent 1-methyl-2-vinylpyridinium trifluoromethanesulfonate to scav-
liquid cultures of P. chrysosporium at tropophase and idiophase. H2O2 and enge GSH. The remaining GSSG was then reduced to GSH with NADPH
superoxide anion (O2) levels were detected by adding 2,7-dichlorodihy- catalyzed by glutathione reductase, and the GSH formed was measured. GSSG
drofluorescein diacetate (13, 37) and dihydroethidium (9, 13), respectively, to the at concentrations from 0 to 1.5 M was used as the standard to calibrate the
cultures. These compounds were added to the cultures at a final concentration of curves. The GSH/GSSG ratio was calculated as follows: GSH/GSSG [GSH
30 M and incubated for 30 min at 37C and 250 rpm. The mycelia were then (2 GSSG)]/GSSG.
harvested, washed with double-distilled water, and treated with 2 ml of 5% Determination of antioxidant enzyme activities. (i) MnSOD activity. The SOD
5-sulfosalicylic acid (vol/vol) for 20 min at 4C. The treated mycelia were cen- activity assay (5) was based on the SOD-mediated increase in the rate of auto-
trifuged at 20,000 g for 15 min at 4C. The production of 2,7-dichlorofluo- oxidation of hematoxylin in aqueous alkaline solution, which yields a chro-
rescein (a fluorescent indicator of peroxide) and ethidium (a fluorescent indica- mophore with maximum absorbance at 560 nm (29). The change in the absor-
6502 BELINKY ET AL. APPL. ENVIRON. MICROBIOL.

bance of the mixture at 560 nm, which measured the auto-oxidation of


hematoxylin, was monitored for 1 min.
The activity of the enzyme in each sample was calculated as the ratio between
the reaction rates of the auto-oxidation of hematoxylin in the presence of sample
(Vsample) and in the absence of sample (Vcontrol). Vsample/Vcontrol was translated
to units of activity (units per milliliter) by means of a calibration curve prepared
with 0 to 400 U of purified MnSOD from E. coli per ml. Actual MnSOD activity
was expressed as relative units.
CuZnSOD activity is sensitive to both H2O2 and cyanide. In contrast, MnSOD
activity is not inhibited by H2O2 or cyanide. To distinguish MnSOD from CuZn
SOD, MnSOD activity was assayed in the presence of 5 mM KCN in all cases.
(ii) Catalase activity. Catalase activity was assayed by measuring the degra-
dation of H2O2. The rate of disappearance of H2O2 was monitored by measuring
A240 (25). One unit of catalase decomposed 1 mol of H2O2 in 1 min (H2O2
39.4 M1 cm1).
(iii) Glutathione reductase activity. Glutathione reductase activity was deter-
mined by monitoring the rate of production of TNB2 at 412 nm. TNB2 is
formed by the reduction of DTNB by GSH, which in turn is formed by the
reduction of GSSG by glutathione reductase (25). One unit of enzyme activity
was defined as the production of 1 mol of TNB2 per min (TNB2 14,150
M1 cm1). FIG. 1. Levels of hydrogen peroxide and superoxide anion in oxy-
(iv) Glutathione peroxidase activity. Glutathione peroxidase activity was as- genated and aerated cultures of P. chrysosporium as a function of
sayed by the method of Chiu et al. (10) with slight modifications. One unit of culture age. Oxygenation was performed by flushing the headspace of
enzyme activity was defined as the oxidation of 1 mol of NADPH per min the culture flasks with pure oxygen gas for 2 min at a flow rate of 1
(NADPH 6,220 M1 cm1). liter/min. Aerated cultures are cultures exposed to free exchange of air.
cAMP assay. Intracellular cAMP was measured with the 3H-labeled cAMP The levels of hydrogen peroxide in oxygenated cultures (F) and aer-
assay kit (catalog no.TRK 432; Amersham, Little Chalfont, Buckinghamshire, ated cultures (E) and the levels of O2 in oxygenated cultures () and
United Kingdom). The cAMP assay is based on the competition between unla- aerated cultures () were determined by fluorometric detection of the
beled cAMP and a fixed quantity of the tritium-labeled compound for binding to oxidation products of 2,7-dichlorodihydrofluorescein diacetate and
a protein which has a high specificity and affinity for cAMP. The amount of dihydroethidium, respectively. FU521, fluorescence units at 521 nm;
labeled protein-cAMP complex formed is inversely related to the amount of FU600, fluorescence units at 600 nm. The means standard deviations
unlabeled cAMP present in the assay sample. (error bars) of eight replicates are shown.
Expression of LIP. (i) LIP activity. Enzyme activity was measured in the
extracellular fluid by the method of Tien and Kirk (41). One unit of activity was
defined as 1 mol of veratryl alcohol oxidized to veratryl aldehyde per min
(iii) LIP heme protein analysis. LIP heme proteins were analyzed as previ-
(veratryl aldehyde 9,300 M1 cm1).
ously reported (36). Heme proteins (isozymes H1 to H10) were identified on the
(ii) LIP transcription. Total RNA was isolated from P. chrysosporium cell
basis of elution properties and the results of activity tests.
cultures with the Tri reagent (Sigma), according to the manufacturers instruc-
tions. LIP mRNA levels were measured by reverse transcription-PCR (RT-PCR)
and compared to P. chrysosporium 18S rRNA levels (1). We measured only the RESULTS
mRNA level of isozyme LIP-H2. First-strand cDNA synthesis and the amplifi-
cation reaction were performed in the same tube (Access RT-PCR System; ROS level. Mycelia produced in cultures treated with oxygen
Promega, Madison, Wis.). The 50-l reaction mixture contained avian myelo- grew and utilized glucose at a rate similar to that of cultures
blastosis virus (AMV) RT/Tfl reaction buffer, 100 g of total RNA, 0.2 mM grown with atmospheric air (not shown).
concentration (total) of a mixture of the four deoxynucleoside triphosphates, 5 U
of AMV reverse transcriptase, and 5 U of the thermostable Tfl DNA polymerase.
In cultures flushed with pure O2, intracellular H2O2 concen-
The mixture was incubated at 48C for 45 min for first-strand cDNA synthesis tration increased gradually with culture age, reaching a maxi-
and then heated to 94C for 2 min to inactivate the reverse transcriptase. The mum during idiophase (almost 10-fold higher than the initial
primer 5-AGAGCGCCGTGAAGATGAACTGGA-3 was used for the first- level), but remained stable during the entire incubation period
strand cDNA synthesis of LIP-H2, and the primer 5-CAACTACGAGCTTTT in the aerated control cultures, with only a slight increase of
TAACTGC-3 was used for the first-strand cDNA synthesis of 18S rRNA.
Second-strand cDNA synthesis and PCR amplification with primers specific
less than twofold (Fig. 1). In contrast, O2 levels were highest
for LIP-H2 (encoded by the lip gene deposited in GenBank under accession at tropophase and decreased during the transition to idiophase
number X15599) (antisense [5-TCAGGTTCTCGCTCGCATGCT-3] and in aerated and oxygenated cultures (Fig. 1).
sense [5-AGAGCGCCGTGAAGATGAACTGGA-3]) were performed in a Antioxidative enzyme activity. ROS measurements alone are
minicycler (MJ Research, Watertown, Mass.) under the following conditions: 40 insufficient to determine the existence of oxidative stress, prob-
cycles of PCR amplification, with 1 cycle consisting of 30 s at 94C, 1 min at 62C,
and 2 min at 68C. 18S cDNA was amplified in a similar manner, except that 10
ably because of the short half-lives of ROS and the fact that the
cycles and a temperature of 55C for annealing were used. Two 18S rRNA- measurements taken are of steady-state concentrations. Cou-
specific primers (antisense [5-CAAATTACCCAATCCCGACAC-3] and sense pling ROS level with the change in activity of antioxidant
[5-CAACTACGAGCTTTTTAACTGC-3]) were used. Forty cycles of amplifi- enzymes and cumulative oxidative damage of macromolecules
cation of the LIP cDNA and 10 cycles of amplification of the 18S cDNA pro- gives a better indication of the existence of stress. MnSOD, but
duced enough PCR products for semiquantitative analysis, while maintaining the
linear relationship between the concentrations of both transcripts. LIP-H2 RNA
not CuZnSOD, activity was detected in fungal cell extracts,
and 18S rRNA levels were precalibrated and diluted to levels below the satura- since no change in activity was observed in the presence of
tion levels after PCR analysis. The short cycling of the rRNA amplicons was not KCN as described by Belinky et al. (5). MnSOD activity in-
at saturation levels. The precalibration identified plateau cycle levels. The PCR creased at similar rates in oxygenated and aerated cultures
products (5 l) were electrophoresed on a 2% agarose gel at 100 V for about 45
during the first 96 h of incubation (Fig. 2). Thereafter, it
min. All reaction mixtures were electrophoresed on the same gel. DNA was
stained with 30 g of ethidium bromide (Sigma) in 100 ml of distilled water and
increased approximately 1 order of magnitude in the oxygen-
then imaged and analyzed with a Fluor-S MultiImager (Bio-Rad, Hercules, ated cultures but remained constant in the aerated cultures.
Calif.). Catalase activity peaked at 96 and 72 h in the oxygenated and
VOL. 69, 2003 ROS INDUCTION OF LIP GENE EXPRESSION 6503

FIG. 2. MnSOD and catalase activities in cell extracts of P. chryso- FIG. 3. Oxidative damage in oxygenated and aerated cultures of P.
sporium as a function of culture age from oxygenated and aerated chrysosporium as a function of culture age. Lipid oxidative damage was
cultures. SOD activity (measured in relative units [rU]) in oxygenated determined in oxygenated cultures () and aerated cultures () by the
cultures () and aerated cultures () was assayed by the auto-oxida- MDA assay. Protein oxidative damage was determined in oxygenated
tion of hematoxylin in the presence of 5 mM KCN. Catalase activity in cultures (F) and aerated cultures (E) by measuring PCC using an
oxygenated cultures (F) and aerated cultures (E) was determined by ELISA-like method. The means standard deviations (error bars) of
monitoring the degradation of H2O2 as measured by the absorbance at eight replicates are shown.
240 nm. The means standard deviations (error bars) of eight repli-
cates are shown.

fold higher) in oxygenated cultures approaching and during


idiophase. Some protein and lipid damage was also detected in
aerated cultures, respectively, although the peak was twice as aerated (control) cultures but at much lower levels. DNA AP
high for oxygenated cultures (Fig. 2). site production measured after 24 and 120 h of growth were 3
Glutathione peroxidase activities were similar during tropo- 0.45 and 7 0.97 AP sites/104 bp, respectively, in oxygen-
phase in oxygenated and aerated cultures (2 to 3 U/g) but ated cultures and 2 0.42 and 3 0.48 AP sites/104 bp,
increased to 15 U/g at idiophase in the oxygenated cultures respectively, in aerated cultures. These results indicate that the
compared to approximately 6 U/g in the aerated cultures (Ta- overall response of the antioxidant system was insufficient to
ble 1). In contrast, glutathione reductase activities were similar prevent macromolecule damage. Although the fungus survived
in both cultures during tropophase and idiophase (Table 1). when flushed with pure O2, the high levels of oxidized products
Although glutathione reductase activity hardly changed over of lipids, proteins, and DNA compared to those in cultures
time, its level was 10-to 85-fold higher than that of glutathione grown with atmospheric air confirmed the development of
peroxidase. The GSH/GSSG ratio was significantly lower in
oxygenated cultures than in aerated cultures and decreased
from tropophase to idiophase in both cultures but exhibited a
more pronounced decline in oxygenated cultures (Table 1).
Cumulative oxidative damage. Lipid oxidation and protein
carbonylation (Fig. 3) were significantly higher (roughly four-

TABLE 1. Glutathione-related functions in cell extracts


of P. chrysosporiuma
Enzyme activity
(U/g of protein) GSH/GSSG
Culture conditions
Glutathione Glutathione ratio
peroxidase reductase

Incubated for 24 h
Aerated (control) 3.3 0.8 173.2 11.6 70.5 9.5
Oxygenated 2.1 0.7 179.8 10.7 49.0 1.1

Incubated for 120 h


Aerated (control) 5.8 0.9 164.7 38.0 25.3 5.8
Oxygenated 15.3 2.2 154.2 39.5 2.71 1.8 FIG. 4. LIP activity in the extracellular fluid of P. chrysosporium
a
Glutathione peroxidase activity was determined by monitoring the oxygen- cultures as a function of culture age. LIP activity in oxygenated cul-
ation of NADPH. Glutathione reductase activity and the concentrations of tures () and aerated cultures (E) was assayed by monitoring the
GSSG and GSH were determined by monitoring the oxidation production of oxidation of veratryl alcohol to veratryl aldehyde. The means stan-
TNB2. The values are means standard deviations of eight replicates. dard deviations (error bars) of eight replicates are shown.
6504 BELINKY ET AL. APPL. ENVIRON. MICROBIOL.

aerated and oxygenated cultures grown without DMSO (Fig.


5). The LIP-H8 isoenzyme might be an exception to this
general pattern. The level of LIP-H8 isoenzyme in the ex-
perimental cultures was very low (perhaps within the base-
line drift), which makes it difficult to determine whether the
H8 isoenzyme is regulated differently, like the other heme
proteins. The levels of the other heme proteins decreased
significantly.
These results indicated that OH might differentially in-
fluence transcription. We tested this hypothesis by measur-
ing mRNA levels of LIP-H2 isozyme and its dephosphory-
lated form, LIP-H1, the predominant LIP isoenzyme
expressed. Significantly higher LIP-H2 transcription oc-
curred in oxygenated cultures than in aerated cultures, and
no LIP transcripts were obtained in the presence of 50 mM
DMSO (Fig. 6A and B). The level of 18S mRNA was con-
stant and did not vary with the different treatments (Fig. 6C
FIG. 5. Production of LIP isozymes by P. chrysosporium. LIP and D). Similar results were obtained with Northern blots
isozymes in oxygenated (solid line) and aerated (broken) cultures in (data not shown). The absence of LIP-H2 activity and tran-
the absence of the hydroxyl radical scavenger DMSO (controls) were
examined. No detection signals were obtained in the presence of
scription in cultures in which hydroxyl radicals were scav-
DMSO in any culture. Fractionation was performed by anion-exchange enged suggests the involvement of this radical as a second
high-performance liquid chromatography analysis, with a MonoQ col- messenger in LIP gene induction. Additional support for the
umn with monitoring at 409 nm. mAU, milli absorbance units. involvement of OH in LIP gene induction was obtained by
measuring LIP-H2 mRNA after in situ generation of OH
via the Fenton reaction (Fig. 7). Higher expression of
severe oxidative stress in oxygenated, i.e., LIP-producing, cul- LIP-H2 occurred in cultures with Fenton reagents than in
tures. regular cultures (with no Fenton reagents), while either
LIP activity and transcription. The pattern of extracellular DMSO or mannitol repressed LIP-H2 expression. Slightly
LIP activity was similar to the patterns of ROS accumulation higher LIP-H2 expression occurred when Fe(II) alone was
and macromolecule damage in oxygenated cultures, peaking at added, while when H2O2 alone was added, LIP-H2 expres-
120 h (Fig. 4). Negligible LIP activity was observed in aerated sion was increased, although its intensity was considerably
cultures. The correspondence between LIP activity and oxida- lower than when both Fenton reagents were added together
tive damage suggests a role for ROS in inducing LIP synthesis. (data not shown). The more pronounced repression ob-
Glucose consumption and biomass development in liquid cul- served with mannitol may be due to a higher OH concen-
tures and radial growth on agar plates containing 50 mM tration scavenged for mannitol than for DMSO. The level of
DMSO, an OH scavenger, were similar to the values for cul- 18S mRNA remained constant across the treatments.
tures and agar plates lacking DMSO. Thus, 50 mM DMSO Intracellular cAMP level. Intracellular cAMP levels re-
does not affect the availability of carbon and the viability of sponded directly to starvation and were almost identical in
fungal cells (not shown). both aerated and oxygenated cultures whether or not LIP
No LIP activity was detected in cultures supplemented synthesis occurred (Fig. 8). In all cases, it peaked near the
with DMSO (i.e., grown in the absence of intracellular OH) transition to the idiophase around day 2 as a function of
under either aerated or oxygenated conditions (not shown). nitrogen starvation. This trend corresponded to a complete
No significant levels of heme protein or LIP isozyme activity depletion of nitrogen during the first 30 h (not shown).
were detected in cultures grown with DMSO, in contrast to These results strongly suggest that even though cAMP is a

FIG. 6. LIP transcription in cell extracts of P. chrysosporium from oxygenated and aerated cultures in the presence and absence of the hydroxyl
radical scavenger DMSO. (A and C) Aerated cultures; (B and D) oxygenated cultures. LIP-H2 and 18S mRNA levels were determined by RT-PCR
analysis after 96 and 120 h of culture. Lanes M, DNA markers.
VOL. 69, 2003 ROS INDUCTION OF LIP GENE EXPRESSION 6505

gested that Mn deficiency increases the level of oxygen radicals


generated within the cell. Zacchi et al. (4446) reported a
major loss in organization of cellular structure, possibly due to
oxygen toxicity, in carbon-limited cultures of P. chrysosporium
exposed to an atmosphere of pure O2, which implies that LIP
may be synthesized in response to oxidative stress.
Oxygen free radicals or ROS mediate cellular activity and
FIG. 7. Effect of in situ generation of OH in LIP transcription in regulate gene expression. Superoxide mediates cell growth and
cell extracts of 96-h-old cultures of P. chrysosporium. Cultures that motility in tumor cells and endothelial cells (31, 32, 39), and
were grown with free exchange of atmospheric air for 96 h were oxygen free radicals and their derivatives help activate mito-
incubated for 1 h at 37C and at 175 rpm with or without Fenton gen-activated protein kinases, which induce phosphorylation
reagents (0.1 mM Fe-EDTA and 0.5 mM H2O2) in the absence or
and dephosphorylation cascades, leading to the conversion of
presence of DMSO or mannitol (50 mM). LIP-H2 and 18S mRNA
levels were determined by RT-PCR. Lane M, DNA markers. extracellular effects into intracellular actions (2). Oxidants me-
diate the mitogenic signaling induced by the Ras oncogene
(18). The increased production of oxygen free radicals induced
prerequisite for LIP expression, a second factor, possibly by high oxygen levels in liquid cultures of P. chrysosporium may
induced by ROS, may also be involved. Indeed, only cultures act as second messengers for the regulation of LIP gene ex-
exposed to pure O2 had significant LIP activity. pression.
It is difficult to directly identify ROS in any biological sample
due to their very short half-lives. A common approach is to add
DISCUSSION radical scavengers that undergo characteristic preferential re-
We found that P. chrysosporium cells produce a high con- actions with ROS over an extended period and then measure
centration of ROS when stimulated by pure O2, which seems to the accumulation of the diagnostic products in the culture (16).
be involved in modulating LIP expression. Oxidative stress was The hydroxyl radical, which has the shortest half-life, is the
confirmed by measuring ROS concentration, the enhancement most reactive and toxic oxygen radical, and it is nonspecific to
of the antioxidant defense system, and oxidative damage of tissues and macromolecules. Scavenging of OH appeared to be
major macromolecules, lipids, proteins, and DNA. The low the most effective way of proving its influence in vivo. DMSO
GSH/GSGG ratio and the high activities of SOD, catalase, and is a well-known scavenger of hydroxyl radicals (3, 15, 17, 27,
glutathione peroxidase in oxygenated cultures of P. chrysospo- 43). At 50 mM, DMSO did not affect the viability of the fungus,
rium could be associated with the neutralization of ROS, in- as evidenced by biomass, glucose consumption, and protein
cluding lipid peroxyl radicals. Resistance of P. chrysogenum to levels; however, it might directly or indirectly alter nutrient
high concentrations of peroxide (12) was explained by high availability and ultimately lip expression. On the basis of our
catalase and glutathione peroxidase activities. The levels of results, we hypothesize that ROS, and in particular hydroxyl
GSSG were higher than those of GSH levels when this fungus radicals, mediate LIP expression. When these radicals are de-
was exposed to the superoxide-generating agent menadione. pleted by DMSO, LIP expression is not induced at either the
Collectively, our results are consistent with recent hypothe- mRNA or protein level, regardless of the type of oxygenation.
ses in which oxidative stress conditions affect LIP synthesis in The lower levels of oxygen in aerated cultures may produce a
P. chrysosporium (35, 36, 4446). Rothschild et al. (36) sug- low concentration of hydroxyl radicals, resulting in a slight
induction of LIP expression. This hypothesis is also supported
by the fact that in situ generation of OH enhances LIP-H2
expression and that this expression was repressed by the OH
scavengers DMSO and mannitol.
The induction of LIP by oxygen may result from reactions of
ROS (hydroxyl radicals) with the cell surface or from the entry
of these species into the cells. Boominathan and Reddy (7)
suggested that transcription from genes encoding certain LIP
enzymes is controlled by cAMP levels, a starvation signal. We
found that intracellular cAMP levels responded to starvation
conditions and that similar cAMP levels occurred with or with-
out oxygenation. However, LIP expression was affected only
when pure O2 was flushed, i.e., under oxidative stress, suggest-
ing that cAMP is necessary, but not sufficient, for LIP expres-
sion, and that high levels of ROS, preferably hydroxyl radicals,
are needed to trigger LIP synthesis in P. chrysosporium.
Taken together, our findings demonstrate that the condi-
tions used to promote LIP synthesis in P. chrysosporium
result in oxidative stress, indicating the involvement of ROS
FIG. 8. Intracellular cAMP level in oxygenated () and aerated
in the induction of LIP gene expression. Although discern-
(E) cultures of P. chrysosporium, as a function of culture age. The ing the role for ROS in the signal transduction pathway of
means standard deviations (error bars) of five replicates are shown. LIP requires further work, the results presented here pro-
6506 BELINKY ET AL. APPL. ENVIRON. MICROBIOL.

vide a firm framework for future studies on the regulation of 22. Kow, Y. W., and A. Dare. 2000. Detection of abasic sites and oxidative DNA
base damage using an ELISA-like assay. Methods 22:164169.
fungal peroxidases.
23. Kwon, S. I., and A. J. Anderson. 2001. Catalase activities of Phanerochaete
chrysosporium are not coordinately produced with ligninolytic metabolism:
ACKNOWLEDGMENTS catalases from a white-rot fungus. Curr. Microbiol. 42:811.
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Colleges of Higher Education from the Sacta-Rashi Foundation and 31273132.
by the Regional R&D Program of the Ministry of Science of Israel. 25. Lee, J. S., Y. C. Hah, and J. H. Roe. 1993. The induction of oxidative enzymes
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