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

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

6500
VOL. 69, 2003
both catalase and SOD decreased at the end of the primary
growth phase before idiophase in liquid cultures of P. chryso-
sporium (CMI 174727) (30).
LIP production in liquid cultures of P. chrysosporium occurs
only when the cultures are flushed with pure O2. The objective
of this study was to determine the mode of action by which this
high O2 requirement triggers LIP synthesis. Our working hy-
pothesis was that ROS induce LIP gene expression. The results
of this work provide new insights in the understanding of the
signal transduction mechanisms triggering LIP synthesis in re-
sponse to high oxygen levels in P. chrysosporium in particular
and in white rot fungi in general.
MATERIALS AND METHODS
Reagents. Hematoxylin, 5,5-dithiobis-2-nitrobenzoic acid (DTNB), NADP
(NADPH), veratryl alcohol, phenylmethanesulfonyl fluoride (PMSF), reduced
glutathione (GSH), oxidized glutathione (GSSG), glutathione reductase, 2,4-
dinitrophenylhydrazine (DNPH), p-nitrophenylphosphate, dimethyl sulfoxide
(DMSO), mannitol, MnSOD from Escherichia coli, calf thymus DNA, N-methyl-
2-phenylindole, and bovine serum albumin were purchased from Sigma Chem-
ical Co. (St. Louis, Mo.). 1-Methyl-2-vinylpyridinium trifluoromethanesulfonate
was purchased from Bioxytech (Oxis International Inc., Portland, Oreg.). Alde-
hyde reactive probe (ARP)-biotinylated alkoxyamine, and 2,7-dichlorodihy-
drofluorescein diacetate were from Molecular Probes (Eugene, Oreg.). Vec-
tastain ABC-alkaline phosphatase reagent was from Vector Laboratories
(Burlingame, Calif.), and dihydroethidium was from Fluka (Buchs, Switzerland).
Fungal strain and culture conditions. The filamentous fungus P. chrysospo-
rium Burds BKM-F-1767 (ATCC 24725) was used for this study. The fungus was
maintained at 4C on 2% (wt/vol) malt extract agar slants and inoculated by the
method of Tien and Kirk (41). The growth medium was prepared as previously
described (35, 36) with initial concentrations of glucose, diammonium tartrate,
and MnSO4 H2O of 56, 2.4, and 0.225 mM, respectively. The fungus was grown
in submerged liquid cultures (90 ml) at 175 rpm and at 37C in 250-ml flasks
sealed with rubber stoppers, and the headspace was flushed twice a day with O2
for 2 min at a flow rate of 1 liter/min (oxygenated cultures). Oxygen gas was of
medical-grade purity. Cultures grown with free exchange of atmospheric air
(flasks sealed with dense paper plugs) (aerated cultures) served as controls.
Unless otherwise specified, the cultures were incubated for 5 days. For determi-
nation of LIP activity, ROS concentration, macromolecule damage, and antiox-
idant enzyme activities, the entire contents of flasks were taken after 24, 48, 72,
96, and 120 h of culture.
To determine the effect of hydroxyl radical (OH ) on LIP synthesis, the fungus
was grown under the same conditions as described above, in oxygenated or
aerated cultures with or without the addition of 50 mM DMSO every 12 h for
120 h (27). The effect of DMSO on cell viability was measured by inoculating
mycelial plugs onto agar plates containing the growth medium supplemented
with various concentrations of DMSO and comparing the amount of radial
growth. For determination of LIP expression (activity, transcription, and heme
protein analyses), the entire contents of flasks were taken after 96 and 120 h of
culture.
The direct effect of OH on LIP expression was studied through the in situ
generation of OH via the Fenton reaction with Fe(II)-EDTA (0.1 mM) and
H2O2 (0.5 mM). The Fenton reagents were added separately or together to
cultures grown for 96 h with free exchange of atmospheric air, and the cultures
were incubated for an additional hour. Cultures that were incubated without
Fenton reagents or OH scavengers served as controls. Cultures incubated with
Fenton reagents in the presence of either 50 mM DMSO or 50 mM mannitol
served as negative controls. After the additional incubation, the sample flasks
were harvested, and LIP transcription was then determined.
ROS determination. Intracellular ROS concentrations were determined in
liquid cultures of P. chrysosporium at tropophase and idiophase. H2O2 and
superoxide anion (O2) levels were detected by adding 2,7-dichlorodihy-
drofluorescein diacetate (13, 37) and dihydroethidium (9, 13), respectively, to the
cultures. These compounds were added to the cultures at a final concentration of
30 M and incubated for 30 min at 37C and 250 rpm. The mycelia were then
harvested, washed with double-distilled water, and treated with 2 ml of 5%
5-sulfosalicylic acid (vol/vol) for 20 min at 4C. The treated mycelia were cen-
trifuged at 20,000 g for 15 min at 4C. The production of 2,7-dichlorofluo-
rescein (a fluorescent indicator of peroxide) and ethidium (a fluorescent indica-
ROS INDUCTION OF LIP GENE EXPRESSION 6501
tor of superoxide) was measured in the extracellular medium and in the cellular
extract with a fluorescence spectrophotometer (F-2000; Hitachi, Tokyo, Japan).
To detect 2,7-dichlorofluorescein, the extinction and emission wavelengths
were 520 6 and 590 10 nm, respectively. To detect ethidium, the extinction
and emission wavelengths were 501 and 521 nm, respectively.
Cell extract preparations. Pellets were separated from extracellular fluid by
filtration through 200-m-pore-size nylon mesh (Amiad Filtration Systems,
Amiad, Israel). Extracellular fluid was used for LIP activity measurements and
the fractionation of heme proteins.
Cell extracts were prepared differently for the four different aims.
(i) Determination of oxidative damage of macromolecules, intracellular enzy-
matic activities, and protein content. Samples were homogenized in the cold for
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
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%
(vol/vol) perchloric acid was added to the samples before homogenization to
prevent enzymatic degradation of cAMP. Homogenization and centrifugation
were performed as described above.
(iii) Measurement of GSH and GSSG content. Two milliliters of 5% (vol/vol)
perchloric acid was added to each sample before homogenization. The homog-
enates were centrifuged at 20,000 g for 15 min at 4C.
(iv) RNA and DNA isolation. Frozen biomass (frozen by treatment with liquid
nitrogen) was ground with a mortar and pestle. The cell extract was separated
from cell debris by centrifugation at 20,000 g for 20 min at 4C.
Measurements of oxidative damage. (i) Oxidized lipids. The levels of oxidized
lipids were detected with a lipid peroxidation assay kit (catalog no. 437634;
Calbiochem, San Diego, Calif.) following the manufacturers instructions. This
kit detects a chromogen that absorbs 586-nm-wavelength light and is formed by
the reaction between malondialdehyde (MDA), an end product of the peroxi-
dation of polyunsaturated fatty acids, and N-methyl-2-phenylindole.
(ii) Oxidized proteins. The levels of oxidized proteins were monitored by
measuring carbonyl accumulation, which serves as an early marker for metal-
catalyzed protein oxidation (40). Redox cycling cations, e.g., Fe2, can bind to
cation-binding sites on proteins and, when combined with H2O2, transform side
chain amine groups on some amino acids into carbonyl groups. Protein carbonyl
content (PCC) in the cellular extracts was measured in an enzyme-linked immu-
nosorbent assay (ELISA), a modified version of the method described by Buss et
al. (8). The hydrazones obtained from derivatization of proteins with DNPH,
were reacted with biotinylated anti-DNPH and quantified using Vectastain ABC-
alkaline phosphatase reagent instead of streptavidin-biotinylated horseradish
peroxidase. A standard curve representing 0 to 4 nmol of PCC/mg of protein was
prepared with oxidized bovine serum albumin (8).
(iii) Oxidized DNA. The principal damage to DNA produced by ROS is the
loss of a base and the production of abasic (AP) sites. Oxidative damage of DNA
molecules was measured as the number of AP sites/104 bp by an ELISA-like
assay (22). A biotinylated aldehyde-specific reagent (ARP-biotinylated alkoxy-
amine) reacted specifically with the aldehyde group present in the open ring in
AP sites, and the amount of biotinylated AP sites was quantified using Vectastain
ABC-alkaline phosphatase reagent. Calf thymus DNA containing 1 to 5 AP
sites/104 bp, was used for the standard calibration curve. DNA was extracted by
a modified version of the rapid method of Raeder and Broda (33).
GSH/GSSG ratio. The GSH/GSSG molar ratio was determined by using the
Bioxytech GSH:GSSG-412 assay kit (catalog no. 21040) according to the man-
ufacturers instructions with slight modifications. This method employs DTNB,
which reacts with GSH to form 2-nitro-5-thiobenzoate anion (TNB2), a product
which can be detected spectrophotometrically with 412-nm-wavelength light with
an extinction coefficient of 14,150 M1 cm1. The concentration of GSH was
calculated from a calibration curve (0 to 3 M GSH). The assay was conducted
on two parallel sets of samples. One set of samples, preincubated with glutha-
thione reductase, gave total glutathione (reduced and oxidized) as GSH. The
second set, used for GSSG determination, was first treated with the thiol-scav-
enging reagent 1-methyl-2-vinylpyridinium trifluoromethanesulfonate to scav-
enge GSH. The remaining GSSG was then reduced to GSH with NADPH
catalyzed by glutathione reductase, and the GSH formed was measured. GSSG
at concentrations from 0 to 1.5 M was used as the standard to calibrate the
curves. The GSH/GSSG ratio was calculated as follows: GSH/GSSG [GSH
(2 GSSG)]/GSSG.
Determination of antioxidant enzyme activities. (i) MnSOD activity. The SOD
activity assay (5) was based on the SOD-mediated increase in the rate of auto-
oxidation of hematoxylin in aqueous alkaline solution, which yields a chro-
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-
sporium as a function of culture age from oxygenated and aerated
cultures. SOD activity (measured in relative units [rU]) in oxygenated
cultures () and aerated cultures () was assayed by the auto-oxida-
tion of hematoxylin in the presence of 5 mM KCN. Catalase activity in
oxygenated cultures (F) and aerated cultures (E) was determined by
monitoring the degradation of H2O2 as measured by the absorbance at
240 nm. The means standard deviations (error bars) of eight repli-
cates are shown.
FIG. 3. Oxidative damage in oxygenated and aerated cultures of P.
chrysosporium as a function of culture age. Lipid oxidative damage was
determined in oxygenated cultures () and aerated cultures () by the
MDA assay. Protein oxidative damage was determined in oxygenated
cultures (F) and aerated cultures (E) by measuring PCC using an
ELISA-like method. The means standard deviations (error bars) of
eight replicates are shown.

fold higher) in oxygenated cultures approaching and during

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

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.
FIG. 5. Production of LIP isozymes by P. chrysosporium. LIP
isozymes in oxygenated (solid line) and aerated (broken) cultures in
the absence of the hydroxyl radical scavenger DMSO (controls) were
examined. No detection signals were obtained in the presence of
DMSO in any culture. Fractionation was performed by anion-exchange
high-performance liquid chromatography analysis, with a MonoQ col-
umn with monitoring at 409 nm. mAU, milli absorbance units.
severe oxidative stress in oxygenated, i.e., LIP-producing, cul-
tures.
LIP activity and transcription. The pattern of extracellular
LIP activity was similar to the patterns of ROS accumulation
and macromolecule damage in oxygenated cultures, peaking at
120 h (Fig. 4). Negligible LIP activity was observed in aerated
cultures. The correspondence between LIP activity and oxida-
tive damage suggests a role for ROS in inducing LIP synthesis.
Glucose consumption and biomass development in liquid cul-
tures and radial growth on agar plates containing 50 mM
DMSO, an OH scavenger, were similar to the values for cul-
tures and agar plates lacking DMSO. Thus, 50 mM DMSO
does not affect the availability of carbon and the viability of
fungal cells (not shown).
No LIP activity was detected in cultures supplemented
with DMSO (i.e., grown in the absence of intracellular OH)
under either aerated or oxygenated conditions (not shown).
No significant levels of heme protein or LIP isozyme activity
were detected in cultures grown with DMSO, in contrast to
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.
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
and D). Similar results were obtained with Northern blots
(data not shown). The absence of LIP-H2 activity and tran-
scription in cultures in which hydroxyl radicals were scav-
enged suggests the involvement of this radical as a second
messenger in LIP gene induction. Additional support for the
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
LIP-H2 occurred in cultures with Fenton reagents than in
regular cultures (with no Fenton reagents), while either
DMSO or mannitol repressed LIP-H2 expression. Slightly
higher LIP-H2 expression occurred when Fe(II) alone was
added, while when H2O2 alone was added, LIP-H2 expres-
sion was increased, although its intensity was considerably
lower than when both Fenton reagents were added together
(data not shown). The more pronounced repression ob-
served with mannitol may be due to a higher OH concen-
tration scavenged for mannitol than for DMSO. The level of
18S mRNA remained constant across the treatments.
Intracellular cAMP level. Intracellular cAMP levels re-
sponded directly to starvation and were almost identical in
both aerated and oxygenated cultures whether or not LIP
synthesis occurred (Fig. 8). In all cases, it peaked near the
transition to the idiophase around day 2 as a function of
nitrogen starvation. This trend corresponded to a complete
depletion of nitrogen during the first 30 h (not shown).
These results strongly suggest that even though cAMP is a
VOL. 69, 2003
FIG. 7. Effect of in situ generation of OH in LIP transcription in
cell extracts of 96-h-old cultures of P. chrysosporium. Cultures that
were grown with free exchange of atmospheric air for 96 h were
incubated for 1 h at 37C and at 175 rpm with or without Fenton
reagents (0.1 mM Fe-EDTA and 0.5 mM H2O2) in the absence or
presence of DMSO or mannitol (50 mM). LIP-H2 and 18S mRNA
levels were determined by RT-PCR. Lane M, DNA markers.
prerequisite for LIP expression, a second factor, possibly
induced by ROS, may also be involved. Indeed, only cultures
exposed to pure O2 had significant LIP activity.
DISCUSSION
We found that P. chrysosporium cells produce a high con-
centration of ROS when stimulated by pure O2, which seems to
be involved in modulating LIP expression. Oxidative stress was
confirmed by measuring ROS concentration, the enhancement
of the antioxidant defense system, and oxidative damage of
major macromolecules, lipids, proteins, and DNA. The low
GSH/GSGG ratio and the high activities of SOD, catalase, and
glutathione peroxidase in oxygenated cultures of P. chrysospo-
rium could be associated with the neutralization of ROS, in-
cluding lipid peroxyl radicals. Resistance of P. chrysogenum to
high concentrations of peroxide (12) was explained by high
catalase and glutathione peroxidase activities. The levels of
GSSG were higher than those of GSH levels when this fungus
was exposed to the superoxide-generating agent menadione.
Collectively, our results are consistent with recent hypothe-
ses in which oxidative stress conditions affect LIP synthesis in
P. chrysosporium (35, 36, 4446). Rothschild et al. (36) sug-
FIG. 8. Intracellular cAMP level in oxygenated () and aerated
(E) cultures of P. chrysosporium, as a function of culture age. The
means standard deviations (error bars) of five replicates are shown.
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
regulate gene expression. Superoxide mediates cell growth and
motility in tumor cells and endothelial cells (31, 32, 39), and
oxygen free radicals and their derivatives help activate mito-
gen-activated protein kinases, which induce phosphorylation
and dephosphorylation cascades, leading to the conversion of
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
by high oxygen levels in liquid cultures of P. chrysosporium may
act as second messengers for the regulation of LIP gene ex-
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
radical scavengers that undergo characteristic preferential re-
actions with ROS over an extended period and then measure
the accumulation of the diagnostic products in the culture (16).
The hydroxyl radical, which has the shortest half-life, is the
most reactive and toxic oxygen radical, and it is nonspecific to
tissues and macromolecules. Scavenging of OH appeared to be
the most effective way of proving its influence in vivo. DMSO
is a well-known scavenger of hydroxyl radicals (3, 15, 17, 27,
43). At 50 mM, DMSO did not affect the viability of the fungus,
as evidenced by biomass, glucose consumption, and protein
levels; however, it might directly or indirectly alter nutrient
availability and ultimately lip expression. On the basis of our
results, we hypothesize that ROS, and in particular hydroxyl
radicals, mediate LIP expression. When these radicals are de-
pleted by DMSO, LIP expression is not induced at either the
mRNA or protein level, regardless of the type of oxygenation.
The lower levels of oxygen in aerated cultures may produce a
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
in the induction of LIP gene expression. Although discern-
ing the role for ROS in the signal transduction pathway of
LIP requires further work, the results presented here pro-
6506 BELINKY ET AL.
vide a firm framework for future studies on the regulation of
fungal peroxidases.
ACKNOWLEDGMENTS
This research was supported in part by the Guastella Fellowship for
Colleges of Higher Education from the Sacta-Rashi Foundation and
by the Regional R&D Program of the Ministry of Science of Israel.
REFERENCES
1. Amoureux, M. C., D. van Gool, M. T. Herrero, R. Dom, F. C. A. Colpaert,
and P. J. Pauwels. 1997. Regulation of metallothionein-III (GIF) mRNA in
the brain of patients with Alzheimer disease is not impaired. Mol. Chem.
Neuropathol. 32:101121.
2. Baas, A. S., and B. C. Berk. 1995. Differential activation of mitogen-activated
protein kinase by H2O2 and O2 in vascular smooth muscles. Circ. Res.
77:2936.
3. Babbs, C. F., J. A. Pham, and R. C. Coolbaugh. 1989. Lethal hydroxyl radical
production in paraquat-treated plants. Plant Physiol. 90:12671270.
4. Bar-Lev, S. S., and T. K. Kirk. 1981. Effects of molecular oxygen on lignin
degradation by Phanerochaete chrysosporium. Biochem. Biophys. Res. Com-
mun. 99:373378.
5. Belinky, P. A., D. Goldberg, B. Krinfeld, M. Burger, N. Rothschild, U.
Cogan, and C. G. Dosoretz. 2002. Manganese-containing superoxide dis-
mutase from the white-rot fungus Phanerochaete chrysosporium: its function,
expression and gene structure. Enzyme Microb. Technol. 31:754764.
6. Bietti, M., E. Baciocchi, and S. Steenken. 1998. Lifetime, reduction potential
and base-induced fragmentation of the veratryl alcohol radical cation in
aqueous solution. Pulse radiolysis studies on a ligninase mediator. J. Phys.
Chem. 102:73377342.
7. Boominathan, K., and C. A. Reddy. 1992. cAMP-mediated differential reg-
ulation of lignin peroxidase and manganese-dependent peroxidase produc-
tion in the white-rot basidiomycete Phanerochaete chrysosporium. Proc. Natl.
Acad. Sci. USA 89:55865590.
8. Buss, H., T. P. Chan, K. B. Sluis, N. M. Domigan, and C. C. Winterbourn.
1997. Protein carbonyl measurement by a sensitive ELISA method. Free
Radic. Biol. Med. 23:361366.
9. Carter, W. O., P. K. Narayanan, and J. P. Robinson. 1994. Intracellular
hydrogen peroxide and superoxide anion detection in endothelial cells.
J. Leukoc. Biol. 55:253258.
10. Chiu, D. T. Y., F. H. Stults, and A. L. Tappel. 1976. Purification and prop-
erties of rat lung soluble glutathione peroxidase. Biochim. Biophys. Acta
445:558566.
11. Dosoretz, C. G., A. H. Chen, and H. E. Grethlein. 1990. Effect of oxygenation
conditions on submerged cultures of Phanerochaete chrysosporium. Appl.
Microbiol. Biotechnol. 34:131137.
12. Emri, T., I. Pocsi, and A. Szentirmai. 1997. Glutathione metabolism and
protection against oxidative stress caused by peroxides in Penicillium chry-
sogenum. Free Radic. Biol. Med. 23:809814.
13. Emri, T., I. Pocsi, A. Szentirmai, and B. Halliwell. 1999. Analysis of the
oxidative stress response of Penicillium chrysogenum to menadione. Free
Radic. Res. 30:125132.
14. Gold, M. H., and M. Alic. 1993. Molecular biology of the lignin-degrading
basidiomycete Phanerochaete chrysosporium. Microbiol. Rev. 57:605622.
15. Gutierrez, P. T. 2000. The metabolism of quinone-containing alkylating
agent radical production and measurement. Front. Biosci. 5:629638.
16. Hammel, K. E., A. N. Kapich, K. A. Jensen, Jr., and Z. C. Ryan. 2002.
Reactive oxygen as agents of wood decay by fungi. Enzyme Microb. Technol.
30:445453.
17. Herdener, M., S. Heigold, M. Saran, and G. Bauer. 2000. Target cell-derived
superoxide anions cause efficiency and selectivity of intercellular induction of
apoptosis. Free Radic. Biol. Med. 29:12601271.
18. Irani, K., Y. Xia, J. L. Zweier, S. J. Sollot, E. R. Fearon, M. Sundearesan, T.
Finkel, and P. J. Goldschmidt-Clermont. 1997. Mitogenic signaling medi-
ated by oxidants in Ras-transformed fibroblasts. Science 275:16491652.
19. Jamieson, D. J. 1995. The effect of oxidative stress on Saccharomyces cer-
evisiae. Redox Rep. 1:8995.
20. Jamieson, D. J., D. W. S. Stephen, and E. C. Terriere. 1996. Analysis of the
adaptive oxidative stress response of Candida albicans. FEMS Microbiol.
Lett. 138:8388.
21. Kersten, P. J., M. Tien, B. Kalyanaraman, and T. K. Kirk. 1985. The
ligninase of Phanerochaete chrysosporium generates cation radicals from me-
thoxybenzenes. J. Biol. Chem. 260:26092612.
APPL. ENVIRON. MICROBIOL.
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.
23. Kwon, S. I., and A. J. Anderson. 2001. Catalase activities of Phanerochaete
chrysosporium are not coordinately produced with ligninolytic metabolism:
catalases from a white-rot fungus. Curr. Microbiol. 42:811.
24. Lee, J., I. W. Dawes, and J. H. Roe. 1995. Adaptive response of Schizosac-
charomyces pombe to hydrogen peroxide and menadione. Microbiology 141:
31273132.
25. Lee, J. S., Y. C. Hah, and J. H. Roe. 1993. The induction of oxidative enzymes
in Streptomyces coelicolor upon hydrogen peroxide treatment. J. Gen. Mi-
crobiol. 139:10131018.
26. Leisola, M. S. A., D. Ulmer, and A. Fietcher. 1984. Factors affecting lignin
degradation in lignocellulose by Phanerochaete chrysosporium. J. Biotechnol.
3:97107.
27. Li, B., P. L. Gutierres, and N. V. Blough. 1998. Trace determination of
hydroxyl radical in biological systems. Anal. Chem. 69:42954302.
28. Li, D., M. Alic, J. A. Brown, and M. Gold. 1995. Regulation of manganese
peroxidase gene transcription by hydrogen peroxide, chemical stress, and
molecular oxygen. Appl. Environ. Microbiol. 61:341345.
29. Martin, J. P., M. Dailey, and E. Sugarman. 1987. Negative and positive
assays of superoxide dismutase based on hematoxylin autoxidation. Arch.
Biochem. Biophys. 255:329336.
30. Morpeth, F. F. 1987. Intracellular oxygen metabolizing enzymes of Phanero-
chaete chrysosporium. J. Gen. Microbiol. 133:35213525.
31. Nonaka, Y., H. Iwagaki, T. Kimura, S. Fuchimoto, and K. Orita. 1993. Effect
of reactive oxygen intermediates on the in vitro invasive capacity of tumor
cells and liver metastasis in mice. Int. J. Cancer 54:983986.
32. Oberley, L. W., and G. R. Buettner. 1979. Role of superoxide in cancer: a
review. Cancer Res. 39:11411149.
33. Raeder, U., and P. Broda. 1985. Rapid DNA isolation from filamentous
fungi. Lett. Appl. Microbiol. 1:1720.
34. Reddy, C. A., and T. M. DSouza. 1994. Physiology and molecular biology of
the lignin peroxidases of Phanerochaete chrysosporium. FEMS Microbiol.
Rev. 13:137152.
35. Rothschild, N., Y. Hadar, and C. G. Dosoretz. 1995. Ligninolytic system
formation by Phanerochaete chrysosporium in air. Appl. Environ. Microbiol.
61:18331838.
36. Rothschild, N., A. Levkowitz, Y. Hadar, and C. G. Dosoretz. 1999. Manga-
nese deficiency can replace high oxygen levels needed for lignin peroxidase
formation by Phanerochaete chrysosporium. Appl. Environ. Microbiol. 65:
483488.
37. Royall, J. A., and H. Ischiropoulos. 1993. Evaluation of 2,7-dichlorofluo-
rescin and dihydrorhodamine 123 as fluorescent probes for intracellular
H2O2 in cultured endothelial cells. Arch. Biochem. Biophys. 302:348355.
38. Ruiz-Duenas, F. J., F. Guillen, S. Camarero, M. Perez-Boada, M. J. Mar-
tinez, and A. T. Martinez. 1999. Regulation of peroxidase transcript levels in
liquid cultures of the ligninolytic fungus Pleurotus eryngii. Appl. Environ.
Microbiol. 65:44584463.
39. Shinkai, K., M. Mukai, and H. Akedo. 1986. Superoxide radical potentiates
invasive capacity of rat ascites hepatoma cells in vitro. Cancer Lett. 32:713.
40. Stadtman, E. R., and B. S. Berlett. 1998. Reactive oxygen-mediated protein
oxidation in aging and disease. Drug Metabol. Rev. 272:225243.
41. Tien, M., and K. T. Kirk. 1988. Lignin peroxidase of Phanerochaete chryso-
sporium. Methods Enzymol. 161:238249.
42. Tran, L. T., T. Miki, M. Kamakura, S. Izawa, Y. Tsujimoto, S. Miyabe, Y.
Inoue, and A. Kimura. 1995. Oxidative stress response in yeast: induction of
glucose-6-phosphate dehydrogenase by lipid hydroperoxide in Hansenula
mrakii. J. Ferment. Bioeng. 80:606609.
43. Ueno, I., M. Hoshito, T. Maitani, S. Kanegasaki, and Y. Ueno. 1993. Lu-
teoskyrin, an anthraquinoid hepatoxin, and ascorbic acid generate hydroxyl
radical in vitro in the presence of a trace amount of ferrous iron. Free Radic.
Res. Commun. 19:95100.
44. Zacchi, L., G. Burla, D. Zuolong, and P. J. Harvey. 2000. Metabolism of
cellulose by Phanerochaete chrysosporium in continuously agitated culture is
associated with enhanced production of lignin peroxidase. J. Biotechnol.
78:185192.
45. Zacchi, L., I. Morris, and P. J. Harvey. 2000. Disordered ultrastructure in
lignin-peroxidase secreting hyphae of the white-rot fungus Phanerochaete
chrysosporium. Microbiology 146:759765.
46. Zacchi, L., J. M. Palmer, and P. J. Harvey. 2000. Respiratory pathways and
oxygen toxicity in Phanerochaete chrysosporium. FEMS Microbiol. Lett. 183:
153157.