Stimulation of the cyanide-resistant alternative respiratory pathway ...

216
Stimulation of the cyanide-resistant alternative
respiratory pathway by oxygen in Acremonium
chrysogenum correlates with the size of the
intracellular peroxide pool
Levente Karaffa, Erzsébet Sándor, Erzsébet Fekete, József Kozma,
Attila Szentirmai, and István Pócsi
Abstract: The relationship between oxygen input and activity of the cyanide-resistant alternative respiration of submerged
cultures of Acremonium crysogenum was investigated. The volumetric oxygen transfer coefficient of the respective
cultures correlated positively within almost two ranges of magnitude with the size of the intracellular peroxide
pool, which in turn, correlated with the activity of the cyanide-resistant alternative respiratory pathway. Increased aeration
also stimulated the glucose uptake rate but had no effect on the total respiration rate or the growth rate. Addition
of the lipid peroxyl radical scavenger DL-α-tocopherol to A. chrysogenum cultures decreased the rate of intracellular
peroxide production as well as glucose uptake. An increase in the cyanide-resistant fraction of total respiration was observed,
while growth and the total respiratory activity remained unchanged. We conclude that intracellular peroxides
may stimulate the alternative respiration in A. chrysogenum.
Key words: Acremonium chrysogenum, alternative respiration, oxygen, peroxide, Kla.
Résumé : Nous avons étudié le lien qui existe entre la consommation d’oxygène et l’activité Karaffarespiratoire et al. auxiliaire résistante
au cyanure de cultures submergées de Acremonium crysogenum. Le coefficient de transfert volumétrique de
l’oxygène des cultures respectives fut corrélé positivement sur près de deux ordres de grandeur avec la taille de la réserve
de peroxydes intracellulaires, qui à son tour était corrélé avec l’activité de la voie respiratoire auxiliaire résistante
au cyanure. Une augmentation de l’aération a également stimulé le taux d’absorption de l’oxygène, mais n’a eu aucun
effet sur la vitesse respiratoire ou le taux de croissance. L’ajout de DL--tocopherol, un agent neutralisant les radicaux
lipidiques peroxydés, aux cultures de A. crysogenum a réduit le taux de production de peroxydes intracellulaires de
même que l’absorption de glucose. Nous avons observé une augmentation de la fraction de la respiration totale qui
était résistante au cyanure, alors que la croissance et l’activité respiratoire totale demeuraient inchangées. Nous concluons
que les peroxydes intracellulaires pourraient stimuler la respiration auxiliaire chez A. crysogenum.
Mots clés : Acremonium crysogenum, respiration auxiliaire, oxygène, peroxyde, Kla.
[Traduit par la Rédaction] 220
Introduction
Mitochondria of many fungi possess a cyanide-resistant alternative
respiratory pathway containing a characteristic
alternative oxidase, which bypasses the sites of energy conservation,
leading to a decrease in the energy yield of cells
(Sluse and Jarmuszkiewicz 1998). Fungal alternative oxidase
Received 3 December 2002. Revision received 4 March 2003.
Accepted 26 March 2003. Published on the NRC Research
Press Web site at http://cjm.nrc.ca on 22 April 2003.
L. Karaffa, 1 E. Sándor, E. Fekete, A. Szentirmai, and I.
Pócsi. Department of Microbiology and Biotechnology,
Faculty of Science, University of Debrecen, H-4010, P.O.
Box 63, Debrecen, Hungary.
J. Kozma. Chemical Works of Gedeon Richter Ltd., H-1103,
Gyömri út 19–21, Budapest, Hungary.
1 Corresponding author (e-mail: karaffal@tigris.klte.hu).
activity is dependent on substrate availability (i.e.,
ubiquinone concentration and its redox state in the membrane
and O 2 concentration in the cell) and is regulated at
the level of gene expression by post-translational modification
and by allosteric activation (Umbach and Siedow 2000).
The production of reactive oxygen species (ROS), such as
O 2 – and H 2 O 2 , is an unavoidable consequence of aerobic metabolism.
In fungal mycelia, as in other respiratory chains,
oxidases of the mitochondrial electron transport chain are
major sites of ROS production. Several studies to date have
demonstrated the capacity of the alternative respiratory pathway
to prevent the production of ROS (Wagner 1995; Millar
and Day 1996; Maxwell et al. 1999; Karaffa et al. 2001).
We previously reported that the alternative respiratory activity
of Acremonium chrysogenum is strongly dependent on
the dissolved oxygen level (Kozma and Karaffa 1996a). We
also showed that the activity of this pathway is responsive to
changes in the intracellular peroxide (IP) levels (Karaffa et
Can. J. Microbiol. 49: 216–220 (2003) doi: 10.1139/W03-029 © 2003 NRC Canada

Karaffa et al. 217
al. 2001). In this study, we will demonstrate that the effect
of oxygen on the cyanide-resistant alternative respiratory
pathway is mediated via changes in the size of the IP pool.
Materials and methods
Fungal strain and cultivation conditions
Acremonium chrysogenum ATCC 46117 was grown at
28°C in 500-mL Erlenmeyer flasks on a NBS orbital shaker
(New Brunswick Scientific Co., Inc., Edison, N.J., U.S.A.)
at 200 rpm (Karaffa et al. 1999). The complete growth medium
was inoculated with a 10% (v/v) 3-day-old seed culture,
as detailed earlier (Kozma and Karaffa 1996a).
For replacement experiments, mycelia grown for 14 h in
complete medium were harvested by filtration on a sintered
glass funnel, washed with cold tap water, and then transferred
into a minimal medium (Karaffa et al. 1997) with
glucose as the sole carbon source. DL-α-Tocopherol was supplied
to the culture media 3 h after the transferring procedure,
to yield a final concentration of 0.2 mM. Samples were taken
after 4hoffurther incubation — a period that was found to
be sufficient to achieve maximal mycelial respiration, including
the activity of the alternative respiratory pathway.
The starting mycelial dry weight was approximately 4.0 g L –1
in each replacement experiment.
Creation of different oxygen input in shake flasks
Although the technical means to modify the oxygen transfer
rate in a series of 500-mL shake flasks are limited compared
with what may be obtained within a fermenter, there
are still a few methods that could result in a gradient of oxygen
input. For example, varying the ratio of flask volume to
medium volume can change the volumetric oxygen transfer
coefficient (Kla). High volumes within flasks lower the specific
oxygen transfer rate. In addition, by putting a porous
cotton cap onto the flask instead of a metal cap, aeration and
Kla may be increased. Finally, the presence of baffles that
serve to disrupt the vortex pattern result in the production of
a larger liquid–air interfacial surface. The resulting turbulent
flow pattern is also beneficial with respect to increasing the
oxygen transfer rate. The aeration-related properties of the
shake-flask cultures used in this study are displayed at Table
1.
Analytical methods
Kla values of the shake flasks (characterized in Table 1)
were determined by the sulphite-oxidation method (Cooper
et al. 1944).
Fungal growth was monitored by recording dry cell
weights (DCW; Sándor et al. 2001). Specific growth rates
were calculated from the increased DCW over the duration
of the experiment (Pirt 1975).
Glucose consumption was monitored by HPLC on a H +
exchange column (Bio-Rad Aminex HPX-H + , Hercules, Calif.),
as described earlier (Sándor et al. 2001).
The measurement of the mycelial respiration rates, including
that of the alternative respiratory pathway, was performed
in an oxygraphic cell (Bahr and Bonner 1973). A 1-
mM concentration of KCN was used to inhibit cytochrome
oxidase of the cytochrome-dependent pathway.
Table 1. Physical properties related to aeration of the shake-flask
cultures used in this study.
Culture
No.
I
II
III
IV
V
IP levels were calculated by the spectrofluorimetric determination
of 2′,7′-dichlorofluorescein (2′,7′-DCF) production
from 2′,7′-dichlorofluorescin diacetate, as described earlier
(Royall and Ischiropoulos 1993; Karaffa et al. 2001). Specific
IP values are related to milligrams of protein, which
was determined by means of a modified Lowry method (Peterson
1983) using bovine serum albumin for calibration.
Reproducibility
All the data presented here are means of at least three independent
experiments. The variations among experiments
were estimated by standard deviations (SDs) for each procedure.
The SD values were always less than 10% of the
means. The significance of changes as a function of Kla values
was assessed using the Student’s t test, with p values
cited in the text.
Chemicals
All chemicals were of analytical grade, and were purchased
from Sigma-Aldrich Kft., Budapest, Hungary, with
the exceptions of 2′,7′-dichlorofluorescin diacetate and 2′,7′-
dichlorofluorescein, which were purchased from Eastport
Kft., Budapest, Hungary.
Results
Aeration properties of the
500-mL shake-flask
cultures
30 mL of aliquots,
baffled, cotton cap
80 mL of aliquots,
baffled, cotton cap
80 mL of aliquots,
unbaffled, cotton cap
80 mL of aliquots,
unbaffled, metal cap
150 mL of aliquots,
unbaffled, metal cap
Volumetric oxygen
transfer coefficient (Kla)
value (min –1 )
11.5
Growth and glucose consumption
Washed and transferred mycelia of A. chrysogenum
quickly adapted to the new environment as indicated by the
glucose uptake and the subsequent biomass production rates.
The growth rate of cultures with different Kla values was not
significantly different (p < 0.1) during the examined period
(Fig. 1A). On the contrary, glucose consumption was progressively
more rapid (p < 0.1) with increasing oxygen
transfer rate (Fig. 1A).
Respiratory activity
Despite the major differences in the respective oxygen
transfer rates of cultures, the total respiratory activity
seemed relatively unaffected (p < 1), very slightly decreasing
with decreasing Kla values. The difference in total oxygen
uptake among cultures with the highest and the lowest
oxygen transfer rates was not more than 8% (Fig. 1B).
7.8
3.2
1.0
0.42
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218 Can. J. Microbiol. Vol. 49, 2003
Fig. 1. Comparison of Acremonium chrysogenum shake-flask cultures
with no DL-α-tocopherol supplementation. (A) Growth rate
() and glucose uptake rate (). (B) Total respiratory activity
() and cyanide-resistant fraction of the total respiration ().
(C) Intracellular peroxide production (). DCW, dry cell weight;
2′,7′-DCF, 2′,7′-dichlorofluorescein.
Fig. 2. Comparison of Acremonium chrysogenum shake-flask cultures
with 0.2 mM DL-α-tocopherol supplementation in all flasks.
(A) Growth rate () and glucose uptake rate (). (B) Total respiratory
activity () and cyanide-resistant fraction of the total
respiration (). (C) Intracellular peroxide production (). DCW,
dry cell weight; 2′,7′-DCF, 2′,7′-dichlorofluorescein.
In contrast, the cyanide-resistant fraction of the respiration
significantly (p < 0.1) increased by increasing the oxygen
transfer rate (Fig. 1B). At Kla = 11.5 min –1 , the cyanideresistant
respiration comprised almost 40% of the total oxygen
consumption as opposed to a mere 16% at Kla =
0.42 min –1 .
Intracellular peroxide levels
Figure 1C shows that the IP levels of cultures were increasing
with increasing Kla (p < 1). At the two most extreme
values, IP values in mycelia growing in a shake flask
were about 50% higher at Kla = 11.5 min –1 than at Kla =
0.42 min –1 .
Effects of DL-α-tocopherol
To provide a correlation between the intensity of the alternative
respiration and the IP levels, the free radical scavenger
DL-α-tocopherol was employed. Comparison of Figs. 1A
and 2A shows that the addition of DL-α-tocopherol in the
medium did not influence growth rate of any of the examined
cultures (p < 0.1), but it decreased the glucose uptake
rate by up to 18–25%. DL-α-Tocopherol also affected both
the alternative respiration (compare Figs. 1B and 2B) and
the IP levels (compare Figs. 1C and 2C), irrespective of the
actual aeration of the culture. DL-α-Tocopherol effectively
(p < 0.1) decreased the IP concentration generated by the
oxygen input and also decreased the activity of the alternative
respiratory pathway. When DL-α-tocopherol was added
to the culture with the lowest Kla value, IP levels declined
until they went down below the lower limit of sensitivity of
the 2′,7′-DCF method. At the same time, cyanide-resistant
respiration also decreased until almost to its detection limit.
All these data indicate that manipulation of the IP concentration
modulates the activity of the alternative respiratory
pathway. Noteworthily, the total respiration rate of the cultures
was not affected (p < 0.1) by the presence of DL-αtocopherol
(Fig. 2B).
Discussion
The present study implies that the requirement of oxygen
for growth or respiratory activity is not particularly high in
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Karaffa et al. 219
A. chrysogenum — in the range of the oxygen transfer rates
studied (defined by the Kla value of the respective shake
flasks), growth was not limited by the oxygen. Roughly
similar specific growth rates were measured in each of the
cultures, including Culture No. I with Kla = 11.5 min –1 , corresponding
to ca. 30% of saturation of dissolved oxygen
(Kozma and Karaffa 1996b). A constant rate of biomass production
was sustained by a practically unchanged total respiratory
activity, rendering all the five cultures apparently similar.
However, several other parameters of the cultures were Kla
dependent. Glucose consumption increased with increased
aeration. This could well be explained by the enhanced activity
of the nonphosphorylating alternative respiratory pathway,
which is able to sink significant amounts of carbon without
ATP production (Lambers 1982). The high oxygen requirement
(ca. 30% of saturation) of the fungal alternative respiratory
pathway compared with the cytochrome-dependent route
is well documented (Kubicek et al. 1980; Kozma and Karaffa
1996a). As a consequence of the enhanced alternative respiratory
activity, more glucose had to be catabolized to restore the
necessary ATP yield.
Glucose uptake data are in good agreement with those obtained
by the addition of exogenous H 2 O 2 to A. chrysogenum
cultures, thereby, stimulating the alternative respiratory route
(Karaffa et al. 2001). Nonetheless, biomass production of
the cultures was not steady in those experiments, but transiently
decreased when H 2 O 2 concentration was above
100 mM. An explanation for this contradiction might be that
the presence of H 2 O 2 in such high concentrations increased
the alternative respiratory activity beyond the level that
could still be balanced by the cytochrome-dependent, ATPgenerating,
and therefore, growth-supporting respiratory pathway.
In this study, that limit was obviously not reached. Indeed,
the highest level of the cyanide-resistant respiratory
activity achieved by the increased oxygen transfer rates was
less than 40% of the total respiration, in contrast to the effect
of 200 mM H 2 O 2 , which resulted in a cyanide-resistant
fraction comprising more than 70% of the total respiration
(Karaffa et al. 2001).
The IP level of mycelia proved to be yet another responsive
parameter to aeration, even if its increase caused by the
higher oxygen input could be counteracted by the presence
of the lipid peroxyl radical scavenger DL-α-tocopherol and
vice versa (Kanno et al. 1996). As obvious as this relationship
may seem, no such correlation has ever been previously
reported. In turn, the activity of the alternative respiratory
pathway correlated with the IP concentrations under all cultivation
conditions.
Since a lipid peroxyl radical scavenger could effectively
hinder the IP production, an overwhelming majority of the
molecules measured by the 2′,7′-DCF method should be of
lipophylic character. Since the primarily produced ROS are
water-soluble, this can be only explained by a secondary,
tertiary, etc., production of peroxides due to the propagation
of lipid peroxidation by peroxyl radicals (Halliwell and
Gutteridge 1998; Karaffa et al. 2001).
It has widely been suggested by several authors that the
noncoupled alternative respiratory pathway contributes to
the prevention of the generation of ROS (Wagner 1995;
Popov et al. 1997; Maxwell et al. 1999; Umbach and Siedow
2000) and that it can be considered as an integral part of the
antioxidant defense system in eucaryotes, including fungi.
ROS are formed when the cytochrome path is impaired and
(or) the intracellular oxygen is in excess. In case the available
capacity of the alternative respiratory pathway is not
sufficient, ROS induce the expression of the alternative
oxidase protein, which is similar to the H 2 O 2 -mediated expression
of the plant-pathogenesis-related proteins (Chen et
al. 1993).
In summary, our data are in compliance with the current
understanding of the nature of the cyanide-resistant alternative
oxidase. Moreover, a well-known feature of this route,
e.g., its high oxygen requirement, was also integrated into
the most accepted model of its regulation.
Acknowledgements
We thank Professor Christian P. Kubicek (Section Microbial
Biochemistry and Gene Technology, Institute of Chemical
Engineering, TU Wien, Austria) for critically reading the
manuscript. The project was aided by grants from the Hungarian
Scientific Research Fund (OTKA F 031985 and F
042602) and from the Fund for Research and Development
in Higher Education (FKFP 0009/2001). Erzsébet Sándor is
a recipient of an OTKA Postdoctoral Fellowship (D 37975).
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