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

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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]

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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, GyömrÅi út 19–21, Budapest, Hungary. 1

Corresponding author (e-mail: [email protected]).

Can. J. Microbiol. 49: 216–220 (2003)

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activity is dependent on substrate availability (i.e., ubiquinone concentration and its redox state in the membrane and O2 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 O2– and H2O2, 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

doi: 10.1139/W03-029

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Karaffa et al.

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

217 Table 1. Physical properties related to aeration of the shake-flask cultures used in this study. Culture No. I

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 4 h of further 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 1mM concentration of KCN was used to inhibit cytochrome oxidase of the cytochrome-dependent pathway.

II III IV V

Aeration properties of the 500-mL shake-flask cultures

Volumetric oxygen transfer coefficient (Kla) value (min–1)

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

11.5 7.8 3.2 1.0 0.42

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 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). © 2003 NRC Canada

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218 Fig. 1. Comparison of Acremonium chrysogenum shake-flask cultures with no DL-α-tocopherol supplementation. (A) Growth rate (䊉) and glucose uptake rate (䊊). (B) Total respiratory activity (䊏) and cyanide-resistant fraction of the total respiration (ⵧ). (C) Intracellular peroxide production (䉱). 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 exam-

Can. J. Microbiol. Vol. 49, 2003 Fig. 2. Comparison of Acremonium chrysogenum shake-flask cultures with 0.2 mM DL-α-tocopherol supplementation in all flasks. (A) Growth rate (䊉) and glucose uptake rate (䊊). (B) Total respiratory activity (䊏) and cyanide-resistant fraction of the total respiration (ⵧ). (C) Intracellular peroxide production (䉱). DCW, dry cell weight; 2′,7′-DCF, 2′,7′-dichlorofluorescein.

ined 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 © 2003 NRC Canada

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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 H2O2 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 H2O2 concentration was above 100 mM. An explanation for this contradiction might be that the presence of H2O2 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 H2O2, 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

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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 H2O2-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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