Cyanide-resistant alternative respiration is strictly correlated to intracellular peroxide levels in Acremonium Chrysogenum

A strict correlation between the intensity of the cyanide-resistant alternative respiratory pathway and the intracellular peroxide levels in the cephalosporin C producer filamentous fungus Acremonium chrysogenum was demonstrated. Intracellular peroxide levels increased in a dose-dependent manner after addition of H₂O₂ to the culture media. A similar phenomenon was observed due to the specific inhibition of catalase by salicylic acid. In both cases, cyanide-resistant respiration was markedly stimulated. On the other hand, both cyanide-resistant respiration and intracellular peroxide levels were effectively suppressed by the lipid peroxyl radical scavenger DL-α-tocopherol, which breaks lipid peroxidation chains effectively. Our findings firmly supported the assumption that there is a connection between the intracellular peroxide levels and the intensity of the alternative respiratory pathway in fungi.     

Cephalosporin-C production, morphology and alternative respiration of Acremonium chrysogenum in glucose-limited chemostat

Summary In this paper, the connection between morphology, cephalosporin-C production and alternative respiration of Acremonium chrysogenum is examined. As demonstrated by chemostat experiments, the ratio of the filamentous and the yeast-like forms depended on the growth rate. The yeast-like form, but not the filamentous form exhibited cyanide-resistant alternative respiration. As a consequence, the yeast-like form was regarded to be more suitable for antibiotic overproduction.     

Alternative respiration and antibiotic production of Acremonium chrysogenum

The influence of potassium cyanide (KCN), dissolved O₂ concentration and medium composition on alternative respiration (AR) of Acremonium chrysogenum were investigated. The respiration of the fungus was only partially inhibited by KCN, but completely inhibited by the combination of KCN with salicylhydroxamic acid. It has been proved by in-situ measurements of the NADH-dependent fluorescence that the AR is active at low dissolved O₂ concentrations. The influence of the medium composition and the age of the fungus on the specific oxygen uptake rate is considered. Correpondence to: K. Schügerl     

Stimulation of the cyanide-resistant alternative respiratory pathway by oxygen in Acremonium chrysogenum correlates with the size of the intracellular peroxide pool

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-alpha-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.     

Cyanide-resistant respiration in Streptomyces citreofluorescens

The capacity of Streptomycetes to carry out cyanide-resistant respiration was investigated. With the model strain, Streptomyces citreofluorescens, it was shown that deprivation of glucose followed by transition from exponential to stationary growth was coupled with declining sensitivity of cellular respiration to cyanide ions. Cyanide-resistant oxidase is located within the cytoplasmic membrane. The occurrence of the cyanide-resistant oxidase did not correspond to qualitative changes of cytochrome spectrum. Cytochrome d is involved neither in cyanide-sensitive nor in cyanide-resistant respiratory chain.     

Cyanide-resistant respiration in Torulopsis candida

The effect of cyanide and the inhibitors of cyanide-resistant oxidase--hydroxamic acids on endogenous respiration and oxidation of a number of substrates by Torulopsis candida resting cells taken at different stages of growth on glucose and hexadecane was studied and made it possible to arrive at the following conclusions. 1. The effect of cyanide on endogenous respiration of T. candida differs during its growth on glucose and hexadecane. On hexadecane, irrespective of the growth phage, cyanide inhibits endogenous respiration by 70--75%. On glucose, cyanide inhibits endogenous respiration not more than by 35% and only at the exponential growth phase whereas it stimulates endogenous respiration in the course of other growth stages. 2. The effect of cyanide on respiration of the resting cells of T. candida which oxidize glucose, hexadecane, primary alcohols and tetradecanoic acid hardly depends on the growth stage. It is determined mainly by the nature of a substrate to be oxidized. 3. Hydroxamic acid have no effect on the cell respiration in the absence of cyanide. However, in its presence, they entirely inhibit both endogenous respiration and oxidation of the aforementioned substrates. 4. Under almost all above experimental conditions, the sensitivity of cell respiration to cyanide changes only slightly at different stages of growth on either glucose or hexadecane. This feature markedly distinguishes T. candida among other cyanide-resistant yeasts.     

Cyanide-resistant respiration is frequent, but confined to yeasts incapable of aerobic fermentation

In Pichia membranifaciens, cyanide-resistant respiration (CRR) sensitive to salicylhydroxamic acid emerged after forced aeration of starved cells for 4 h. Surveying a large number of species by this simple methodology, we found that CRR is very frequent among yeasts. Remarkably, considering our results together with previous data in the literature, CRR was present in 24 out of 28 non-fermentative or Crabtree-negative yeasts and absent in 10 out of 12 Crabtree-positive yeasts. We submit that, as alternatives to cytochromic respiration, yeasts developed two strategies: either aerobic fermentation in Crabtree-positive yeasts or CRR in non-fermentative or Crabtree-negative yeasts.     

Specific cephalosporin C production of Acremonium chrysogenum is independent of the culture density

Specific cephalosporin C production of Acremonium chrysogenum grown on a glucose-based minimal medium using conventional batch and dialysis membrane reactor systems was independent of the cell density in the range of 0.4 to 40 g biomass l^–1.     

Cyanide-resistant respiration in light- and dark-grown soybean cotyledons

Measurements of respiration were made on intact tissue and mitochondria isolated from soybean (Glycine max [L.] Merr. cv `Corsoy') cotyledons from seedlings of different ages grown in light and darkness. Effects of cyanide (KCN) and salicylhydroxamic acid (SHAM) on O₂ uptake rates were determined. O₂ uptake was faster in light-grown tissue and was inhibited by both KCN and SHAM in all except light-grown tissue older than 9 days. Both inhibitors stimulated O₂ uptake in tissues more than 9 days old. Mitochondria in which O₂ uptake was coupled to ATP synthesis were isolated from all tissues. O₂ uptake by mitochondrial preparations from light- and dark-grown cotyledons was equally sensitive to KCN. Similarly, age did not affect KCN sensitivity, but sensitivity to SHAM declined with age both in the presence and absence of KCN. Estimated capacities of the cytochrome and alternative pathways of the mitochondrial preparations indicated considerably larger cytochrome than alternative pathway capacities. The cytochrome pathway capacities paralleled the state 3 mitochondrial respiration rates, which increased from day 5 to day 7 then declined thereafter. The alternative pathway capacities were not affected by light. The uncoupler, p-trifluoromethoxycarbonylcyanide phenylhydrazone (FCCP), increased the flow of electrons through the cytochrome pathway at the expense of flow through the alternative pathway in isolated mitochondria. However, the combined capacities did not exceed the rate in the presence of FCCP. The results are interpreted to indicate that the stimulation of respiration by KCN and SHAM observed in the 12-day-old green cotyledons and previously observed in older soybean leaves is not explained by characteristics of the mitochondria.     

Cyanide-resistant respiration of bacteria and its relation to metabolite oversynthesis

The phenomenon of cyanide-resistant respiration was studied in bacteria as well as its relation to the oversynthesis of metabolites. Cyanide-resistant respiration was shown to be common of aerobic and facultative anaerobic bacteria. The resistance of respiration to cyanide was observed when the growth of a microbial culture was inhibited or ceased entirely, provided the culture lacked the oversynthesis of metabolites. When a culture was capable of metabolites oversynthesis under such conditions, cyanide-resistant respiration (if any) declined. Therefore, there is a competition between metabolites oversynthesis and cyanide-resistant respiration, which is typical of many microorganisms.     

Cyanide-resistant respiration of Physarum polycephalum in course of starvation

The inhibition of respiration of $\text{Physarum polycephalum}$ microplasmodia by KCN varies between 30% and 70% of initial rate of O₂ uptake, depending on the time of starvation. The kinetics of the development of cyanide-resistant respiration and its sensitivity toward salicylhydroxamic acid (SHAM) point out that CN-resistant respiration represents the activity of the alternative pathway of the electron transport. There is no evidence that during starvation the alternative pathway of respiration is active in the absence of cyanide.     

Cyanide-resistant respiration in photosynthetic organs of freshwater aquatic plants

The rate and sensitivity to inhibitors (KCN and salicylhydroxamic acid[SHAM]) of respiratory oxygen uptake has been investigated in photosynthetic organs of several freshwater aquatic plant species: six angiosperms, two bryophytes, and an alga. The oxygen uptake rates on a dry weight basis of angiosperm leaves were generally higher than those of the corresponding stems. Leaves also had a higher chlorophyll content than stems. Respiration of leaves and stems of aquatic angiosperms was generally cyanide-resistant, the percentage of resistance being higher than 50% with very few exceptions. The cyanide resistance of respiration of whole shoots of two aquatic bryophytes and an alga was lower and ranged between 25 and 50%. These results suggested that the photosynthetic tissues of aquatic plants have a considerable alternative pathway capacity. The angiosperm leaves generally showed the largest alternative path capacity. In all cases, the respiration rate of the aquatic plants studied was inhibited by SHAM alone by about 13 to 31%. These results were used for calculating the actual activities of the cytochrome and alternative pathways. These activities were generally higher in the leaves of angiosperms. The basal oxygen uptake rate of Myriophyllum spicatum leaves was not stimulated by sucrose, malate or glycine in the absence of the uncoupler carbonylcyanide-m-chlorophenylhydrazone (CCCP), but was greatly increased by CCCP, either in the presence or in the absence of substrates. These results suggest that respiration was limited by the adenylate system, and not by substrate availability. The increase in the respiratory rate by CCCP was due to a large increase in the activities of both the cytochrome and alternative pathways. The respiration rate of M. spicatum leaves in the presence of substrates was little inhibited by SHAM alone, but the SHAM-resistant rate (that is, the cytochrome path) was greatly stimulated by the further addition of CCCP. Similarly, the cyanide-resistant rate of O₂ uptake was also increased by the uncoupler.     

The role of the alternative respiratory pathway in the stimulation of cephalosporin C formation by soybean oil in Acremonium chrysogenum

Addition of soybean oil to Acremonium chrysogenum cultures growing on sugars doubled the specific production of cephalosporin C during the idiophase of growth. While the addition of soybean oil had no effect on the total rate of respiration, the respiration that proceeded via the alternative, cyanide-insensitive pathway exhibited a more than twofold increase. Addition of soybean oil also stimulated the formation of isocitrate lyase activities. Inhibition of oxidative metabolism of one of the products of isocitrate lyase (succinate) by thenoyltrifluoroacetone completely inhibited the alternative respiratory pathway. The role of soybean-oil-stimulated alternative respiration in the stimulation of cephalosporin C production and the role of isocitrate lyase are discussed. Received: 13 October 1998 / Revised revision: 14 January 1999 / Accepted: 22 January 1999     

Cyanide-resistant respiration,a very frequent metabolic pathway in yeasts

It has recently been shown that cyanide-resistant respiration (CRR) is very common in Crabtree-negative yeasts (incapable of aerobic fermentation) and in non-fermentative yeasts. It is conferred by a salicylhydroxamic acid-sensitive alternative oxidase that transfers electrons from ubiquinol to oxygen, bypassing the cytochrome chain. An interesting finding is that, in general, whenever CRR is present, complex I is also present. In this article we briefly review the occurrence of CRR, the biochemistry and molecular biology of the alternative oxidase, and summarise the putative functions that have been attributed to this ubiquitous metabolic pathway, whose usefulness for the yeast cells still remains obscure.