Wolfgang Burgstaller

Characterization of the Penicillium chrysogenum antifungal protein PAF.

The filamentous fungus Penicillium chrysogenum abundantly secretes the small, highly basic and cysteine-rich protein PAF (Penicillium antifungal protein). In this study, the antifungal activity of PAF is described. PAF inhibited the growth of a variety of filamentous fungi, including opportunistic human pathogenic and phytopathogenic fungi, whereas bacterial and yeast cells were unaffected. PAF reduced the conidial germination and hyphal extension rates in a dose-dependent manner and induced severe changes in cell morphology that resulted in crippled and distorted hyphae and atypical branching. Growth-affected hyphae suffered from oxidative stress, plasma membrane leakage, and metabolic inactivity, which points to an induction of multifactorial effects in sensitive fungi. In contrast to other known antifungal proteins, the effects of PAF were only partially antagonized by cations.

Influence of medium components and metabolic inhibitors on citric acid production by Penicillium simplicissimum.

Penicillium simplicissimum excreted more than 100 mmol citric acid l-1 [2.9 mmol (g dry wt)-1; 9 d] if an industrial filter dust (> 50% ZnO) providing a high extracellular buffering capacity was present in the medium. A similar specific [2 mmol (g dry wt)-1], but lower absolute (26 mmol l-1), citric acid excretion occurred in the absence of an extracellular buffer and if amino acids or urea were used as nitrogen source. P. simplicissimum excreted no citric acid under conditions where Aspergillus niger produces citric acid (deficiency of trace elements, low pH and reduced biomass formation). Citric acid excretion by P. simplicissimum always paralleled biomass formation and occurred in a pH range between 4 and 7. This indicated that different imbalances of metabolism were responsible for citric acid excretion in A. niger and P. simplicissimum. However, provided a high extracellular buffering capacity was present, the response of the Penicillium system to different carbon and nitrogen sources was similar to the Aspergillus system. In contrast, the metals iron and copper had virtually no effect on citric acid excretion compared with A. niger. Estimation of intracellular citric acid, as well as the effects of the uncoupler 2,4-dinitrophenol, and the H(+)-ATPase inhibitor sodium orthovanadate, led to the conclusion that the buffer-stimulated citric acid efflux was dependent on metabolic energy and an energized plasma membrane, respectively. Despite similarities to the Aspergillus system, a different mechanism for buffer-stimulated citric acid excretion by P. simplicissimum seems probable.

Leaching of zinc from an industrial filter dust withPenicillium, Pseudomonas andCorynebacterium: Citric acid is the leaching agent rather than amino acids

SummaryHeterotrophic microorganisms are able to solubilize metals via excreted metabolites-most often di- or tricarboxylic acids but also amino acids. With amino acids Cu, Zn, Au, Ni, U, Hg and Sb have been solubilized from metal oxides, metal sulfides or elementary metals. In this work it was investigated if excreted amino acids play a role in the leaching of zinc from a zinc oxide containing industrial filter dust. Two bacteria-Pseudomonas putida andCorynebacterium glutamicum-and a fungus-Penicillium simplicissimum were used.P. putida andP. Simplicissimum have already been used to solubilize zinc oxide, whereasC. glutamicum was used because of its known ability to excrete amino acids. Amino acids in culture fluids were analyzed via derivatization with phenyl isothiocyanate, separation on a RP-18 column and UV-detection. All three microorganisms solubilized zinc from the filter dust and excreted much more citric acid than amino acids. Thus citric acid rather than amino acids was regarded to be the leaching agent. Of the two bacteriaP. putida was more resistant towards the heavy metalcontaining filter dust.

Efflux of organic acids in Penicillium simplicissimum is an energy-spilling process, adjusting the catabolic carbon flow to the nutrient supply and the activity of catabolic pathways.

Continuous cultivation was used to study the effect of glucose, ammonium, nitrate or phosphate limitation on the excretion of tricarboxylic acid (TCA) cycle intermediates by Penicillium simplicissimum. Additionally, the effect of benzoic acid, salicylhydroxamic acid (SHAM) and 2,4-dinitrophenol on TCA cycle intermediates was studied. The physiological state of the fungus was characterized by its glucose and O(2) consumption, its CO(2) production, its intra- and extracellular concentrations of TCA cycle intermediates, as well as by its biomass yield, its maintenance coefficient and its respiratory quotient. The excretion of TCA cycle intermediates was observed during ammonium-, nitrate- and phosphate-limited growth. The highest productivity was found with phosphate-limited growth. The respiratory quotient was 1.3 under ammonium limitation and 0.7 under phosphate limitation. Citrate was always the main excreted intermediate. This justifies calling this excretion an energy-spilling process, because citrate excretion avoids the synthesis of too much NADH. The addition of benzoic acid further increased the excretion of TCA cycle intermediates by ammonium-limited hyphae. A SHAM-sensitive respiration was constitutively present during ammonium-limited growth of the fungus. The sum of the excreted organic acids was negatively correlated with the biomass yield (Y(GlcX)).

Buffer-stimulated citrate efflux in Penicillium simplicissimum: an alternative charge balancing ion flow in case of reduced proton backflow?

Organic acids excreted by filamentous fungi may be used to win metals from industrial secondary raw materials. For a future commercial use a high production rate of organic acids is necessary. The conditions under which the commercially used fungus Aspergillus niger excretes high amounts of citric acid can not be maintained in metal leaching processes. However, Penicillium simplicissimum showed an enhanced citric acid efflux in the presence of an industrial filter dust containing 50% zinc oxide. Because Good buffers of high molarity were able to mimic the effect of zinc oxide, the high buffering capacity of zinc oxide and not an effect of the zinc ions was held responsible for the enhanced citric acid efflux. The presence of ammonium and trace elements reduced this buffer-stimulated citric acid efflux, whereas the plant hormone auxine canceled this reduction. This citric acid efflux was influenced by a depolarization of the membrane: the freely permeable compound tetraphenylphosphoniumbromide decreased the citric acid efflux, without decreasing intracellular citric acid or consumption of glucose and oxygen. Vanadate, an inhibitor of the plasma membrane H+-ATPase also reduced the buffer-stimulated citric acid efflux. The role of the efflux of citrate anions as an alternative charge balancing ion flow in case of impaired backflow of extruded protons because of a high extracellular buffering capacity is discussed.

Formation of exudate droplets by Metarhizium anisopliae and the presence of destruxins

Nutritional conditions causing droplet exudation by Metarhizium anisopliae var. anisopliae were studied. Exudation in droplets occurred only on media with more than one carbon source and was highly dependent on the ratio of a well metabolized sugar such as trehalose and a nonpreferred sugar, in particular arabinose. Exuded droplets contained destruxin A, B and E in concentrations similar to those on submerged culture on Czapek Dox medium with equivalent C : N ratios but was clearly less than previously reported on standard Czapek Dox or Sabouraud dextrose broth. Destruxins also were found in agar samples from directly below mycelium and from up to 2 cm from the colony edge. Exudates retrieved from different media were proven to have Pr1 protease-related enzyme activity. Additional HPLC analysis indicated that droplets from diverse media did not differ in their sugar and acid content. A hypothesis is presented regarding the trigger for guttation in Metarhizium during growth under these conditions.

Net efflux of citrate in Penicillium simplicissimum is mediated by a transport protein

Penicillium simplicissimum excreted citrate, isocitrate, and succinate when grown in a strongly buffered medium [1 M Mes (pH 6) or 1 M Hepes (pH 7.3)]. Growth in a weakly buffered medium did not lead to citrate excretion despite a similar intracellular citrate concentration. When nongrowing, citrate-excreting hyphae were aerated in a glucose solution, the following steady-state intracellular concentrations of organic acids were measured: succinate (25 mM); citrate, isocitrate, malate, and fumarate (all less than 5 mM). After 2 h of incubation, the extracellular concentrations of these acids were [μmol (g dry wt.)–1]: isocitrate [100], citrate [60], succinate [30], and malate, fumarate, and α-ketoglutarate [<5]. The excretion of citrate was due neither to an unspecific change in the permeability of the plasma membrane nor to simple diffusion of undissociated citric acid. The involvement of a transport protein in citrate excretion was indicated because N-ethylmaleimide and sodium azide inhibited citrate excretion strongly despite an unchanged outward-directed citrate gradient. Arguments are given why efflux via a citrate uptake carrier is not considered probable. These results indicate that citrate is excreted by P. simplicissimum via a transport protein that probably specifically mediates the efflux of citrate.

Metabolite Toxicology of Fungal Biocontrol Agents

Up to 300,000 potentially unique mycotoxins were reported by the Council of Agricultural Science Technology in 2003. Nevertheless, little information is available on fungal biological control agents (FBCAs) that have been developed or are being developed. The knowledge on fungal secondary metabolites and their toxicological significance depends on what is already known in the published scientific literature or in few cases becomes apparent by chance from high throughput screening programmes during product development. The purpose of this chapter is to describe what data basis is most frequently used for a routine evaluation of fungal metabolites and their residues in FBCAs. A decision scheme is discussed, which should be used to assess metabolite toxicity in the context of a worst-case scenario testing. This risk assessment procedure will help to identify the low risk, if any, of old and newly developed microbial pest control agents, and give support to applicants to market their FBCAs.

Algorithm‐based design of synthetic growth media stimulating virulence properties of Metarhizium anisopliae conidia

Aims:  Synthetic media should be designed for the production of Metarhizium anisopliae conidia with improved virulence properties.

Characteristics of glucose uptake by glucose- and NH4-limited grown Penicillium ochrochloron at low, medium and high glucose concentration☆

Glucose uptake by Penicillium ochrochloron (formerly Penicillium simplicissimum) was studied from 0.01 to 400 mM glucose using chemostat culture and bioreactor batch culture. The characteristics of glucose uptake varied considerably with the conditions of growth, harvest and uptake assay. Glucose-limited grown mycelium showed one saturable transport system [K(S) below 0.01 mM; v(max) 1.1-1.2 mmol (g dry weight)(-1)h(-1)] plus a first order process (permeability P=1.2x10(-7)cm s(-1)). Ammonium-limited grown mycelium showed only one saturable transport system [K(S) 0.3-0.7 mM; v(max) 0.5-0.8 mmol (g dry weight)(-1)h(-1)]. During exponential growth at high glucose concentration (300-400 mM) a first order process was found with a P value of 5.6-9.3x10(-7)cm s(-1). After ammonium exhaustion a second first order phase showed a lower P value (6.1-9.3x10(-8)cm s(-1)). A similar change in permeability was also found after a re-evaluation of published data for Gibberella fujikuroi, Aspergillus niger, Aspergillus awamori and Saccharomycopsis lipolytica. For the first order processes simple diffusion was ruled out as a mechanism for glucose uptake. Glucose uptake by P. ochrochloron was controlled more strongly by metabolism than by transport and was not rate limiting for overflow metabolism.