The entomopathogenic fungus Metarhizium anisopliae alters ambient pH, allowing extracellular protease production and activity

Abstract

Ambient pH regulates the expression of virulence genes of Metarhizium anisopliae, but it was unknown if M. anisopliae can regulate ambient pH. Mutants of M. anisopliae altered in production of oxalic acid were evaluated for the interrelationship of ambient pH, buffering capacity added to media, growth, and generation of extracellular proteases and ammonia. Wild-type and acid-overproducing mutants [Acid(M)] grew almost as well at pH 8 as at pH 6, but acid-non-producing [Acid(N)] mutants showed limited growth at pH 8, indicating that acid production is linked to the ability to grow at higher pH. Production of ammonia by M. anisopliae was strongly stimulated by low levels of amino acids in the medium when cells were derepressed for nitrogen and carbon. Likewise, although Aspergillus fumigatus and Neurospora crassa produced some ammonia in minimal media, addition of low levels of amino acids enhanced production. Ammonia production by A. fumigatus, N. crassa and M. anisopliae increased the pH of the medium and allowed production of subtilisin proteases, whose activities are observed only at basic pH. In contrast, protease production by the Acid(M) mutants of M. anisopliae was greatly reduced because of the acidification of the medium. This suggests that alkalinization by ammonia production is adaptive by facilitating the utilization of proteinaceous nutrients. Collectively, the data imply that ammonia may have functions related to regulation of the microenvironment and that it represents a previously unconsidered virulence factor in diverse fungi with the potential to harm tissues and disturb the host's immune system.

Ubiquity of Insect-Derived Nitrogen Transfer to Plants by Endophytic Insect-Pathogenic Fungi: an Additional Branch of the Soil Nitrogen Cycle

“…The inability of M. robertsii to furnish soybean with significant levels of insect-derived nitrogen may be directly related to the pH of the soil. M. robertsii can potentially alter the pH of the rhizosphere (19). This pH change can directly affect the activity of H ϩ -ATPase or the proton permeability of the plant cell membrane and conse- quently influence the amount of nitrogen absorbed by the plant (18).…”

Yeast Infection in a Beached Southern Right Whale (Eubalaena Australis) Neonate

“…It was found that these signals are essential to the virulence and survival of the pathogen during infection. The virulence factors of these organisms also include secretory hydrolases, particularly proteases, which could facilitate penetration into host tissue (St Leger et al, 1999). We tested for the production of protease on a medium containing milk powder and bromocresol purple as pH indicator.…”

“…YEPD Medium (without agar) containing cycloheximide (0.1% and 0.01%) (SigmaAldrich Chemie Gmbh, Steinheim, Germany) was used to test the ability of these isolates to grow in the presence of cycloheximide. Screening for protease activity among the isolated yeasts was conducted by growing them on solid medium containing skimmed milk and a pH indicator (0.1% yeast extract, 1% skim milk, 2% bacteriological agar, and 0.01% Bromocresol purple Indicator (Riedel De Haë n AG Zeelze, Hannover, Germany, pH 5.2) and then observing the plates for the formation of clearing zones (St Leger et al, 1999).…”

Development of thermotolerant isolates of Beauveria bassiana (Bals.-Criv.) Vuill. with ethyl methanesulfonate

“…Typically, EMS induces base-pair substitution, changing GC to AT and AT to CG, but sometimes EMS induces base-pair insertion or base-pair deletion (Sega 1984). Reports of EMS inducing mutagenesis in fungi include induced oxalic acid production in Metarhizium anisopliae (Leger 1999) and temperature-sensitive mutants in S. cerevisiae (Momose and Gregory 1998). In this study, EMS was used to induce thermotolerance of B. bassiana.…”