Comparative phenomics and targeted use of genomics reveals variation in carbon and nitrogen assimilation among different Brettanomyces bruxellensis strains.

Recent studies have suggested a correlation between genotype groups of Brettanomyces bruxellensis and their source of isolation. To further explore this relationship, the objective of this study was to assess metabolic differences in carbon and nitrogen assimilation between different B. bruxellensis strains from three beverages, including beer, wine, and soft drink, using Biolog Phenotype Microarrays. While some similarities of physiology were noted, many traits were variable among strains. Interestingly, some phenotypes were found that could be linked to strain origin, especially for the assimilation of particular α- and ß-glycosides as well as α- and ß-substituted monosaccharides. Based upon gene presence or absence, an α-glucosidase and ß-glucosidase were found explaining the observed phenotypes. Further, using a PCR screen on a large number of isolates, we have been able to specifically link a genomic deletion to the beer strains, suggesting that this region may have a fitness cost for B. bruxellensis in certain fermentation systems such as brewing. More specifically, none of the beer strains were found to contain a ß-glucosidase, which may have direct impacts on the ability for these strains to compete with other microbes or on flavor production.

Mutational analysis of a plant defensin from radish (Raphanus sativus L.) reveals two adjacent sites important for antifungal activity.

Mutational analysis of Rs-AFP2, a radish antifungal peptide belonging to a family of peptides referred to as plant defensins, was performed using polymerase chain reaction-based site-directed mutagenesis and yeast as a system for heterologous expression. The strategy followed to select candidate amino acid residues for substitution was based on sequence comparison of Rs-AFP2 with other plant defensins exhibiting differential antifungal properties. Several mutations giving rise to peptide variants with reduced antifungal activity against Fusarium culmorum were identified. In parallel, an attempt was made to construct variants with enhanced antifungal activity by substituting single amino acids by arginine. Two arginine substitution variants were found to be more active than wild-type Rs-AFP2 in media with high ionic strength. Our data suggest that Rs-AFP2 possesses two adjacent sites that appear to be important for antifungal activity, namely the region around the type VI beta-turn connecting beta-strands 2 and 3, on the one hand, and the region formed by residues on the loop connecting beta-strand 1 and the alpha-helix and contiguous residues on the alpha-helix and beta-strand 3, on the other hand. When added to F. culmorum in a high ionic strength medium, Rs-AFP2 stimulated Ca2+ uptake by up to 20-fold. An arginine substitution variant with enhanced antifungal activity caused increased Ca2+ uptake by up to 50-fold, whereas a variant that was virtually devoid of antifungal activity did not stimulate Ca2+ uptake.

Pathogen-induced systemic activation of a plant defensin gene in Arabidopsis follows a salicylic acid-independent pathway.

A 5-kD plant defensin was purified from Arabidopsis leaves challenged with the fungus Alternaria brassicicola and shown to possess antifungal properties in vitro. The corresponding plant defensin gene was induced after treatment of leaves with methyl jasmonate or ethylene but not with salicylic acid or 2,6-dichloroisonicotinic acid. When challenged with A. brassicicola, the levels of the plant defensin protein and mRNA rose both in inoculated leaves and in nontreated leaves of inoculated plants (systemic leaves). These events coincided with an increase in the endogenous jasmonic acid content of both types of leaves. Systemic pathogen-induced expression of the plant defensin gene was unaffected in Arabidopsis transformants (nahG) or mutants (npr1 and cpr1) affected in the salicylic acid response but was strongly reduced in the Arabidopsis mutants eln2 and col1 that are blocked in their response to ethylene and methyl jasmonate, respectively. Our results indicate that systemic pathogen-induced expression of the plant defensin gene in Arabidopsis is independent of salicylic acid but requires components of the ethylene and jasmonic acid response.

Antifungal activity of synthetic 15-mer peptides based on the Rs-AFP2 (Raphanus sativus antifungal protein 2) sequence.

Plant defensins are a class of cysteine-rich peptides of which several members have been shown to be potent inhibitors of fungal growth. A series of overlapping 15-mer peptides based on the amino acid sequence of the radish antifungal protein Rs-AFP2 have been synthesized. Peptides 6, 7, 8 and 9, comprising the region from cysteine 27 to cysteine 47 of Rs-AFP2 showed substantial antifungal activity against several fungal species (minimal inhibitory concentrations of 30-60 micrograms/mL), but no activity towards bacteria (except peptide 6 at 100 micrograms/mL). The active peptides were shown to be sensitive to the presence of cations in the medium and to the composition and pH of the medium. When present at a subinhibitory concentration (20 micrograms/mL), peptides 1, 7, 8 and 10 potentiated the activity of Rs-AFP2 from 2.3-fold to 2.8-fold. By mapping the characteristics of the active peptide on the structure of Rs-AFP2 as determined by nuclear magnetic resonance, the active region of the antifungal protein appears to involve beta-strands 2 and 3 in combination with the loop connecting those strands. A cyclized synthetic mimic of the loop, cysteine 36 to cysteine 45, was shown to have antifungal activity. Substitution of tyrosine 38 by alanine in the cyclic peptide substantially reduced the antifungal activity, indicating the importance of this residue for the activity of Rs-AFP2 as demonstrated carrier by mutational analysis.

Fungal membrane responses induced by plant defensins and thionins.

Treatment of hyphae of Neurospora crassa with antifungal plant defensins, i.e. Rs-AFP2 and Dm-AMP1 isolated from radish and dahlia seed, respectively, induced a rapid K+ efflux, Ca2+ uptake, and alkalinization of the incubation medium. The Rs-AFP2-induced alkalinization of the incubation medium could be inhibited with G-protein inhibitors. alpha-Hordothionin, an antifungal thionin from barley seed, caused a sustained increased Ca2+ uptake at subinhibitory concentrations but only a transient increased uptake at inhibitory concentrations. alpha-Hordothionin also caused increased K+ efflux and alkalinization of the medium, but these fluxes occurred more rapidly compared to those caused by plant defensins. Furthermore, alpha-hordothionin caused permeabilization of fungal hyphae to the non-metabolite alpha-aminoisobutyric acid and, in addition, altered the electrical properties of artificial lipid bilayers, consistently leading to rupture of the lipid bilayers. The plant defensins did not form ion-permeable pores in artificial membranes and did not exhibit substantial hyphal membrane permeabilization activity. Our results are consistent with the notion that thionins inhibit fungal growth as a result of direct protein-membrane interactions, whereas plant defensins might act via a different, possibly receptor-mediated, mechanism.

Isolation and characterisation of plant defensins from seeds of Asteraceae, Fabaceae, Hippocastanaceae and Saxifragaceae.

From seeds of Aesculus hippocastanum, Clitoria ternatea, Dahlia merckii and Heuchera sanguinea five antifungal proteins were isolated and shown to be homologous to plant defensins previously characterised from radish seeds and gamma-thionins from Poaceae seeds. Based on the spectrum of their antimicrobial activity and the morphological distortions they induce on fungi the peptides can be divided into two classes. The peptides did not inhibit any of three different alpha-amylases.

Expression of functional Raphanus sativus antifungal protein in yeast.

Rs-AFP2 is a 51 amino acid cysteine-rich peptide isolated from radish (Raphanus sativus) seeds that exhibits potent inhibitory activity against filamentous fungi. A cDNA clone encoding the Rs-AFP2 preprotein was modified by recombinant DNA methods to allow expression in the yeast Saccharomyces cerevisiae. This peptide was expressed in yeast as a fusion protein carrying at its N-terminus the prepro-sequences derived from the precursor of the yeast pheromone mating factor alpha 1. These sequences allow secretion of the biologically active peptide in a correctly processed form. Deletion of the mating factor alpha 1 pro-peptide drastically reduced the expression level of the peptide.