Cobalamin is a cofactor present in essential metabolic pathways in animals and one of the water-soluble vitamins. It is a complex compound synthesized solely by prokaryotes. Cobalamin dependence is scattered across the tree of life. In particular, fungi and plants were deemed devoid of cobalamin. We demonstrate that cobalamin is utilized by all non-Dikarya fungi lineages. This observation is supported by the genomic presence of both B12-dependent enzymes and cobalamin modifying enzymes. Fungal cobalamin-dependent enzymes are highly similar to their animal homologs. Phylogenetic analyses support a scenario of vertical inheritance of the cobalamin usage with several losses. Cobalamin usage was probably lost in Mucorinae and at the base of Dikarya which groups most of the model organisms and which hindered B12-dependent metabolism discovery in fungi. Our results indicate that cobalamin dependence was a widely distributed trait at least in Opisthokonta, across diverse microbial eukaryotes and was likely present in the LECA.

Mucoromycotina are often considered mainly in pathogenic context but their biology remains understudied. We describe the genomes of six Mucoromycotina fungi representing distant saprotrophic lineages within the subphylum (i.e., Umbelopsidales and Mucorales). We selected two Umbelopsis isolates from soil (i.e., U. isabellina, U. vinacea), two soil-derived Mucor isolates (i.e., M. circinatus, M. plumbeus), and two Mucorales representatives with extended proteolytic activity (i.e., Thamnidium elegans and Mucor saturninus). We complement computational genome annotation with experimental characteristics of their digestive capabilities, cell wall carbohydrate composition, and extensive total lipid profiles. These traits inferred from genome composition, e.g., in terms of identified encoded enzymes, are in accordance with experimental results. Finally, we link the presence of associated bacteria with observed characteristics. Thamnidium elegans genome harbors an additional, complete genome of an associated bacterium classified to Paenibacillus sp. This fungus displays multiple altered traits compared to the remaining isolates, regardless of their evolutionary distance. For instance, it has expanded carbon assimilation capabilities, e.g., efficiently degrades carboxylic acids, and has a higher diacylglycerol:triacylglycerol ratio and skewed phospholipid composition which suggests a more rigid cellular membrane. The bacterium can complement the host enzymatic capabilities, alter the fungal metabolism, cell membrane composition but does not change the composition of the cell wall of the fungus. Comparison of early-diverging Umbelopsidales with evolutionary younger Mucorales points at several subtle differences particularly in their carbon source preferences and encoded carbohydrate repertoire. Nevertheless, all tested Mucoromycotina share features including the ability to produce 18:3 gamma-linoleic acid, use TAG as the storage lipid and have fucose as a cell wall component.

Increasing resistance to conventional antibiotics among microorganisms is one of the leading problems of medicine nowadays. Antimicrobial peptides are compounds exhibiting both antibacterial and antifungal activities. However, it is difficult to predict whether a designed new compound would exhibit any biological activity. Moreover, purification of the peptides is one of the most time-consuming and expensive steps of the synthesis that sometimes leads to unnecessary loss of solvents and reagents. In our study we have developed a thin-layer chromatography (TLC) direct bioautography technique for rapid determination of antimicrobial activity of peptides without the necessity of high-performance liquid chromatography purification. In this assay, crude peptides were applied and separated on a TLC plate. Then, pre-prepared plates were dipped into microbial suspension and incubated under optimum conditions for bacteria and fungi as well. The activity of the tested compounds was visualized by spraying the TLC plates with a cell viability reagent, resazurin (7-hydroxy-3H-phenoxazin-3-one 10-oxide). Effectiveness of this assay was compared with minimal inhibitory concentration results obtained by broth microdilution assay. Interestingly, so far such a screening method has not been applied for this group of compounds.

Early-diverging fungi (EDF) are ubiquitous and versatile. Their diversity is reflected in their genome sizes and complexity. For instance, multiple protein families have been reported to expand or disappear either in particular genomes or even whole lineages. The most commonly mentioned are CAZymes (carbohydrate-active enzymes), peptidases and transporters that serve multiple biological roles connected to, e.g., metabolism and nutrients intake. In order to study the link between ecology and its genomic underpinnings in a more comprehensive manner, we carried out a systematic in silico survey of protein family expansions and losses among EDF with diverse lifestyles. We found that 86 protein families are represented differently according to EDF ecological features (assessed by median count differences). Among these there are 19 families of proteases, 43 CAZymes and 24 transporters. Some of these protein families have been recognized before as serine and metallopeptidases, cellulases and other nutrition-related enzymes. Other clearly pronounced differences refer to cell wall remodelling and glycosylation. We hypothesize that these protein families altogether define the preliminary fungal adaptasome. However, our findings need experimental validation. Many of the protein families have never been characterized in fungi and are discussed in the light of fungal ecology for the first time.

Cobalamin dependence is scattered across the tree of life. In particular, fungi and plants have been deemed devoid of cobalamin. We demonstrate that cobalamin is utilized by all phyla of non-Dikarya fungal lineages. This observation is supported by the identification of B12-dependent enzymes and cobalamin modifying enzymes in published fungal genomes. We found that cobalamin dependence was a widely distributed trait at least in Opisthokonta, across diverse microbial eukaryotes and likely in the LECA (last eukaryotic common ancestor). Moreover, the genes identified are actively transcribed in many taxa.