On the biology and evolution of fungi from soda soils

Summary to the thesis “On the biology and evolution of fungi from soda soils”

Alexey Grum-Grzhimaylo

The presented thesis addresses aspects of biology and evolution of fungi that were recovered from saline soda soils. The work highlights the fact that saline soda soils are populated by a large diversity of fungi capable of withstanding high salts content and high pH. Some of these fungi have been shown to require exceptionally high pH and salts to grow optimally and therefore are called alkaliphiles.

Introductory CHAPTER 1 provides examples of seemingly inhabitable environments and some of its most prominent tenants, with the emphasis on soda lakes ecosystem and alkaliphilic organisms. Aspects of physiology and major adaptive strategies to high pH and salts found in bacteria are portrayed. To our knowledge, there are no studies devoted to the fungi inhabiting saline soda lakes making this work a starting point towards further explorations in the field.

In CHAPTER 2, I show that fungi are actually present in saline soils and focus closely on the fungus that dominated across all our soda soils samples. This fungus displayed a rare obligate alkaliphilic phenotype – it was capable of growing at pH 11.4 on agar plates, with the optimum of around 9–10 and no ability to grow at pH 5.2. Using a combination of morphological and phylogenetic approaches, I describe it as a new name Sodiomyces alkalinus (previously known as Heleococcum alkalinum). We looked at the morphological details of its life cycle and tested for the capacity of utilizing various carbon sources. Given its unique extreme physiology, dominance across the soil samples, and partly for historical reasons, S. alkalinus has become our model organism that found considerable attention across this thesis.

Inspired by the fact that saline soda soils harbour new fungi, I moved on to the investigation of another set of isolates we obtained from soda soils, which belong to the Emericellopsis group (Hypocreales). CHAPTER 3 presents an investigation of the Emericellopsis isolates that showed a much broader pH preference tagging them as facultative alkaliphiles. Here again, combined morphological, phylogenetic, and physiological data allowed us to set this group apart from the rest and described it as a new species – Emericellopsis alkalina. This species is genetically unrelated to S. alkalinus, which provides evidence for the alkaliphilic trait to be polyphyletic, i.e. arisen several times throughout evolutionary history. I showed E. alkalina to be genetically closer to marine-bourne isolates than typical terrestrial species. Such a result provides evidence for the origin of alkaliphilic trait in this group from the marine-bourne fungi, as sea and soda soils environmental factors coincide.

CHAPTER 4 is devoted to a systematic study of our whole collection of fungi recovered from saline soda soils across the world. I investigate over a hundred isolates morphologically, phylogenetically, and test them for growth pH preference. These data confirms the notion that alkaliphily is polyphyletic and has emerged in several lineages of the fungal phylogenetic tree. Detailed morphological descriptions and phylogenetic reconstructions gave me confidence in describing several more new species. A prominent finding is the discovery of two additional Sodiomyces species that also show the obligate alkaliphilic adaptation. Systematic approaches let me to link certain morphological characters of the species to the alkaliphilic phenotype they possess. Although a substantial part of fungi from soda soils indeed displayed alkaliphilic capabilities, we detected typical neutrophilic species that presumably are transient or reside in a dormant state as spores or survival structures.

The next chapters of the thesis are focused on S. alkalinus, chosen as a model organism for studying alkaliphily that we sequenced in collaboration with Joint Genome Institute (Walnut Creek, USA). CHAPTER 5 looks into the aspects of the hydrolytic capabilities of S. alkalinus. The genome and transcriptome provide immense body of data that gave insight on the enzyme sets encoded in the genome involved in the degradation of carbohydrate compounds (so-called CAZymes). Such in silico analysis was backed-up by the enzyme bioassays carried out at various pH and substrates. In S. alkalinus, I found cellulolytic and hemicellulolytic enzymes that act at high pH, as opposed to neutrophilic A. oryzae enzymes that were active mostly at pH 6. Another prominent finding was the detection of strong proteolytic enzymes acting optimally at pH 8. Based on the genome data and bioassays patterns, I speculate on the ecological role of S. alkalinus in soda soils.

CHAPTER 6 addresses the aspects of the PacC transcription factor, a key player in mediating the gene expression under different ambient pH. I sought to find differences in the primary structure of PacC or detecting the multicopiness of the pacC gene, given its function under extreme alkaline conditions. It turned out that the primary structure of the PacC was the same as in other fungi and the pacC gene is presented in a single copy in S. alkalinus genome. However, I noted a shifted expression and proteolytic activation pattern of PacC if compared to neutrophilic fungi. This results provides evidence for the re-tuned pH-sensors on the plasma membrane, however we could not convincingly detect signs of positive selection affecting the PalH sensors that would change its threshold to trigger the downstream molecular cascade.

CHAPTER 7 gives insights into a quite unexpected finding – the presence of viruses in several of the S. alkalinus isolates. I show their effective vertical but not horizontal transmission. Possession of dsRNA as genetic material, icosahedral shapes, and symptomless phenotypes are common characters for a mycovirus. The virus I studied in S. alkalinus exhibits these exact same features. Curiously, no other alkaliphiles from our collection nor known sister species harboured mycoviruses, making this the first instance of mycoviruses detected in an alkaliphilic filamentous fungus.

CHAPTER 8 focuses on another peculiar finding – a bacterial gene in the genome of S. alkalinus. Presumably introduced by a horizontal gene transfer event, this gene encodes for a DD-peptidase homologue commonly found in bacteria, but only in very few eukaryotes. I found only three fungi that possess this gene; two are alkaliphilic – S. alkalinus and its sister species Acremonium alcalophilum. This suggests the importance of this gene for alkaliphily in those species. The DD-peptidase gene appears to be functional and its peak expression was observed at pH 8. Comparative analysis showed this fungal DD-peptidase to be closely related to the homologues derived from halophilic and alkaliphilic bacteria, rather than from neutrophilic ones. I speculate on the putative function of this unusual gene, including the role in the build-up of exo-cellular matrix or defense against dense communities of prokaryotes in soda soils.

The discussion in CHAPTER 9 contemplates on the results obtained throughout the thesis and provides future perspectives on the topic.