Turning to extremes

A taste for pollution, extremophilic cooperation, and genetic clustering – a deep dive into the molecular biology of a rather special fungus led one group of researchers to a few surprises…

We are used to the world of microorganisms giving us unusual species with extraordinary growing habits – here I’d like to introduce you a new one.

The first isolation of Cladophialophora immunda – yes, the name already gives a hint as to its weird tastes – dates back to more than 15 years ago, when the fungus popped up during an experiment involving a polluted soil sample from a gasoline station in The Netherlands.

The species belongs to a very special Ascomycota group ­– the black yeasts – whose members share, among other things, the characteristic of being tolerant to several extreme chemo-physical conditions. Extreme temperatures, pH, salinity, UV radiation and the presence of toxic compounds are usual growing conditions for these fungi, hence they are frequently the only life forms in extremophilic environments like desert, saltern, nuclear power plants and polluted sites.

For the community of scientists that studies this outstanding fungal group, the isolation of a black yeast from a polluted site represented not a shock, nevertheless, C. immunda only afterwards revealed its alternative nutritional tastes: incredibly, the fungus is able to feed on toluene, one of the main industrial waste stream pollutants.

How is that possible? How can it metabolise toluene?

In the fungal Kingdom, the ability to degrade aromatic (such as toluene) and poly-aromatic compounds was already known in ligninolytic fungi, although in these species it is linked to an unspecific and high redox potential of enzymes (laccases and peroxidases) involved in the lignin degradation. In the black yeast, though, there exists a defined pathway which leads to the complete mineralisation of these compounds, which represents an alternative carbon and energy source for the fungi. The environmental factors driving the acquirement of such specific pathway are however not yet fully understood.

Turn to the Krebs At the Department of Biotechnology of the BOKU University in Vienna, the team of Professor Katja Sterflinger, which has always worked with extremophilic fungal species, tried to uncover the secrets of C. immunda with genomic and transcriptomic approaches.

Myself – a molecular biologist ­– and bioinformatician Dr Hakim Tafer analysed the transcriptome of the fungus grown in liquid culture with the air phase saturated with toluene fumes. The toluene degrading pathway, so far characterised by enzymatic and activity assays in the same Genus, was confirmed with the investigation of the genome of the fungus (sequenced by the same team in 2015¹). The pathway consists of a total of fifteen enzymatic reactions (with two possible alternative ways, Figure 1) over which the toluene molecule leads to succinyl-CoA. This then takes part in the Krebs cycle, the set of reactions leading to energy production in all aerobic organisms.

Interestingly, it was observed that some of the genes of the toluene pathway are clustered together, and if this is quite commonly happening in fungi, it had yet to be reported in relation to hydrocarbon degradation. But the surprises from this fungus were not over. When the genome of C. immunda was searched for genes of the bacterial toluene-degradation pathway (which differs from the fungal one), a total of eight genes with high sequence similarity to genes from Pseudomonas putida and P. mendocina were found.

HGT-Finder, a bioinformatics tool to calculate the likelihood of an event of Horizontal Gene Transfer, revealed that four of those genes were indeed transferred from bacteria. While HGTs are common in fungi, they were only recently reported in the group of black yeasts, – in Exophiala dermatitidis. Of special interest among these HGT genes are PCAD (Figure 1) and xylJ (an exclusive gene associated with the bacterial toluene degradation pathway), which are uniquely present in the genome and catalyze two reactions otherwise impossible for the fungus. The fact that the homologues to bacterial enzymes partially cover additional toluene-degradation pathway might be evidence that in a toluene-contaminated environment the fungus cooperates with bacterial species in metabolizing toluene, similar to what has already been demonstrated for poly-aromatic hydrocarbons².

Going deep A deep comparative genomic analysis of the gene conservation and gene family evolution was also performed and it was very useful to get additional insights into the ecology of C. immunda. The genome of the fungus was compared to ten other hydrocarbon degrading and/or pathogenic fungal species, making possible the identification of genes significantly enriched in C. immunda. Among them, for instance, are genes belonging to carbohydrate degradation and sugar transporters. The hypothesis behind their outstanding abundance in the genome is that this was driven by the selective pressure of the polluted sites, which are typically oligotrophic environments.

A very important gene family in fungi, Cytochrome P450, is also highly enriched in the genome of C. immunda, where over two-hundred gene copies were identified. These enzymes play a pivotal role in several cellular process in fungi and they are involved of a series of physiological responses which contribute to the fitness of a species in a particular ecological niche – for example being involved in the biosynthesis of mycotoxins and so in fungal pathogenicity, or the metabolism of xenobiotic compounds. In the toluene degradation pathway, this enzyme is responsible of the first attack on the toluene molecule, producing benzyl alcohol, both a less toxic and less reactive compound. Therefore, most probably, the high presence in the genome is due to the fact that the fungus evolved in an oligotrophic and polluted environment.

What surprised us the most perhaps, was the finding that C. immunda´s genome contains something like sixty-five copies of genes coding for trichothecene efflux pumps, a much higher number than is found in fungal species known to metabolize the compound. Trichothecenes are a class of mycotoxins whose main biological action is the inhibition of protein synthesis. Another recent study demonstrated that this family expanded in the common ancestor of the black fungi, probably being important for colonizing an environment contaminated with this toxin, allowing their expulsion out of the cell³.

Toxic brew The other open question was: how does the cell cope with the toluene and its toxic effects?

The transcriptomic data revealed that, even though the fungus “likes” toluene, its presence still represent a stress both at the cellular and molecular level. The gene expression of the fungus grown in presence of toluene was compared to the growth with glucose and it shows that most cellular metabolism was impaired. The fungus cut main cellular processes like respiration and protein translation down to the minimum requirement, but at the same time it is able to activate several kinds of defense mechanisms. The transcriptome revealed the activation of an antioxidative stress response, together with an induced production of cell detoxifying molecules (Glutathione transferase, Ascorbate peroxidase and Carotenoid oxygenases) and DNA repairing enzymes.

Last but not least, melanin production is also induced by the toluene. As previous studies already demonstrated, melanin represents for the black fungi an important chemo-physical barrier to several kinds of stresses and even an active defense against the immune response of the human host. In particular, when the cell is exposed to a toxic hydrocarbon, one of the first expected actions is the disassembling of double phospholipidic layer of the cell membrane, due to the chemical affinity between the two. No dramatic response was observed at the level of cell membrane metabolism, leading the researchers to think once again that melanin is a key defense in black yeasts.

In summary, when peeking into its cells, C. immunda unexpectedly turned out to be a relatively ordinary living organism. Though, its hunger for toluene makes it definitely a worthy partner in fighting environmental pollution.

Original publication:

Genomic and transcriptomic analysis of the toluene degrading black yeast Cladophialophora immunda. B. Blasi, H. Tafer, C. Kustor, C. Poyntner, K. Lopandic & Katja Sterflinger, Scientific Reports, 7: 11436, DOI:10.1038/s41598-017-11807-8.

References:

1. Sterflinger K, Lopandic K, Blasi B, Poynter C, de Hoog S, et al. (2015) Draft Genome of Cladophialophora immunda, a Black Yeast and Efficient Degrader of Polyaromatic Hydrocarbons. Genome Announc 3: e01283-14. doi:10.1128/genomeA.01283-14.

2. Peng R-H, Xiong A-S, Xue Y, Fu X-Y, Gao F, et al. (2008) Microbial biodegradation of polyaromatic hydrocarbons. FEMS Microbiol Rev 32: 927–955. doi:10.1111/j.1574-6976.2008.00127.x.

3. Teixeira MM, Moreno LF, Stielow BJ, Muszewska A, Hainaut M, et al. (2017) Exploring the genomic diversity of black yeasts and relatives (Chaetothyriales, Ascomycota). Stud Mycol 86: 1–28. doi:10.1016/j.simyco.2017.01.001.

Author:

Dr Barbara Blasi is a molecular biologist at the Department of Biotechnology of the VIBT-Extremophile center, University of Natural Resources and Life Sciences in Vienna