Figure 1: A) Colony morphology of Crusty grown on MEA + 10 mg/mL Tetracycline. Colonies are black, small, and no bacterial growth is present. B) Colony morphology of Crusty and Pinky when Crusty is grown from freezer stocks on MEA without antibiotics. Pinky colonies are able to grow as a result of the lack of antibiotics, due to burst cells from frozen stock. C) Liquid growth of Crusty in MEA. Crusty forms extensive biofilms at the air-liquid interface, that which forms distinct honeycomb-like patterns. In the liquid itself, Crusty does not grow planktonically but stays in its clumped morphology. D) Crusty cell morphology when grown on a plate of MEA. Perfectly round cells form, and high amounts of melanin are observed. E) Lipid droplets come out of large clumps of Crusty when the coverslip is placed over the cells. F) Crusty grown in BBM for 20 days showed unique morphologies: black arrows are large round cells; asterisks are multi-septated meristematic cells; white arrows are curved cells. All images from Erin C. Carr.

Neophaeococcomyces sp. JF2 08-2F Crusty

JF2 08-2F “Crusty”, is a novel melanized polyextremotolerant fungus isolated from a biological soil crust in British Columbia, Canada. Its ITS sequence matched 92.91% identity with Neophaeococcomyces placitae CBS 121716 (e-value 0.0), 92.61% identity with N. oklahomaensis EMSL 3313 (e-value 0.0), and 91.84% identity with N. aloes CBS 136431 (e-value 0.0). Therefore, it is believed that this fungus is a new species of the genus Neophaeococcomyces. Colony morphology of Crusty grown on malt extract medium is dark black, irregular and undulate in shape, does not form inter-agar hyphae, and has a crumbly texture almost like sand (Figure 1A). Growth in liquid medium (malt extract) maintains the cell clumps that are inoculated unless physically disturbed, and forms an extensive air-liquid interface biofilm (Figure 1C). Cells of Crusty are polymorphic in nature, ranging from perfect spheres typical of the microcolonial fungi group of polyextremotolerant fungi (Figure 1D), all the way to the formation of yeast and pseudohyphal cells (Figure 1F). Other times, very large spherical cells can be seen with no observable septa, which are possibly chlamydospores (Figure 1F; black arrow), and additional curved cells are frequently observed (Figure 1F; white arrow). Crusty’s cell division in liquid culture is similar to that of Hortaea werneckii and Phaeotheca salicorniae, in that it seems to both perform budding and binary fission in some aspects, and also form meristematic cells (Figure 1F; asterisk) and hyphae in other parts of budding cells (Mitchison-Field et al. 2019, Sterflinger 2006). JF2 08-2F Crusty is capable of utilizing many sources of carbon and nitrogen and is resistant to multiple metals and UV-C due to its melanized cell wall. Spontaneous pink (albino) mutants of JF2 08-2F Crusty can be easily recovered, which is similar to observations made by Tesei et al. in the closely related fungus Knufia petricola (2017).

The most unique feature of JF2 08-2F Crusty is that it harbors Methylobacterium spp. bacterial associations, called “Pinky” (Figure 1B). Pinky-free cultures of Crusty cannot be recovered following serial exposure to the antibiotics tetracycline and gentamycin. However, when exposed to antibiotics that kill or stop the growth of Pinky, growth of Crusty is also significantly stunted, implying that actively growing Pinky symbionts are needed for Crusty’s optimal growth. Surface sterilization of Crusty using a Chloramine-T method from Mondo et al. (2012) also showed that sterilizing the surface of Crusty maintained 16S rDNA amplification, whereas the same method did not amplify 16S rDNA from surface-sterilized Saccharomyces cerevisiae co-cultured with Pinky. This indicates possible endosymbiosis of these Methylobacterium spp. inside of Crusty. The Crusty-Pinky symbiosis also seems to be able to perform active metabolism (via XTT assay) in carbonless medium, which is presumably due to Pinky’s ability to perform aerobic anoxygenic photosynthesis.

REFERENCES:

Mitchison-Field, L. M. Y., J. M. Vargas-Muñiz, B. M. Stormo, E. J. D. Vogt, S. Van Dierdonck, J. F. Pelletier, C. Ehrlich, D. J. Lew, C. M. Field & A. S. Gladfelter (2019) Unconventional Cell Division Cycles from Marine-Derived Yeasts. Current Biology, 29, 3439-3456.e5.

Mondo, S. J., K. H. Toomer, J. B. Morton, Y. Lekberg & T. E. Pawlowska (2012) Evolutionary stability in a 400-million-year-old heritable facultative mutualism. Evolution, 66, 2564-76.

Sterflinger, K. 2006. Black yeasts and meristematic fungi: ecology, diversity and identification. In Biodiversity and ecophysiology of yeasts, 501-514. Springer.

Tesei, D., H. Tafer, C. Poyntner, G. Piñar, K. Lopandic & K. Sterflinger (2017) Draft Genome Sequences of the Black Rock Fungus Knufia petricola and Its Spontaneous Nonmelanized Mutant. Genome Announc, 5.