Genomic Characterization of Parengyodontium torokii sp. nov., a Biofilm-Forming Fungus Isolated from Mars 2020 Assembly Facility

Abstract

A fungal strain (FJII-L10-SW-P1) was isolated from the Mars 2020 spacecraft assembly facility and exhibited biofilm formation on spacecraft-qualified Teflon surfaces. The reconstruction of a six-loci gene tree (ITS, LSU, SSU, RPB1 and RPB2, and TEF1) using multi-locus sequence typing (MLST) analyses of the strain FJII-L10-SW-P1 supported a close relationship to other known Parengyodontium album subclade 3 isolates while being phylogenetically distinct from subclade 1 strains. The zig-zag rachides morphology of the conidiogenous cells and spindle-shaped conidia were the distinct morphological characteristics of the P. album subclade 3 strains. The MLST data and morphological analysis supported the conclusion that the P. album subclade 3 strains could be classified as a new species of the genus Parengyodontium and placed in the family Cordycipitaceae. The name Parengyodontium torokii sp. nov. is proposed to accommodate the strain, with FJII-L10-SW-P1 as the holotype. The genome of the FJII-L10-SW-P1 strain was sequenced, annotated, and the secondary metabolite clusters were identified. Genes predicted to be responsible for biofilm formation and adhesion to surfaces were identified. Homology-based assignment of gene ontologies to the predicted proteome of P. torokii revealed the presence of gene clusters responsible for synthesizing several metabolic compounds, including a cytochalasin that was also verified using traditional metabolomic analysis.

Keywords: biofilm; fungi; genomics; mars 2020 mission; metabolomics; morphological analysis; phylogenetic analysis.

Figure 1

Macro and micromorphology of Parengyodontium torokii. Colony surface of FJ11-L10-SW-P1 after 21 days of incubation at room temperature (23 °C) in standard 9 cm petri dishes on (A) PDA media and (B) OMA media. (C) Conidia produced at each bent point of the zigzag rachides of the fertile conidiogenous cells. (D) Whorl of two conidiogenus cells with conidia attached at the zigzag rachides. Scale Bars (C–E) = 20 µm. (E) Scanning electron microscopy images of Parengyodontium torokii from ex-type strain FJ11-L10-SW-P1 with whorl of conidiogenous cells showing butt-shaped denticles and (F) subcylindrical to ellipsoidal, hyaline single-celled conidia. Scale bar for all microscopy is 10 µm.

Figure 2

Confocal analysis of biofilm formation.Parengyodontium torokii isolate FJII-L10-SW-P1 was grown in PDB in the presence of untreated (A,C) and treated (B,D) Teflon coupons. Each scan was 1161 µm × 1336 µm and was taken at a representative location on the coupon surface. Composite confocal fungal space-filling structure is color-coded based on relative distance from the coupon surface with blue/violet being proximal to the surface and orange/red being distal. (A,B) are plan views of the region that was imaged (100 µm scale bars) while (C,D) are orthogonal views of the biofilm looking length-wise through the biomass (100 µm scale bars). The comparative biofilm distribution as compared to distance from the coupon surface is presented in (E). The biofilm formed on the uncoated Teflon is smaller and the median height is closer to the surface while the biofilm from the coated Teflon coupon is larger and has a higher median height (94 µm and 107 µm, respectively).

Figure 3

Multi Locus Sequence Typing (MLST) of Parengyodontium torokii. Gene sequences from the ITS region rRNA gene, D1/D2 domain of large subunit (LSU) rRNA gene, small subunit (SSU) rRNA gene, RNA polymerase II (RPB1 and RPB2), and translation elongation factor 1-α (TEF1) were used to investigate phylogenetic placement of the FJII-L10-SW-P1 among the main Cordycipitaceae groups. We used 59 taxa and 4617 nucleotide sites to build up a Maximum Likelihood tree on the IQTREE2 software. The branches are proportional to the number of mutations and 1000 ultrafast bootstraps and SH-like approximate likelihood ratio test (aLRT) was used to test branch support and added to each corresponding branch of the tree. The tree was rooted with the Simplicillium sp.

Figure 4

Phylogenomic analyses of Parengyodontium torokii. A total of 5334 single copy orthologous genes were used to build up a Maximum Likelihood tree among 9 Cordycipitaceae fungi using the IQTREE2 software. Simplicillium aogashimaense was set as the outgroup and the branches are proportional to the number of mutations. Branch fidelity used two different approaches, Ultrafast bootstraps and Gene Concordance Factors, which were added next to its corresponding branches.

Figure 5

Comparative genomic analyses of Cordycipitaceae fungi. (A) Carbohydrate-active enzymes (CAZymes) and (B) proteases (MEROPS). Each category with a standard deviation > 1 was plotted in the heat map. Asterisks represent the more prominent changes in Parengyodontium lineage in both CAZy and MEROPS classes. The numerical number “1” represents for the major families shown in bar plot and “2” depicts the significantly enriched or depleted functional traits.

Figure 6

Fungal-specific transcriptional factors among the Cordycipitaceae species. Fungal transcription factors were screened in 9 Cordycipitaceae genomes and compared. Factors with a standard deviation >1 was plotted in a heat map.

Figure 7

GO-enrichment of the Parengyodontium lineage. The Venn diagram shows shared orthologs and unique groups of genes of the Parengyodontium species using either Lecanicillium fungicola 150-1 (A) or Simplicillium aogashimaense 72-15.1 (B) as outer species for comparisons. (C) The GO categories shared in both scenarios enriched for either Parengyodontium sp., Parengyodontium torokii, or Parengyodontium americanum are listed with its respective function, domain, number of counts, and associated p-value.

Figure 8

LC-MS comparison of FJ11-L10-SW-P1 grown on oatmeal and rice media. (A) The fungus was grown on two different media to examine its secondary metabolite production. Extracts of the fungus were examined through high-resolution mass spectrometry (HRMS). The base peak chromatogram along with light absorbance data collected through Photodiode-Array Detection (PDA) was compared. The extracts showed high similarities in their metabolomic profiling. (B) The fungal extracts were screened for natural compounds that are known to be expressed by the identified biosynthetic gene clusters. (C) The accurate mass of cytochalasins K was identified through extracted-ion chromatogram (XIC) within 5 ppm of the compound’s accurate mass value. Thus, cytochalasin K was putatively biosynthesized by this fungus under the used growth conditions. (D) Predicted cluster of cytochalasin K identified in the P. torokii FJ11-L10-SW-P1 genome. The scaffold and positions spanning the biosynthetic cluster as well the genes are displayed along the schematic representation. The same cluster was identified for a series of other filamentous fungi, which are also shown.