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. 2024 Aug 27;74(8):006496. doi: 10.1099/ijsem.0.006496

Mycovorax composti gen. nov., sp. nov., a member of the family Chitinophagaceae isolated from button mushroom compost

Meghann Thai 1, Tina L Bell 1, Michael A Kertesz 1,*
PMCID: PMC11349053  PMID: 39190453

Abstract

Two Gram-stain-negative, aerobic, rod-shaped, orange-coloured bacterial strains, designated strain C216T and strain M2295, were isolated from mature mushroom compost from composting facilities in Victoria and South Australia, Australia, respectively. External structures such as flagella or pili were not observed on the cells under scanning electron microscopy. Optimal growth was found to occur at 45 °C, at pH 7.25 and in the absence of NaCl on Emerson’s 350 YpSs medium. The genome sequence of strain C216T was 3 342 126 bp long with a G+C content of 40.5 mol%. Functional analysis of the genome of strain C216T revealed genes encoding chitinolytic and hemi-cellulolytic functions, with 166 predicted genes associated with carbohydrate metabolism (8.9% of the predicted genes). These functions are important for survival in the mushroom compost environment, which is rich in hemicelluloses. No antibiotic resistance genes were found in the genome sequence. The major fatty acids of strain C216T were iso-C15 : 0 (56.7%), iso-C17 : 0 3-OH (15.6%), C16 : 1 ω7c/iso-C15 : 0 2-OH (7.3%) and iso-C15 : 1 G (6.1%). The only respiratory quinone was MK-7. The major polar lipid of strain C216T was phosphatidylethanolamine, but three unidentified phospholipids, four unidentified aminophospholipids/aminolipids and one unidentified glycolipid were also detected. Phylogenetic analysis based on proteins encoded by the core genome (bac120, 120 conserved bacterial genes) showed that strain C216T forms a distinct lineage in the family Chitinophagaceae and that the closest identified relative is Niabella soli (69.69% ANI). These data demonstrate that strain C216T represents a novel genus and novel species within the family Chitinophagaceae, for which we propose the name Mycovorax composti. The type strain is C216T (=DSM 114558T=LMG 32998T).

Keywords: Agaricus bisporus, Chitin, compost, Phase II, thermophiles

Introduction

Mushroom compost is a highly selective substrate that is used to support the growth of button mushrooms (Agaricus bisporus). It is produced from wheat straw, poultry manure and gypsum, which are transformed into compost by the bacteria and fungi that colonize these feedstocks. Composting starts with wetting the wheat straw for a period of 3–10 days, followed by addition of poultry manure and gypsum [1]. The mixture is then turned periodically to homogenize and aerate the compost over 6–14 days (phase I) [1,2]. During phase I, heat is rapidly generated by the microbial activity, and peak compost temperatures are as high as 80 °C [3]. This is followed by phase II, in which the partially fermented compost is pasteurized in enclosed tunnels at 58–60 °C for 8 h [1] and then conditioned by decreasing compost temperatures slowly from 58 to 45 °C over 4 days [4,5]. The microbial dynamics during conditioning are of particular interest, as 50–60% of the compost carbohydrates are degraded during this period [6] and incorporated into microbial biomass or respired. When A. bisporus is added to the compost it exploits the bacterial and fungal biomass of this community as a key nutrition source during mycelial proliferation [7]. Chitinophagaceae have been found in various compost systems [5,8,10], and this family is one of the more abundant taxa found during the conditioning stage in phase II compost [5].

The family Chitinophagaceae is part of the phylum Bacteroidota and was first described by Kämpfer et al. [11]. The type genus is Chitinophaga, which was first described by Sangkhobol and Skerman [12] as a chitinolytic myxobacterium, but was later transferred to the family Chitinophagaceae [11]. More than 40 other genera belong to the family Chitinophagaceae, including Compostibacter [13], Filimonas [14], and Pseudocnuella [15]. Cells from genera in the family Chitinophagaceae contain menaquinone MK-7 as the major quinone and the major fatty acids are iso-C15 : 0, iso-C17 : 0 3-OH, and iso-C15 : 1G [11]. Cells are usually thin, rod-shaped, and non-motile; however, swarming motility may be observed [11]. They are usually mesophilic and aerobic or facultatively anaerobic, and some members of this family have limited fermentative capabilities [11].

Two bacterial strains, designated C216T (=DSM 114558T=LMG 32998T) and M2295 (=DSM 114559=LMG 32997), were isolated from button mushroom compost at the end of phase II. These strains demonstrated chitinolytic activity in vitro, and during growth in laboratory media they were antagonistic to Mycothermus thermophilus, a thermophilic cellulolytic ascomycete fungus that is dominant in mushroom compost.

Isolation and ecology

End-phase II compost was collected from commercial mushroom compost yards in Victoria and South Australia, Australia. Compost samples (approximately 1 g) were suspended in 9 ml of Reasoner's 2A (R2A) broth (HiMedia Laboratories) and incubated at 40 °C for 48 h with shaking (200 r.p.m.). Samples were then plated on R2A agar plates (Oxoid), and round orange colonies with entire margins were purified by subculturing on R2A agar (45 °C, 48 h). The bacterial strain isolated from mushroom compost from the compost yard in Victoria was designated C216T, and the bacterial strain isolated from the compost yard in South Australia was designated M2295. Both strains were routinely cultured on Emerson’s YpSs agar [16] at 45 °C and preserved at −80 °C in glycerol suspension (25% v/v).

Prior to isolation, this bacterial taxon had been detected in a comprehensive study of microbial succession during mushroom composting at a commercial compost yard in New South Wales, Australia [5], using 16S diversity profiling with Illumina sequencing. The organism was part of a stable bacterial community that developed in phase II mushroom compost, dominated by Pseudoxanthomonas, Steroidobacter, and the novel genus from the family Chitinophagaceae reported here. The main fungal taxon associated with these bacteria was M. thermophilus, which is dominant in Phase II mushroom compost [5,17] and has been extensively studied for its role in promoting the growth of A. bisporus [18]. When strain C216T was grown in co-culture with M. thermophilus, the bacterium exhibited chitinolytic activity on the hyphal tips of the fungus (see below), suggesting that it plays an important ecological role in mushroom compost.

16S rRNA phylogeny

Genomic DNA was extracted using the CTAB method, according to the method of Wilson [19]. Briefly, late exponential phase bacterial cells were harvested and resuspended in Tris-ethylenediaminetetraacetic acid (TE) buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0; 100 µl) with 1 mg ml−1 lysozyme (Sigma-Aldrich) and incubated at 37 °C for 30 min without agitation. Hexadecyltrimethylammonium bromide (CTAB) extraction buffer (2% CTAB (w/v), 100 mM Tris-HCl, 20 mM EDTA, 1.4 M NaCl; 500 µl) was added and the mixture was incubated at 50 °C for 60 min without agitation. Chloroform:isoamyl alcohol (24 : 1) was added in equal volume and the solution was emulsified before centrifuging at 14 000 g for 10 min to separate the two phases. The top aqueous phase (400 µl) was transferred to a fresh tube and cold isopropanol (320 µl) was added. The precipitated DNA was collected by centrifugation (20 000 g for 20 min) and washed twice with 80% (v/v) ethanol. The DNA obtained was resuspended in TE buffer (pH 8.0) and stored at −20 °C.

The full 16S rRNA gene sequence of strain C216T was obtained from the genome sequence (see below). The partial 16S rRNA gene sequence of strain M2295 was determined by Macrogen Inc. (Seoul, Republic of Korea) following their standard protocol. Pairwise alignment of the 16S rRNA gene sequences of strains C216T and M2295 showed that they were 99.93% identical. 16S rRNA gene sequences of closely related genera were obtained from the National Center for Biotechnology Information (NCBI) [20]. The 16S rRNA gene sequences were aligned using Clustal W [21], and a phylogenetic tree was reconstructed using the software package mega-X (version 10.2.2) [22]. Distances were determined using the Kimura two-parameter model and clustering was done with the neighbour-joining and maximum-likelihood methods. The reliability of the trees obtained was confirmed using bootstrap values based on 1000 replicates. The neighbour-joining tree (Fig. 1) revealed that strain C216T was closely related to members of the family Chitinophagaceae within the phylum Bacteroidota, with strains C216T and M2295 forming a separate branch next to the genera Niabella and Terrimonas. The maximum-likelihood tree showed similar topology. Pairwise comparison of the full 16S rRNA gene sequence of strain C216T (1 526 bp) with those of Niabella aquatica and Terrimonas ferruginea revealed 92.0–93.5 % 16S rRNA gene sequence similarity to the type strains of these species.

Fig. 1. Neighbour-joining phylogenetic tree for Mycovorax composti strains C216T and M2295, and related strains based on partial 16S rRNA gene sequences. Filled circles (●) indicate nodes that were the same in the maximum-likelihood tree. Numbers at nodes indicates the percentages of bootstrap support based on 1000 replications. Bar, 0.05 substitutions per nucleotide position. Flavobacterium aquatile NBRC 15052T, Flexibacter flexilis NBRC 15060T and Cytophaga aurantiaca JM110T were used as outgroups.

Fig. 1.

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Genome features and phylogeny

Whole genome sequencing of the C216T genomic DNA was done by the Microbial Genome Sequencing Center (Pittsburgh, USA), using their standard Illumina protocols (NextSeq 2000; 2×150 bp). Long-read sequencing was done using an R10.4.1 flow cell on minION apparatus (Oxford Nanopore Technology). The resulting FastQ files were analysed using the Galaxy software package [23]. Long-read sequence data were filtered and trimmed using Porechop (version 0.2.4) [24] and filtlong (version 0.2.1) [25], with 90% adapter sequence similarity and 500 bp minimum sequence length, respectively. Trimmed reads were assembled using Flye (version 2.9.1) [26]. Genome closure was achieved by aligning the Illumina sequencing reads to the draft genome using BWA-MEM (version 0.7.17.2) [27] and the genome was polished using pilon (version 1.20.1) [28]. Genome annotation was done in Galaxy using Barrnap and Prokka [29,30]. Functional analysis of the genome of strain C216T was done using eggNOG (version 5.0.2) [31] available in the Galaxy software package, and rast with the RASTtk annotation pipeline [32,34]. DNA G+C content of strain C216T was determined from the genome sequence.

The complete genome size of strain C216T was 3 342 126 bp. DNA G+C content of strain C216T was 40.5 mol%. The genome was assembled into one contig which contained 2886 predicted genes, including 2839 coding DNA sequences, six rRNA regions, 40 tRNA regions and one tmRNA region. Barrnap [30] annotation revealed that the genome of strain C216T contained two identical copies of the 16S rRNA gene, 23S rRNA gene and 5S rRNA genes. There were no antibiotic resistance genes identified in the genome. A relatively high proportion (54.1%) of the protein-coding genes could be assigned putative functions by Prokka [29], while the remaining genes were annotated as hypothetical proteins. The genome sequence is available with accession number CP144143. Pairwise analysis of the whole genome of strain C216T was done using the Type Strain Genome Server [35], and revealed that Myroides aquimaris CGMCC 1.10825T was the most closely related strain, with 34.6% similarity in terms of digital DNA–DNA hybridization (dDDH; formula d4). Niabella aurantiaca DSM 17617T had only 19.4% pairwise similarity with strain C216T when comparing dDDH (d4).

Both strains were isolated from wheat straw compost, and therefore genes that are responsible for cellulose and hemi-cellulose degradation were of particular interest. Xylan is a type of hemicellulose commonly found in wheat straw, and is constructed of a β-1,4-xylose backbone with α-l-arabinofuranosyl, acetyl and 4-O-methylglucuronyl branched units [6,36]. Analysis of the C216T genome revealed putative genes encoding endo-1,4-β-xylanase (EC 3.2.1.8), β-d-xylosidase (EC 3.2.1.37), α-l-arabinofuranosidase (EC 3.2.1.55) and acetylxylan esterase (EC 3.1.1.72), suggesting that strain C216T degrades xylan found in wheat straw. No genes for cellulose degradation were found. The functional genes found in strain C216T therefore suggest that the strain is highly specialized for the breakdown of hemi-celluloses in the thermophilic, wheat straw compost environment in which it is found. Further genome features for strain C216T and its six closest phylogenetic neighbours are provided in Tables S1 and S2.

Another feature of strain C216T was its interaction with Mycothermus thermophilus, which it antagonized in vitro by breaking down the fungal hyphae (Fig. S1, available in the online version of this article). This is likely due to the presence of chitin degrading enzymes since chitin is the main structural polymer in fungal cell walls. No genes encoding endo-chitinases (EC 3.2.1.14) were found in the genome of strain C216T, but predicted genes for exo-N-acetylglucosaminidases (EC 3.2.1.52) were present. Exo-N-acetylglucosaminidases have been shown to cleave chitin oligosaccharides such as chitobiose and chitotriose [37].

rast annotation software assigned 239 groups of functional genes, known as subsystems, from the 2922 protein coding sequences within the genome of C216T. The highest number of assigned genes was associated with biosynthesis and metabolism of amino acids (178), followed by carbohydrate subsystems (166) and protein metabolism (124), respectively (Fig. S2). Within the carbohydrate subsystem, several genes involved with chitin and xylose utilization were annotated. COG analysis of strain C216T proteins showed similar annotations to the rast annotation. The majority of the functional genes were assigned to unknown functions (22%), while 8.9% of the functional genes were associated with carbohydrate metabolism. Several proteins associated with chaperones and heat tolerance were also found in the C216T genome, allowing it to survive the high temperatures in phase II mushroom compost.

CAZy analysis from the eggNOG [31] package in Galaxy [23], showed that 127 proteins were associated with glycoside hydrolases (GHs), glucosyltransferases (GTs), carbohydrate binding modules (CBMs), carbohydrate esterases (CE) and polysaccharide lyases (PLs) families in strain C216T. Most of these proteins were associated with GH families (57%), followed by GT families (28%) and CE, CBM and PL families (6, 4, 4%, respectively). No proteins were assigned to the auxiliary activities (AA) family. Most of the enzymes in the GH family were associated with cleavage of β-1,4-linked glycosidic bonds in a broad range of carbohydrates. The genome of strain C216T was compared with the genome of 36 type species within the family Chitinophagaceae, using genome sequences sourced from NCBI [20]. A phylogenetic tree based on 120 concatenated conserved bacterial protein amino acid sequences [38] was reconstructed using the dataset provided by GTDB-tk (version 2.2.2, release 207.2) [39] on Galaxy Australia [23]. A maximum-likelihood tree using these core sequences was reconstructed in Galaxy using FASTTree (version 2.1.10) using model LG+CAT [40]. The phylogenetic tree shows that strain C216T formed a separate branch between the genera Niabella and Terrimonas, clearly indicating that strain C216T represents a novel genus within the family Chitinophagaceae (Fig. 2).

Fig. 2. Maximum-likelihood phylogenetic tree of the famiy Chitinophagaceae based on concatenated amino acid sequences of 120 conserved bacterial proteins. Cytophaga hutchinsonii ATCC 33406T and Flexibacter flexilis DSM 6793T were used as outgroups. Numbers at nodes are percentages of bootstrap support based on 1000 replications. Scale bar, 0.1 substitutions per nucleotide position.

Fig. 2.

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Average nucleotide identity (ANI) values were calculated using OrthoANI, the online tool from EzBioCloud [41]. Average amino acid identity (AAI) values were calculated with the online tool provided by the Environmental Microbial Genomics Laboratory [42]. The ANI and AAI values between strain C216T and its most closely related species were 69.5 and 64.7%, respectively (Table 1). These values are well below the cutoff value for ANI that has been proposed to delineate species (95–96%) and the AAI value (74–76%) used to distinguish genera [43,44].

Table 1. AAI and ANI values between strain C216T and other related type species in the family Chitinophagaceae.

Strain GenBank accession no. AAI (%) ANI (%)
Niabella ginsenosidivorans BS26T GCA_001654455.1 64.78 69.52
Niabella aurantiaca DSM 17617T GCA_000374125.1 64.76 69.42
Niabella soli DSM 19437T GCA_000243115.3 64.72 69.69
Niabella drilacis DSM 25811T GCA_900101395.1 64.15 69.46
Terrimonas ferruginea DSM 30193T GCA_000425585.1 59.85 68.15
Lacibacter cauensis CGMCC 1.7271T GCA_007830055.1 56.26 67.89
Pinibacter aurantiacus MAH-26T GCA_019130065.1 56.06 68.09
Paracnuella aquatica N24T GCA_003332885.2 55.94 67.66
Paraflavitalea soli 5GH32-13T GCA_003555545.1 55.82 67.84
Pseudobacter ginsenosidimutans Gsoil 221T GCA_007970185.1 55.76 67.21
Agriteriibacter humi YJ03T GCA_009192765.1 55.65 67.66
Parafilimonas terrae DSM 28286T GCA_900115755.1 55.41 67.78
Hydrotalea flava CCUG 51397T GCA_001623405.1 55.31 68.04
Panacibacter ginsenosidivorans Gsoil1550T GCA_007971225.1 55.23 67.9
Cnuella takakiae RG1-1T GCA_001953305.1 55.22 67.54
Niastella koreensis GR20-10T GCA_000246855.1 55.21 67.64
Foetidibacter luteolus YG09T GCA_009650435.1 55.00 67.89
Sediminibacterium salmoneum NBRC 103935T GCA_000511175.1 54.93 68.13
Ilymonas limi 17mud1-8T GCA_005233845.1 54.92 67.92
Segetibacter koreensis DSM 18137T GCA_000374045.1 54.8 67.47
Limnovirga soli KCS-6T GCA_013106755.1 54.7 67.97
Flavihumibacter petaseus NBRC 106054T GCA_000974785.1 54.69 67.2
Phnomibacter ginsenosidimutans SB-02T GCA_009740285.1 54.59 67.58
Deminuibacter soli K23C18032701T GCA_003412455.1 54.53 67.8
Gynurincola endophyticus HBUM179779T GCA_003991195.1 54.49 67.63
Hanamia caeni BO-59T GCA_003721595.1 54.45 67.1
Filimonas lacunae NBRC 104114T GCA_002355595.1 54.39 67.71
Ginsengibacter hankyongi BR5-29T GCA_008710285.1 54.34 67.12
Puia dinguensis CGMCC 1.15448T GCA_014642875.1 54.07 67.07
Arachidicoccus rhizosphaerae Vu-144T GCA_900107765.1 53.05 66.85
Dinghuibacter silviterrae DSM 100059T GCA_004366355.1 52.95 66.44
Chitinophaga caeni 13T GCA_002557795.1 52.82 67.01
Chitinophaga pinensis DSM 2588T GCA_000024005.1 52.59 66.46
Chitinophaga dinghuensis DSM 29821T GCA_003259435.1 52.02 66.55
Thermoflavifilum aggregans DSM 27268T GCA_002797735.1 51.08 66.37
Edaphocola aurantiacus H2T GCA_019797745.1 49.41 66.62
Flavipsychrobacter stenotrophus RB1R16T GCA_002954265.1 48.71 66.81
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Physiology and chemotaxonomy

Cell morphology was determined using cells grown in Emerson’s YpSs broth for 24 h at 45 °C and viewed using a scanning electron microscope (Sigma VP HD, Zeiss). Growth was tested at different temperatures (30, 37, 40, 45, 50, 55 and 60 °C) and at pH values between pH 4.0 and 9.0 (in increments of 0.25 pH units) for 72 h. Salt tolerance was tested in R2A liquid medium supplemented with 0, 1, 2, 3, 4, 5, 6 and 9% (w/v) NaCl. Motility was assessed using the hanging drop method using a light microscope at ×400 magnification. Gram staining was done according to Smibert [45]. Catalase activity was determined by observing bubble production in 3% (v/v) H2O2. Oxidase activity was determined by swatching a single bacterial colony (grown on R2A agar for 48 h at 45 °C) onto paper saturated with 1% (w/v) tetramethyl-para-phenylenediamine HCl. The presence of flexirubin-type pigments was tested by flooding the plate with 20% KOH (w/v) and observing colour change [46]. Biochemical characterization was done using the API 20NE (bioMérieux) kit, following the manufacturer’s instructions.

The cellular fatty acid profile was determined using cells grown in Emerson’s YpSs broth for 24 h at 45 °C. Analysis of cellular fatty acids was done by the Identification Service, Leibniz-Institut DSMZ – Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany. Cellular fatty acids were analysed after conversion into fatty acid methyl esters by saponification, methylation, and extraction according to the protocol of Sherlock Microbial Identification System (midi, Microbial ID, Delaware USA). Analysis of respiratory quinones were carried out by DSMZ services, Leibniz-Institut DSMZ – Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Braunschweig, Germany.

Polar lipids for strain C216T were determined from fresh cells and visualized using two-dimensional thin layer chromatography on silica gel 60 according to Nguyen and Kim [47]. Total polar lipids were detected using 5% ethanolic phosphomolybdic acid, aminolipids were detected with ninhydrin, phospholipids were detected with bromthymol blue, and glycolipids were detected with diphenylamine [48].

Cells of strains C216T and M2295 were Gram-stain-negative, short rod shaped, aerobic, non-motile and non-spore forming. Colonies of strains C216T and M2295 grown on R2A, TSA (tryptic soy agar) and Emerson’s YpSs agar at 45 °C for 48 h were circular, convex, opaque, and orange in colour. Growth did not occur on MacConkey agar. Cells were approximately 0.8–1.2 µm long and 0.4–0.5 µm wide (Fig. 3). Growth on R2A medium occurred at 37–55 °C and at pH 6.5–8.0. Tolerance to NaCl was 0–2% (w/v). Dark red to brown flexirubin-type pigments were observed when the plate was flooded with 20% KOH (w/v). Phenotypic and chemotaxonomic characteristics that differentiate this strain from closely related taxa are listed in Table 2.

Fig. 3. Scanning electron microscope image of C216T cell morphology. The longer cell on the right is an elongated cell that is about to divide. No flagella or other external structures can be seen on the cell.

Fig. 3.

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Table 2. Phenotypic and chemotaxonomic characteristics of Mycovorax composti strains C216T and M2295, and type strains of closely related genera in the family Chitinophagaceae.

Strains: 1, C216T; 2, M2295; 3, Niabella aurantiaca R2A15-11T; 4, Hydrotalea flava CGUG 51397T; 5, Compostibacter hankyongensis BS27T; 6, Terrimonas ferruginea ATCC 13524T; 7, Niastella koreensis GR20-10T; 8, Chitinophaca pinensis UQM 2034T. +, Positive; −, negative; w, weak; nd, no data available.

Characteristic 1 2 3 4 5 6 7 8
Colony colour Orange Orange Orange Orange-yellow Milky white Salmon red Light yellow Yellow
Cell morphology Short rod Short rod Rods Thin rods Short rod Rods Filamentous Rod
Gram stain − − − − − − − −
Relationship to O2 Aerobic Aerobic Aerobic Aerobic Aerobic Strictly aerobic Aerobic Aerobic
Gliding motility − − − − − − + +
Growth temperature range (°C) 37–55 37–55 10–30 20–37 15–37 10–37 10–37 12–37
Optimum growth temperature (°C) 40–50 40–50 25–30 nd 30 25–32 nd 24
pH range for growth 6.5–8.0 6.5–8.0 5.0–8.0 nd 6.0–9.0 nd 5.0–8.0 4–10
Salinity range for growth (%) 0–2.0 0–2.0 0–3.0 0–4.0 0–1.5 0–1.0 nd 0–1.5
Flexirubin production + + + − − nd − nd
Catalase w w + + + w − +
Oxidase + + − + − + − −
Indole − − + nd − − − −
H2S − − nd − nd − nd −
Hydrolysis of:                
 Starch + + − nd − + − −
 Chitin + + − nd nd − + +
 CM-Cellulose − − − nd − nd + −
 Xylan − − − nd − nd nd nd
 Casein − − + nd − nd + +
 Gelatin − − − nd − + + +
 Urea − − − nd − − − +
Voges–Proskauer − − nd nd nd + nd nd
Nitrate reduction − − − − + + − −
DNA G+C content (mol%) 40.5 nd 45.0 42.0 53.0 48.9 45.8 44.6
Assimilation of sugars:
 Amygdalin − − nd − nd nd nd nd
 Arabinose + + nd − − nd nd nd
 Cellobiose + + nd − nd + nd nd
 Glucose + + − − + + − +
 Maltose + + nd − + nd nd
 Mannose − − nd − + + nd nd
 Melibiose − − nd − + + nd nd
 Mannitol
 Rhamnose − − nd − nd + nd nd
 Sorbitol − − nd − + − nd nd
 Sucrose − − nd − + − nd +
 Xylose + + nd nd nd + nd nd
 N-Acetyl-glucosamine + + nd nd nd nd nd nd
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Data in columns 3–8 were taken from [11,13,49]((; (; (; (; [] and [].+, Positive; -, negative; w, weak; , no data available

The DNA G+C content of strain C216T was 40.5 mol%, which was less than that of Niabella species (45.0 mol%) and Terrimonas species (48.9 mol%) (Table 2). Menaquinone-7 (MK-7) was detected as the only respiratory quinone. The major fatty acids (>5 %) identified in strain C216T were iso-C15 : 0 (56.7%), iso-C17 : 0 3-OH (15.6%), summed feature 3 (C16 : 1 ω7c and/or iso-C15 : 0 2-OH, 7.3%) and iso-C15 : 1 G (6.1%). The total fatty acid profile of strain C216T in comparison to closely related genera is summarized in Table 3. Much higher levels of fatty acid iso-C15 : 0 (56.7%) clearly distinguished strain C216T from Niabella and Terrimonas species and contribute to the thermotolerant/thermophilic nature of this strain.

Table 3. Fatty acid profile of strain C216T and related type strains of genera in the family Chitinophagaceae.

Strains: 1, C216T; 2, Niabella aurantiaca R2A15-11T; 3, Hydrotalea flava CGUG 51397T; 4, Compostibacter hankyongensis BS27T; 5, Terrimonas ferruginea ATCC 13524T; 6, Niastella koreensis GR20-10T; 7, Chitinophaga pinensis UQM 2034T. Data for strains 2–7 were taken from [11,13,49]. –, Not detected; tr, trace amounts.

Fatty acid 1 2 3 4 5 6 7
Saturated
C14 : 0 0.8 – – 1.0 0.5 – 1.0
C15 : 0 0.7 – 0.6 – 2.7 – –
C16 : 0 3.5 3.5 0.9 1.9 1.7 2.6 1.9
Branched
iso-C13 : 0 0.1 – 4.2 – 0.5 – 1.1
iso-C13 : 0 3-OH – – – – – – –
iso-C14 : 0 – – – – 1.1 – tr
iso-C14 : 0 3-OH 0.1 – – – – – –
C14 : 0 2-OH 0.1 – – – – – –
iso-C15 : 0 56.7 33.7 34.8 29.7 28.4 26.8 29.0
iso-C15 : 0 3-OH 2.0 2.9 4.0 4.4 2.2 1.3 3.2
anteiso-C15 : 0 0.4 1.6 1.7 3.7 0.6 4.9 0.5
C15 : 0 2-OH 0.3 – 0.4 – 0.8 – –
iso-C16 : 0 0.1 – 0.7 – 1.4 1 0.8
iso-C16 : 0 3-OH 0.4 – 1.2 – 0.7 – –
C16 : 0 2-OH 1.4 – – 2.6 – – –
C16 : 0 3-OH 1.6 2.4 0.9 2.4 1.3 – 2
iso-C17 : 0 0.6 – – 7.4 – 1.6 tr
anteiso-C17 : 0 – – – – – – –
iso-C17 : 0 3-OH 15.6 – 16.9 22.7 – 29.4 11.3
C17 : 0 2-OH 0.1 – 0.5 – – 3.5 –
C17 : 0 3-OH – 15.5 – – 15.3 1.2 –
Unsaturated – –
C15 : 1 ω6c – – – – – – –
iso-C15 : 1G 6.1 22.3 8.2 – 26.2 15.6 15.6
anteiso-C15 : 1A – – – – – – tr
C16 : 1 ω5c – – – 1.7 – – 6.0
C17 : 1 ω6c 0.1 – – – – – –
iso-C17 : 1 ω9c – – 5.9 – – – –
iso-C17 : 1 ω10c – – – – – – –
Summed features*
1 – – – –
2 0.1 – – – – – –
3 7.3 10.6 4.7 13.1 11.2 4.3 23
4 – – – 9.4 – – –
Unknown
11.543 0.1 – 0.5 – – – –
13.565 1.0 – 8.4 – 1.3 – –
16.582 1.1 1.2 1.7 – 1.3 1.4 –
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*Summed feature 1 is C13 : 0 3-OH and/or iso-C15 : 1 h. Summed feature 2 is C12 : 0 aldehyde, C14 : 0 3-OH and/or iso-C16 : 1I. Summed feature 3 is C16 : 1 ω7c and/or iso-C15 : 0 2-OH. Summed feature 4 is iso-C17 : 1I and/or anteiso-C17 : 1B.

The major polar lipids of strain C216T were phosphatidylethanolamine, three unidentified phospholipids, three unidentified aminophospholipids, one unidentified aminolipid and one unidentified glycolipid (Fig. S3).

Strains C216T and M2295 were positive for utilization of aesculin and β-galactosidase and negative for protease activity. Both strains were positive for assimilation of d-glucose, l-arabinose, d-mannose, N-acetyl-glucosamine, and maltose. Both strains were negative for indole production. Assimilation of d-mannitol, potassium gluconate, capric acid, adipic acid, malic acid, trisodium citrate, and phenylacetic acid was not observed. Neither strain had the ability to reduce nitrates to nitrites or nitrogen. Fermentation of glucose, urease and arginine dehydrogenase activity was not observed under anaerobic conditions.

In conclusion, from the data and observations described, strains C216T and M2295 represent members of a novel genus within the phylum Bacteroidota, for which the name Mycovorax gen. nov. is proposed, and a novel species within this genus which we propose as Mycovorax composti sp. nov.

Description of Mycovorax gen. nov.

Mycovorax (My.co.vo’rax. Gr. masc. n. mykes, mushroom; L. masc. adj. vorax, voracious; N.L. masc. n. Mycovorax, devourer of fungi)

Named for the strain’s interaction with Mycothermus thermophilus, an ascomycete fungus, where the bacterium degraded the fungal mycelium of M. thermophilus. Gram-stain-negative, aerobic, and non-motile. The major fatty acids are iso-C15 : 0, iso-C17 : 0 3-OH, C16 : 1 ω7c/iso-C15 : 0 2-OH and iso-C15 : 1G. The only respiratory quinone is MK-7. Positioned phylogenetically within the phylum Bacteroidota. The type species is Mycovorax composti.

Description of Mycovorax composti sp. nov.

Mycovorax composti (com.pos’ti. N.L. gen. n composti, of compost).

Isolated from button mushroom compost. Colonies are orange, 2 mm in size, circular, opaque, and convex with entire margins when grown on 350 Emerson YPSs agar at 45 °C for 2 days. Positive for flexirubin-type pigments. Cells are strictly aerobic, Gram-stain-negative, non-motile and non-spore forming short rods that are 0.8–1.2 µm long and 0.4–0.5 µm wide. Grows at 37–55 °C (optimum, 45–50 °C), at pH 6.5–8.0 (optimum, pH 7.0) and in the presence of 0–2 % (w/v) NaCl (optimum, 0–1 % NaCl). Grows on 350 Emerson YpSs, R2A, TSA and LB. Does not grow on MacConkey agar. Weakly positive for catalase activity and positive for oxidase activity. Indole and H2S are not produced. Negative for urease, glucose fermentation, arginine dihydrolase, and protease. Negative for reduction of nitrates to nitrites and nitrates to nitrogen. Positive for chitinase, xylanase, α-N-acetylgalactosaminidase, β-glucosidase, β-galactosidase, and amylase. Hydrolyses gelatin after 48 h. Positive for the assimilation of d-glucose, l-arabinose, d-mannose, maltose, and N-acetyl-glucosamine. The only isoprenoid quinone is MK-7. The major fatty acids are iso-C15 : 0 (56.7%), iso-C17 : 0 3-OH (15.6%), C16 : 1 ω7c/iso-C15 : 0 2-OH (7.3%) and iso-C15 : 1G (6.1%). When grown in co-culture with the thermophilic ascomycete M. thermophilus, Mycovorax composti strain C216T degrades the fungal mycelium and exhibits anti-fungal activity by inhibiting mycelial growth.

Two strains of Mycovorax composti, C216T (=DSM 114558T=LMG 32998T) and M2295 (=DSM 114559=LMG 32997), were isolated from phase II mushroom compost in Australia. The type strain is C216T, which was isolated from end-phase II mushroom compost from Victoria, Australia. The DNA G+C content of the type strain is 40.5 mol%. The GenBank accession numbers for the 16S gene sequence and the genomic assembly of strain C216T are OP379917 and CP144143, respectively. Strain M2295 was isolated from end-phase II compost from South Australia, Australia. The GenBank accession number of the 16S gene sequence of strain M2295 is OP379911.

supplementary material

Uncited Supplementary Material 1.
ijsem-74-06496-s001.pdf (418.5KB, pdf)
DOI: 10.1099/ijsem.0.006496
Uncited Table S1.
ijsem-74-06496-s002.xlsx (312.7KB, xlsx)
DOI: 10.1099/ijsem.0.006496

Acknowledgements

We thank the participating mushroom composters and the Australian Mushroom Growers Association (AMGA) for discussions and support. We thank Paul Roche and Bob Cowan for their linguistic advice regarding genus and species names, and Dr. Vera Thiel from DSMZ for her expertise and help with transfer of strains between collections. We also thank Dr. Jacob Downs and Dr. Peri Tobias for producing the long read sequencing data. We acknowledge the technical and scientific assistance of Sydney Microscopy and Microanalysis, The University of Sydney node of Microscopy Australia.

Abbreviations

AAI

average amino acid identity

ANI

average nucleotide identity

CAZy

carbohydrate-active enzymes

CBM

carbohydrate binding module

CE

carbohydrate esterase

COG

clusters of orthologous groups

CTAB

hexadecyltrimethylammonium bromide

dDDH

digital DNA–DNA hybridization

DSMZ

Deutsche Sammlung von Mikroorganismen und Zellkulturen

GH

glycoside hydrolase

GT

glucosyl transferase

GTDB

genome taxonomy database

PL

polysaccharide lyase

RAST

rapid annotations using subsystems technology

Footnotes

Funding: This research was funded by Hort Innovation, using the Australian Mushroom Research and Development Levy and contributions from the Australian Government. Hort Innovation is the grower-owned, not-for-profit research and development corporation for Australian horticulture.

Accession No: Accession numbers available at NCBI C216T WGS: GCA_028485475.1, CP144143. C216T 16S rRNA sequence: OP379917. M2295 16S rRNA sequence: OP379911.

Author contributions: M.K. and M.T. designed the study. M.T. did the experimental work. M.T. drafted the manuscript. M.K. and T.B. reviewed and edited the manuscript.

Contributor Information

Meghann Thai, Email: meghann.thai@sydney.edu.au.

Tina L. Bell, Email: tina.bell@sydney.edu.au.

Michael A. Kertesz, Email: michael.kertesz@sydney.edu.au.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Uncited Supplementary Material 1.
ijsem-74-06496-s001.pdf (418.5KB, pdf)
DOI: 10.1099/ijsem.0.006496
Uncited Table S1.
ijsem-74-06496-s002.xlsx (312.7KB, xlsx)
DOI: 10.1099/ijsem.0.006496

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