The family Isosphaeraceae was recognized in 2016 based on comparative analysis of 16S rRNA gene sequences of cultured members of the phylum Planctomycetota (Kulichevskaya et al., 2016). To date, it is the only family of the order Isosphaerales (class Planctomycetia). The family includes plancomycetes with spherical cells that can be assembled in short chains or long filaments, or else form shapeless aggregates. The cells feature crateriform structures all over their surface. Cell division occurs by budding; daughter cells are nonmotile. Members of Isosphaeraceae are chemoorganotrophic aerobes, although some of them are capable of growing under microaerobic conditions. The type genus of this family is Isosphaera; its only member, Isosphaera pallida IS1BT (Giovannoni et al., 1987), was isolated from a hot spring in North America. In addition to Isosphaera, the family Isosphaeraceae includes the genera Singulisphaera (Kulichevskaya et al., 2008), Aquisphaera (Bondoso et al., 2011), Paludisphaera (Kulichevskaya et al., 2016), Tundrisphaera (Kulichevskaya et al., 2017), and Tautonia (Kovaleva et al., 2019). One more genus, Candidatus Nostocoida, is represented by filamentous planctomycetes that have not been obtained in pure cultures yet (Liu et al., 2001; Kulichevskaya et al., 2012).

The genome sizes of Isosphaeraceae members range from 5.53 Mb in Isosphaera pallida IS1BT to 9.74 Mb in Singulisphaera acidiphila DSM 18658T (Göker et al., 2011; Ivanova et al., 2017). Noteworthy, the complete genomes of all these bacteria were found to include one to four plasmids (Ivanova et al., 2017).

A characteristic feature of Isosphaeraceae planctomycetes is their hydrolytic potential. The taxonomically described members of this family can utilize a broad range of polysaccharides of plant and microbial origin (Dedysh and Ivanova, 2019). The genomes of these bacteria contain a large number of glycoside hydrolase genes, many of which do not belong to any of the known families according to the CAZy classification (Ivanova et al., 2017).

Planctomycetes of the family Isosphaeraceae inhabit a broad range of terrestrial and aquatic ecosystems rich in organic matter. According to the data of molecular studies, Isosphaeraceae is one of the most abundant planctomycete groups in boreal and subarctic peat bogs (Serkebaeva et al., 2013; Moore et al., 2015; Dedysh and Ivanova, 2019). Studies aimed at isolation of new microorganisms from these ecosystems led to description of three new genera of this family (Kulichevskaya et al., 2008, 2016, 2017).

In the present work, we investigated the planctomycete strain Pla2T, which was isolated from a boreal fen in Russia and identified as a member of the genus Paludisphaera based on analysis of the 16S rRNA gene. To date, this genus includes three taxonomically described members: P. borealis PX4T isolated from peat of a raised bog (Kulichevskaya et al., 2016), ‘P. soli’ JC670T from soil of high-altitude Himalaya (Kaushik et al., 2020), and ‘P. rhizosphaerae’ JC665T from rhizosphere soil (Lhingjakim et al., 2022).

The goal of this work was to study the complex of morphological, ecophysiological, and genomic characteristics of the new strain Pla2T that distinguish it from the previously described members of Paludisphaera and to establish its taxonomic status.

MATERIALS AND METHODS

Source of isolation and culture conditions. Strain Pla2T was isolated from a sample of peat soil (pH 7.1) collected in August 2019 from the depth of 5‒10 cm of the Charozerskoe fen profile (Vologda oblast; 60°30′42′′ N, 38°38′59′′ E). The plant community at the sample collection site was represented by an association of Carex lasiocarpa and Campylium stellatum. The electrical conductivity of fen water ranged from 236 to 279 μS/cm. The total organic carbon content in the peat sample was 66.2%, and the total nitrogen content was 2.4%. A more detailed characterization of the sample collection site was provided in our previous work (Dedysh et al., 2020).

The collected peat sample was used to obtain an enrichment culture of microorganisms that participate in hydrolysis of xylan, one of the key biopolymers of wetland ecosystems. For this purpose, 2 g of peat fragmented with sterile shears and 0.1% birch xylan (Sigma-Aldrich, United States) were placed into 160‑mL glass flasks and filled with 30 mL of native fen water. The enrichment culture was incubated at room temperature (20‒22°С) for 8 weeks. The isolate, strain Pla2T, was obtained by plating a 0.2-mL aliquot of the enrichment on medium M31 of the following composition (g/L distilled water): KH2PO4, 0.1; N-acetylglucosamine, 0.5; peptone, 0.1; yeast extract, 0.1; Hutner’s salt solution, 20 mL; vitamin solution no. 6 (Staley et al., 1992), 1 mL; pH 7.0. For the solid medium, 1% solution of PhytaGel (Fluka, Germany), a polysaccharide of microbial origin, was added. The isolate was purified by plating on the same medium supplemented with ampicillin (200 mg/L). The plates were incubated at 22°С for 3 weeks.

Identification of the isolate. Total DNA was isolated from Pla2T cells using a FastDNA SPIN kit for soil (Biol 101, United States) according to the manufacturer’s instructions. The obtained DNA was used as a template for PCR amplification of the 16S rRNA gene with the standard bacterial primers 9F/1492R (Lane, 1991). The PCR products were purified using a Wizard® SV Gel and a PCR Clean-Up System (Promega, United States). Sequencing was carried out in the Core Facility “Bioengineering” of the Research Center of Biotechnology. The obtained nucleotide sequences were edited using the BioEdit software. Phylogenetic trees were constructed using the maximum likelihood approach with the MEGAX software (Kumar et al., 2018). Statistical significance of the trees was assessed by bootstrap analysis with construction of 100 alternative trees. The obtained sequence of the 16S rRNA gene of strain Pla2T was deposited in GenBank under accession number OQ533426.

Genome sequencing and annotation. DNA for genome sequencing was isolated using the standard CTAB–phenol/chloroform procedure. A part of the obtained genomic DNA was sequenced in a R9.4 cell of a MinION device (Oxford Nanopore, United Kingdom) using the Ligation Sequencing kit 1D according to the manufacturer’s instructions. The rest of genomic DNA was sequenced on the Illumina MiSeq platform. Library preparation and sequencing were performed commercially by the ReaGen company (Russia). Hybrid assembly of Illumina and Nanopore reads was carried out using the Unicycler software (Wick et al., 2017). The genome sequence of strain Pla2T was deposited in GenBank under accession number PRJNA940391.

Genome annotation was performed with the program packages PROKKA (Seemann, 2014) and BLASTKoala (Kanehisa et al., 2016). The search for secondary metabolite gene clusters was performed with the AntiSmash software (Medema et al., 2011). The genome tree was constructed by multiple alignment of 120 marker genes using the GTDB-Tk program (Chaumeil et al., 2020). The analysis also included the genomes of other characterized members of Isosphaeraceae.

Search for genes of glycoside hydrolases and their homologs. In this work, we employed the CAZy classification of glycoside hydrolases, which is based on homology of the amino acid sequences of their catalytic domains (Drula et al., 2022). A search for potential glycoside hydrolases among the proteins encoded in the genome of Pla2T was carried out on the dbCAN3 server (https://bcb.unl.edu/dbCAN2/ index.php) by means of three algorithms: HMMER:dbCAN, DIAMOND:CAZy, and HMMER:dbCAN-sub. For all proteins identified by at least one of the algorithms and recognized as glycoside hydrolases of the families GH1–GH173 or as unclassified glycoside hydrolases (provisional family GH0 or GH_NC), their affiliation with the families GH1–GH180 was verified manually. The list of glycoside hydrolases of strain Pla2T discussed in this work is provided in Table S1. The blastp algorithm on the NCBI website (http://www.ncbi.nlm.nih.gov/) was applied to search the pool of protein sequences encoded in the Pla2T genome using 37 planctomycete proteins as queries. The proteins represented 24 families: GH1, GH3, GH9, GH17, GH23, GH25, GH26, GH35, GH55, GH62, GH65, GH73, GH76, GH79, GH88, GH92, GH104, GH108, GH125, GH133, GH135, GH136, GH137, and GH144.

Assessment of phenotype characteristics. The growth characteristics of the isolate Pla2T were determined by culturing bacteria in the liquid medium described above on a shaker (120 rpm). The range of the tested temperatures was from 4 to 35°С (with an increment of 5°С), pH values ranged from 4 to 8 (increment, 0.5), and the NaCl concentration in the medium ranged from 0 to 3.0% (increment, 1%). Oxidative and enzymatic carbon utilization was assessed using an API 20NE kit (bioMérieux, France). Enzymatic activity was evaluated using an API ZYM kit (bioMérieux). The range of utilized carbon sources was determined in mineral medium MM of the following composition (g/L distilled water): KH2PO4, 0.1; (NH4)2SO4, 0.1; MgSO4·7H2O, 0.1; CaCl2·2H2O, 0.02; solution 44 (Staley et al., 1992), 1 mL, and vitamin solution no. 6 (Staley et al., 1992), 1 mL. The test cultures were grown in three replicates in 100-mL flasks containing 10 mL of the medium. The substrates tested as potential carbon sources included sugars (glucose, sucrose, xylose, lactose, mannose, raffinose, and melibiose) and polysaccharides (phytagel, starch, pectin, xylan, xanthan gum, chitin, and microcrystalline cellulose); they were added in a concentration of 0.5 g/L.

The nitrogen sources utilized by Pla2T were identified by growing experimental cultures in liquid MM medium where (NH4)2SO4 was replaced by one of the following compounds in a concentration of 0.01%: KNO3, NaNO2, urea, yeast extract, N-acetylglucosamine, peptone, ammonium chloride, or one of the amino acids: isoleucine, alanine, threonine, tryptophan, proline, valine, phenylalanine, glutamine, lysine, asparagine, arginine, or glycine.

The growth of strain Pla2T was monitored by measuring the optical density of the culture at 600 nm using an Eppendorf Biophotometer AG 22331 (Eppendorf, Germany). Prior to measurements, the cultures were homogenized by vigorous shaking. Antibiotic sensitivity of strain Pla2T was tested on solid medium by placing test discs with various antibiotics on the bacterial lawn and subsequently measuring the resulting growth suppression zones.

RESULTS AND DISCUSSION

Isolation, morphological analysis, and identification of the new strain. The isolate Pla2T obtained from peat of a boreal fen grew in liquid medium M31 in the form of a pink-colored slime aggregate (Fig. 1a). On the solid medium with phytagel, this strain formed pale pink colonies of dense consistency that were slightly submerged in the medium (Fig. 1b). A similar effect of phytagel degradation around the growing colonies was previously described for Paludisphaera borealis PX4T (Kulichevskaya et al., 2016). Microscopic analysis showed that the colonies were formed by spherical nonmotile cells 1.5‒2.8 μm in diameter that proliferated by budding (Fig. 1c). In culture, single cells were rarely found; as a rule, bacteria developed as dense clusters in the form of shapeless aggregates. In some older cultures (more than 3 weeks of incubation), the cells were observed to develop amorphous extracellular projection structures, which apparently served for their attachment to various surfaces, but we could not determine the conditions of their formation (Fig. 1d).

Fig. 1.
figure 1

Growth features and cell morphology of strain Pla2T. (a) Culture growth in a liquid medium with formation of a slime aggregate; (b) Pla2T growth on a solid medium with phytagel (colonies partially submerged in the medium are indicated with arrows); (c), cell morphology in a 10-day-old culture of strain Pla2T on a solid medium; (d) cells with projection structures in an old liquid Pla2T culture. Scale bar, 10 μm.

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Based on sequencing and comparative analysis of the 16S rRNA gene of strain Pla2T, it was identified as a member of the genus Paludisphaera (Fig. 2). The closest phylogenetic relative of strain Pla2T with 97.9% identity of 16S rRNA gene sequences was the planctomycete ‘Paludisphaera soli’ JC670T isolated from soil in a high-altitude region of the Western chain of the Himalayas (Kaushik et al., 2020). Comparison with other members of Paludisphaera showed that the level of 16S rRNA gene identity was 97.0% for ‘Paludisphaera rhizosphaera’ JC665T from the rhizosphere of Erianthus ravennae (Lhingjakim et al., 2022) and 93.8% for Paludisphaera borealis PX4T isolated from peat of a raised bog (Kulichevskaya et al., 2016).

Fig. 2.
figure 2

Phylogenetic tree constructed using the maximum likelihood approach based on comparative analysis of the nucleotide sequences of the 16S rRNA genes of strain Pla2T and other members of the phylum Planctomycetota. The sequences of the 16S rRNA genes of five anammox planctomycetes (AF375994, AF375995, AY254883, AY254882, AY257181) were used as an outgroup. Bootstrap support values of >60 are shown.

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Physiological characteristics. The growth of strain Pla2T was observed for medium pH values ranging from 6 to 8 (optimum, pH 5.5‒6.5) and salinities from 0 to 2%. The temperature range of its growth was 4‒28°С, with an optimum at 15‒20°С. Experiments on oxygen dependence showed that the strain Pla2T was strictly aerobic. Our attempts to culture it under anaerobic conditions were unsuccessful.

Enzymatic analysis of the strain Pla2T using the standard API ZYM kit (bioMérieux) revealed the activity of several enzymes, including esterase (C4), lipase (C8), leucine-, valine-, and cystine-arylamidases, trypsin, α-chymotrypsin, acid phosphatase, and naphthol-AS-BI-phosphohydrolase. The activities of alkaline phosphatase, α- and β-glucosidase, α‑ and β-galactosidase, β-glucuronidase, N-acetyl-β-glucosaminidase, α-mannosidase, and α-fucosidase were not detected. According to the results of the API NE tests, the new planctomycete is not capable of fermentation nor of reducing nitrates to nitrites and molecular nitrogen.

Evaluation of the spectrum of utilizable substrates showed that the preferred sources of carbon and energy for strain Pla2T were the polysaccharides xylan, phytagel, and xanthan gum. Among simple sugars, the new isolate exhibited good growth on glucose, mannose, xylose, and melibiose. The possible nitrogen sources for this planctomycete were nitrate, peptone, yeast extract, N-acetylglucosamine, lysine, glycine, asparagine, aspartate, valine, proline, tryptophan, threonine, isoleucine, and arginine.

Tests for antibiotic sensitivity of Pla2T revealed resistance to chloramphenicol, gentamicin, ampicillin, kanamycin, and imipenem; at the same time, the strain was sensitive to tetracycline.

Genome characteristics. The sequencing of Pla2T DNA on the Nanopore platform produced 140 874 reads with a total length of 1.4 Gb. An additional round of sequencing on the Illumina MiSeq platform produced altogether 10 517 034 paired reads with an average length of 150 bp. As a result of hybrid genome assembly, we obtained nine contigs from 15 502 to 7 626 770 bp long. The G + C content in the Pla2T genome was 67 mol % (Table 1). The genome annotator Prokka predicted about 6500 putative protein-coding sequences, 3 copies of the rRNA operon, and 93 tRNA genes. Based on the obtained assembled genome, we were unable to determine exactly the number of plasmids. However, we revealed a similarity between some contigs of the Pla2T genome and plasmid sequences from other members of Isosphaeraceae. The highest coverage and similarity levels were observed for the PALBO1 plasmid from Paludisphaera borealis PX4T (78.6%) and two plasmids from the uncharacterized strain SH-PL62 (84.5 and 86.9%).

Table 1. Characteristics of the strain Pla2T in comparison to previously described species of Paludisphaera
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DNA‒DNA hybridization in silico showed the following levels of similarity between strain Pla2T and three members of the genus Paludisphaera: 24.4 ± 2.3% with ‘P. soli’ JC670T, 22.0 ± 2.3% with ‘P. rhizosphaerae’ JC665T, and 21.0 ± 2.3% with P. borealis PX4T. Based on the results obtained with the ANI (average nucleotide identity) calculator, the levels of similarity between the Pla2T genome sequence and the genomes of Paludisphaera members were 82.6% (‘P. soli’ JC670T), 80.8% (‘P. rhizosphaera’ JC665T), and 79.4% (P. borealis PX4T). According to the currently accepted standards, these ANI values indicate that the isolate represents a new species (Chun et al., 2018). A similar conclusion was suggested by the results of phylogenomic analysis, which showed that the strain Pla2T formed a common cluster with other members of the genus Paludisphaera within the family Isosphaeraceae (Fig. 3).

Fig. 3.
figure 3

Phylogenomic tree constructed based on comparative analysis of 120 concatenated sequences of conserved marker proteins of strain Pla2T and other members of the family Isosphaeraceae. Genomes of anammox planctomycetes available in the GTDB database were used as an outgroup (Parks et al., 2022).

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The genome of Pla2T contains genes that encode the enzymes of the principal metabolic pathways of chemoorganotrophic bacteria, such as glycolysis, tricarboxylic acid cycle, pentose-phosphate pathway, and oxidative phosphorylation. This planctomycete possesses the genomic potential for synthesis of all amino acids. The genome of Pla2T also contains most genes responsible for chemotaxis, including cheB, cheR, and cheW.

Analysis of genes encoding the synthesis of secondary metabolites. Planctomycete genomes contain a large number of gene clusters responsible for the synthesis of secondary metabolites. This property makes this microbial group a promising object of biotechnological research (Wiegand et al., 2018). Using the Antismash software, we performed a search for such gene clusters in the genome of Pla2T. As a result, six gene clusters potentially encoding enzymes for secondary metabolite synthesis were detected. Among them, three clusters were responsible for the synthesis of polyketide synthases (PKSs), one cluster determined the synthesis of indole, and two clusters determined the synthesis of terpenes. PKSs are key enzymes in the synthesis of biologically active polyketides (Donadio et al., 2007). The closest homologs of PKSs encoded by the Pla2T genome were found in the planctomycete P. borealis PX4T (100% similar) and in the cyanobacterium Scytonema hofmannii (8% identity).

Indole, a nitrogen-containing heterocyclic aromatic compound, is ubiquitously present in nature. In bacteria, indole affects spore formation, virulence, and biofilm formation. It is used in industry as a taste enhancer and a flavoring agent (Ferrer et al., 2022). The closest homolog of the indole gene cluster in Pla2T was found in the planctomycete Aquisphaera giovannonii OF2T. Terpenes are a class of natural compounds with the highest structural diversity. Accordingly, they perform a broad range of functional roles, including vitamins, carotenoids, hormones, aromatic compounds, and protective metabolites (Helfrich et al., 2019). The closest homolog of the two terpene clusters of Pla2T was the gene cluster from the strain SH-PL62, an uncharacterized planctomycete of the family Isosphaeraceae (50 and 100% similarity).

Repertoire of glycoside hydrolases encoded in the Pla2T genome. A dbCAN3-based search for putative glycoside hydrolases and their subsequent verification resulted in identification of 108 proteins that represented 40 families: GH2 (2 proteins), GH5 (11), GH10 (3), GH13 (11), GH15 (2), GH16 (3), GH18 (2), GH20 (1), GH27 (2), GH29 (3), GH32 (3), GH33 (4), GH36 (2), GH38 (2), GH39 (4), GH43 (5), GH50 (1), GH51 (2), GH57 (4), GH77 (1), GH78 (3), GH94 (2), GH95 (1), GH97 (1), GH99 (1), GH106 (1), GH116 (1), GH127 (5), GH130 (2), GH140 (2), GH141 (2), GH146 (1), GH148 (3), GH151 (1), GH163 (2), GH165 (1), GH171 (1), GH172 (3), GH176 (1), and GH177 (6). By comparing this list of families with the lists of families present in six planctomycetes of the family Isosphaeraceae (A. giovannonii OJF2T, I. pallida ATCC 43644T, P. borealis PX4T, S. acidiphila DSM 18658T, T. plasticadhaerens ElPT, and the strain SH-PL62) available on the CAZy site, we identified 24 families of glycoside hydrolases that were not detected by the dbCAN3 algorithms in strain Pla2T. The corresponding 37 planctomycete proteins were used as queries in a screening of Pla2T protein sequences, and the screening did not detect any close homologs in any of these cases.

The families GH27 and GH36 of glycoside hydrolases are closely related (clan GH-D); almost all their members are enzymes with α-D-galactosidase (EC 3.2.1.22) activity (Naumoff, 2004, 2011). Genes that encode proteins of these two families are extremely rarely found in planctomycete genomes. Among 101 genomes present in the CAZy database, only 11 genomes encode proteins of the GH27 family and 16 genomes encode GH36 proteins. Among the members of Isosphaeraceae, only the strain A. giovannonii OJF2T encodes a protein of the family GH27 (GenPept, QEH34490.1). It has 48 and 28% identity to the proteins Pla2_02372 and Pla2_03258 from Pla2T, respectively (these proteins have 30% identity to each other). The proteins Pla2_03686 and Pla2_05159 from Pla2T belong to the family GH36. Their N- and C-terminal fragments have 41 and 37% identity, respectively; in Pla2_05159, the terminal fragments are separated by a long insert. Both proteins belong to the same subfamily GH36A. The low level of similarity of their amino acid sequences suggests that all five α‑galactosidase genes were acquired by planctomycetes of the family Isosphaeraceae as a result of independent lateral transfer events. The fact that strain Pla2T accumulated four α-galactosidase genes certainly has adaptive significance, but its nature currently remains unclear. Our experiments confirmed that Pla2T was capable of growing on melibiose; similar data were previously reported for the strain A. giovannonii OJF2T (Bondoso et al., 2011).

The family GH10 of glycoside hydrolases is represented in planctomycetes by two clearly distinct subfamilies (Naumoff, 2016; Naumoff et al., 2022). Genes of the first subfamily are present as a single copy in each genome; they form a compact cluster in the phylogenetic tree, and their amino acid sequences lack two catalytically important Glu residues, suggesting that they do not possess any enzymatic activity. This subfamily includes Pla2_04228 from strain Pla2T. Genes of the second subfamily are present in only a few planctomycete strains, but they usually exist in several copies. This group includes the proteins Pla2_03346 and Pla2_05951 from Pla2T, which have 71% amino acid sequence identity and retain the catalytically significant residues. They probably possess endo-β-xylanase activity and are partially responsible for the strain’s capacity of growing on xylan.

The family GH32 of glycoside hydrolases comprises various β-fructosidases. Genes encoding these proteins are present in 43 of 101 planctomycete genomes included in the CAZy database. Previously, we proposed to divide this family in several subfamilies (Naumoff, 2001). The proteins Pla2_05839 and Pla2_05968 with 38% identity contain both catalytically significant residues and belong to the subfamily GH32b; bacterial enzymes from this subfamily typically possess endo-β-fructosidase activity (digestion of fructose polymers). Pla2_05993 does not belong to any of the previously determined subfamilies, but it is closely related (71% identity) to the protein from P. borealis PX4T (GenPept, APW62066.1). Considering the unusual structure of the putative active center of the latter protein, we previously supposed that it probably lacked catalytic activity (Ivanova et al., 2017). The low levels of similarity among these three proteins from strain Pla2T suggest that they have independent evolutionary histories and rule out the possibility of a recent gene duplication.

Investigation of the morphological, physiological, biochemical, and genetic characteristics of the new isolate Pla2T and their comparison to the traits of the previously described members of the genus Paludisphaera revealed a number of differences, which are listed in Table 1. Phylogenetically, the planctomycete ‘Paludisphaera soli’ JC670T isolated from soil is the closest relative of the new strain, but Pla2T differs from it in that it has a growth optimum at 15‒20°С and prefers media with lower pH values. There are also certain differences in their ability to grow on various sources of carbon and nitrogen. A comparison with ‘P. rhizosphaerae’ JC665T also revealed differences in the temperature and pH growth ranges, as well as in the spectrum of growth substrates. According to the taxonomic classification standards (Chun et al., 2018), the values of DNA‒DNA hybridization and the ANI calculation suggest that strain Pla2T should be classified as a new species of the genus Paludisphaera.

Novel species diagnosis: Paludisphaera mucosa sp. nov.

Paludisphaera mucosa sp. nov. (mu.co′sa. L. fem. adj. mucosa mucous). Spherical nonmotile cells 1.5–2.8 μm in diameter, individual or clustered in shapeless aggregates. Colonies with pink pigmentation. In liquid culture cells grow as slime aggregates. An obligate aerobe. A psychrotolerant mesophile and neutrophile with a growth optimum at 15‒20°С and pH 5.5‒6. Preferable growth substrates are polysaccharides, in particular xylan, phytagel, and xanthan gum. Good growth was observed on glucose, xylose, mannose, and melibiose. Activity was observed for the following enzymes: esterase (С4), lipase (С8), leucine-, valine-, and cystine-arylamidases, trypsin, α‑chymotrypsin, acidic phosphatase, and naphtol-AS-BI-phosphohydolase. No activity was detected for alkaline phosphatase, α- and β-glucosidases, α- and β-galactosidases, β-glycuronidase, N-acetyl-β-glucosaminidase, α-mannosidase, and α-fucosidase. The type strain of the species is Pla2T (=KCTC92668T = VKM B-3698T). The habitat of the species is fens.