Draft genome sequence of a predatory bacterium from northern peatland soil

Soil sampling, isolation and cultivation conditions

A soil sample was collected from peatland in the Pallas area, Northern Finland, in September 2022 (N67°59’ E24°13’, Figure 1A). The vegetation was mainly sedges ( Figure 1B). The sample was collected from a depth of 5 cm with sterile instruments and stored at 4°C. The pure culture of Mucilaginibacter cryoferens FT3.2 (Kumar et al. 2025), was used as the prey for isolating predatory bacteria from the soil sample. Bacteria were cultivated using R2A medium (Neogen, NCM0188A), which contained 0.5 g L⁻¹ yeast extract, 0.5 g L⁻¹ meat peptone, 0.5 g L⁻¹ casamino acid, 0.5 g L⁻¹ glucose, 0.5 g L⁻¹ starch, 0.3 g L⁻¹ K₂PO₄, 0.05 g L⁻¹ MgSO₄, and 0.3 g L⁻¹ C₃H₃NaO₃, and was adjusted to pH 6. For solid and top agar, 15 and 4 g L⁻¹ of agar (Sigma-Aldrich, A4550) were added, respectively. The cultures were grown aerobically at room temperature (RT).

Figure 1. (A, B) Sampling location, Pallas, and (C) strain 1-FT3.2 growing on plate.

In (A), Kilpisjärvi, the original isolation location for the prey strain, Mucilaginibacter cryoferens FT3.2, is additionally shown. Map modified from Wikimedia Commons (NordNordWest). In (C), a representative plate with lysis zones on the M. cryoferens FT3.2 lawn after 14 days of incubation is shown, scale bar, 1 cm.

For the isolation, 5 g of the soil sample (wet weight) was resuspended in 50 ml of R2A broth and incubated on a shaker (~200 rpm) at RT for two weeks for the sample enrichment. The enriched sample was centrifuged (ThermoScientific F15-6x100y, 30 min, 2,500 g, 20°C) and 100 μl of non-diluted supernatant plated with 300 μl of the M. cryoferens FT3.2 liquid culture and 3 ml of R2A soft agar (46°C) as a top layer on R2A solid agar plates. The plates were incubated aerobically at RT. The observed growth inhibition/lysis zone was picked up with a sterile pipette tip, resuspended in R2A broth, and plated in a top agar layer as before, which was repeated three consecutive times.

DNA extraction and sequencing

The top agar layers of the semi-confluent plates were collected and resuspended in R2A broth (3 ml per plate), incubated with shaking (~200 rpm) at RT for one hour and centrifuged (ThermoScientific F15-6x100y, 30 min, 10,000 g, 4°C). The supernatant was collected and stored at 4°C. The stock titers were determined by plating serial dilutions in a top agar layer as described above. DNA was extracted with the GeneJET Genomic DNA Purification Kit (Thermo Scientific, K0721) using the manufacturer’s protocol for Gram-negative bacteria and 20 ml of the agar stock as input. Note that the agar stocks contained cells from both M. cryoferens FT3.2 and the new strain.

For sequencing, 100 ng of genomic DNA was converted to a sequencing library using the Illumina DNA prep. Samples were dual indexed using the sequencing core unit’s own Nextera primers. Seven cycles were used in the PCR step and DNA was pooled and purified using Illumina’s SPB bead purification. The Library pool was sequenced at 12 pM on the AVITI sequencer (Element Biosciences) using the AVITI 2x150 Sequencing kit Cloudbreak FreeStyle High Output. Sequencing was performed at the DNA Sequencing and Genomics Laboratory (supported by HiLIFE and Biocenter Finland funding), Institute of Biotechnology, University of Helsinki.

Genome annotation

FastQC v. 0.11.9 (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) was used to assess the quality of reads. Raw reads were trimmed and adaptors removed with Cutadapt v. 2.7 (-m 50 --nextseq-trim 20) (Martin 2011). Read-based taxonomic profiling was performed using PhyloFlash v. 3.4.2 and SILVA138.1.eukmod database (Gruber-Vodicka et al. 2020). Since the sample contained a mixed culture of the prey strain M. cryoferens FT3.2 and a new potentially predatory strain, SPAdes v. 3.15.5 was used in the --meta mode for genome assembly (Bankevich et al. 2012). BBTools Stats was used for assessing the assembly statistics, Reformat for sorting scaffolds by their GC content, and Dedupe for dereplicating scaffolds (minidentity = 95 absorbrc = t absorbmatch = t sort = length) (sourceforge.net/projects/bbmap/). The full-length SSU rRNA gene sequences obtained from the PhyloFlash run and the assembled scaffolds of ≥10 kbp in length were searched with BLASTN (Altschul et al. 1990) against the NCBI nt database using an E-value cutoff of 0.001. The quality of the draft genome of a new strain was assessed with CheckM2 v. 1.0.1 (Chklovski et al. 2023). GTDB-Tk v. 2.3.2 with GTDB release 226 database (Chaumeil et al. 2022) was used for assigning a taxonomic classification. For the genome annotation, DRAM v. 0.1.2 (Shaffer et al. 2020) was used at KBase (Arkin et al. 2018). Putative (pro)viral sequences were predicted by geNomad v. 1.7 (Camargo et al. 2023) and their quality and completeness were assessed with CheckV v. 0.8.1 (Nayfach et al. 2021). Bowtie2 v. 2.5.3 was used for the additional mapping of reads to putative viral sequences (Langmead and Salzberg 2012).

Isolation

After about two weeks of incubating the plates, growth inhibition/lysis areas of 4-5 mm were observed. In subsequent platings, the size of lytic zones reached up to ~1 cm ( Figure 1C). The central parts of these zones were clear, while edges were hazier. Agar stocks produced lysis zones on the M. cryoferens FT3.2 lawn when diluted up to 10000-fold, but no lysis zones could be observed when titrating filtered stocks (0.22 and 0.45 μm PES LLG-Syringe filters Spheros), suggesting that the origin of the observed lytic zones was not viral. Very small, almost transparent or whitish colonies growing over the lysis zones were observed ( Figure 1C), but no aggregated structures like fruiting bodies were seen. Despite our attempts, these tiny colonies could not be transferred to a fresh plate for independent growth. An alternative cultivation approach using the myxobacterium-suited CY-C10 medium ((Karwowski et al. 1996) modified by omitting antibiotics) and higher incubation temperature (28°C) for stock titration did not improve colony growth visibility. We named the strain causing lytic zones on M. cryoferens FT3.2 as 1-FT3.2.

Genome sequencing and assembly

Sequencing genomic DNA of a mixed culture resulted in 245,936,278 raw read pairs (150 bp + 150 bp), of which 245,436,350 pairs were retained after read trimming and quality control. With the read-based profiling by PhyloFlash, 225,532 reads (0.092% of all reads) could be mapped to SSU rRNA sequences in SILVA database. Of the mapped reads, 212,996 (94%) were assigned to the order Sphingobacteriales (Bacteroidota), where the genus Mucilaginibacter belongs to, and 9,050 (4%) were assigned to the order Polyangiales (Myxococcota). The rest of the hits constituted less than 0.01% of mapped reads each. Thus, read-based profiling suggested two strains present in the sample, comprising about 98% of reads together. Furthermore, full-length SSU rRNA gene sequences assembled by SPAdes, matched to SILVA database, were only two OTUs with the closest-matching references of Mucilaginibacter sp. M20-56 (Sphingobacteriales; GenBank acc. no.: KP899210.1, 99% id., 100% cov.) and Phaselicystis metagenome (Polyangiales; GenBank acc. no.: FPLS01001412.1, 95% id., 99% cov.). Additional BLASTN searches of the two detected OTUs against the NCBI nt database resulted in hits to 16S rRNA gene sequences of Mucilaginibacter sp. strain FT3.2 (100% id., 100% cov., 0 E-value) and the members of the order Polyangiales (the genera Minicystis, Sorangium, Chondromyces, Labilithrix, Polyangium, and uncultured bacterium, 91-92% id., 100% cov., 0 E-value), respectively.

The assembly of the mixed culture consisted of 6,318 scaffolds, of which 140 scaffolds were longer than 10 kbp and represented 95% of the total length of all scaffolds ( Table 1). Most scaffolds longer than 10 kb were characterised by a GC content of either 41-43% (71 scaffolds) or 64-66% (56 scaffolds) ( Figure 2). The Mucilaginibacter cryoferens FT3.2 genome GC content is known to be 42.1 % (Genbank acc. no. CP183228.1). Therefore, 56 scaffolds with a GC content of 64-66% were separated from the rest of the assembly, representing strain 1-FT3.2. Dedupe run confirmed the non-redundancy of the assembled draft genome. The total length of the 1-FT3.2 draft genome was 7,202,438 bp with the scaffolds ranging from 13,622 to 664,534 bp in length ( Table 2). Based on the CheckM2 assessment, the genome is 81.3% complete and 0.5% contaminated.

Figure 2. The distribution of GC content across assembled scaffolds longer than 10 kbp.

Genome classification and annotation

In the BLASTN search, the 1-FT3.2 draft genome scaffolds recruited numerous hits to sequences representing the phylum Myxococcota. GTDB-Tk run on the draft genome suggested classifying 1-FT3.2 within the family Polyangiaceae, order Polyangiales, class Polyangia, phylum Myxococcota. With DRAM, no rRNA encoding genes were identified in the draft genome scaffolds. DRAM-based annotations ( Figure 3) revealed a few complete metabolic modules: pentose phosphate cycle, citrate cycle (TCA cycle), glyoxylate cycle, cytochrome c oxidase, and F-type ATPase, as well as a near-complete (8/9) glycolysis module, suggesting robust central carbon metabolism and aerobic respiration. Also, arsenate reductase (glutaredoxin), acetyl-CoA synthetase, acetate kinase, and alcohol dehydrogenase were predicted, but no CAZy enzymes. The incomplete nature of the draft genome sequence precludes full understanding of metabolic capacities or the lack of those in 1-FT3.2. Among other DRAM predictions, several different CRISPR-Cas system proteins were identified (Cas1, Cas2, Cas3, CasA, CasB, CasC, CasD, CasE, Cmr1, Cmr2, Cmr3, Cmr4, Cmr5, and Cmr6). About 36% of all predicted proteins had no significant hits to any DRAM database.

Figure 3. Metabolic functions of 1-FT3.2 strain predicted with DRAM.

Using geNomad with all scaffolds from the mixed-culture assembly resulted in the prediction of two proviral sequences on scaffolds that belonged to the 1-FT3.2 draft genome: at coordinates 58-30,107 nt in NODE_10_length_239568_cov_144.087670 and 23,906-79,669 nt in NODE_67_length_79671_cov_165.578627. These proviral elements were medium-quality (80 and 53 % complete, respectively) and both assigned as tailed phages within the class Caudoviricetes. In addition, three other short scaffolds (0.2, 5.4, and 6.9 kbp), were identified as viral by geNomad, although the presence of viral genes could be confirmed by CheckV only for one of them. Mapping reads to these three short scaffolds resulted in an overall alignment rate of only 0.00002%, confirming that the nature of the observed lysis zones is unlikely to be viral.