ABSTRACT
The bacterial strains Pseudomonas sp. strain MM221 and Pseudoarthrobacter sp. strain MM222 were isolated from a sandy soil sample. Here, we report on their complete genome sequences, including a circular plasmid for MM221, which were assembled after sequencing with an Oxford Nanopore Technologies flow cell.
ANNOUNCEMENT
Microbial communities are major determinants in the soil environment. Their composition, diversity, and function are highly complex and poorly understood (1). To add genomic information on soilborne coinhabiting bacteria, we sequenced two independent bacterial isolates, MM221 and MM222, from a field sample.
We isolated both strains from a sandy soil sample that was collected in a meadow in Bielefeld, North Rhine-Westphalia, Germany (52°2′23.75016″N, 8°29′45.71592″E). The sample was taken from 5 cm below the surface of the ground, suspended in 0.9% (wt/vol) NaCl, filtered using a cellulose filter (pore size, 4 to 12 μm; product number 431015; Macherey-Nagel, Düren, Germany), and centrifuged. The cell pellet was resuspended in fresh 0.9% NaCl solution. Various dilutions were plated on agar medium (1.5% agar, 1% soy peptone, 0.3% NaCl, 0.1% sucrose, 0.1% cellulose, 0.1% xylan, 0.1% chitin, 0.05% Tris-HCl), and plates were incubated at 30°C for 7 days. Colonies of MM221 appeared yellow, and colonies of MM222 appeared white. Single colonies were picked, streaked on agar medium for propagation, and used for DNA isolation using the NucleoSpin microbial DNA kit (Macherey-Nagel) with RNA digestion according to the manufacturer’s manual. Barcoding of the genomic DNA was performed using a rapid barcoding kit (SQK-RBK004; Oxford Nanopore Technologies [ONT], Oxford, UK) according to the manufacturer’s protocol. For sequencing, one R9.4.1 flow cell was run for 15 h on a GridION system, and bases were called using the super-accurate base-calling model of MinKNOW v22.05.7 (all from ONT). The sequencing reads were processed for each barcode. All programs were run with default parameters unless otherwise specified. Adapters were trimmed with Porechop v0.2.3 (2), and BLAST was used to determine the expected genome sizes (3). The genome sequence was assembled as a single contig each with Canu v2.2 (4), using a genome size of 6.2 Mbp for Pseudomonas sp. strain MM221 and a genome size of 4.5 Mbp for Pseudoarthrobacter sp. strain MM222. For MM221, an additional contig for a plasmid was obtained. Assemblies were polished with Racon v1.5.0 (5), Minimap v2.24-r1122 with parameter setting –ax map-ont (6), and Medaka v1.6.0 with parameter setting –m r941_min_sup_g507 (ONT). Overlaps were trimmed with Berokka v0.2 (https://github.com/tseemann/berokka), and contigs were oriented according to dnaA with Circlator v1.5.5 (7). Genome completeness was examined with benchmarking universal single-copy orthologs (BUSCO) v5.4.3 with parameter setting –augustus (8). Genes were predicted with Prokka v1.14.5 (9). The organism and strain type were identified with TYGS (10). A metabolic pathway analysis was performed with KAAS (11), and potential antibiotic resistances were identified with the Comprehensive Antibiotic Resistance Database (CARD) (12). Relevant statistics for the raw reads and the genome sequences are listed in Table 1.
TABLE 1.
Sequencing and assembly statistics for Pseudomonas sp. strain MM221 and Pseudoarthrobacter sp. strain MM222
| Parameter | Finding for: |
|
|---|---|---|
| Pseudomonas sp. strain MM221 | Pseudoarthrobacter sp. strain MM222 | |
| Raw sequencing reads | ||
| No. of reads | 74,793 | 48,962 |
| Total length (bp) | 487,682,423 | 311,527,256 |
| N50 (bp) | 11,033 | 11,142 |
| Genome sequence | ||
| Length (bp) | 6,728,688 | 4,287,032 |
| GC content (%) | 61.95 | 66.46 |
| Genome coverage (×) | 72.48 | 72.67 |
| Gene annotation | ||
| Total no. of genes | 6,534 | 3,934 |
| No. of protein-coding genes | 6,432 | 3,865 |
| No. of rRNAs | 22 | 15 |
| No. of tRNAs | 79 | 53 |
| No. of transfer-messenger RNAs | 1 | 1 |
| BUSCO results (%)a | ||
| Complete | 93.3 | 99.8 |
| Single copy | 93.2 | 99.4 |
| Duplicated | 0.1 | 0.4 |
| Fragmented | 4.9 | 0.2 |
| Missing | 1.8 | 0.0 |
| Plasmid | ||
| Length (bp) | 2,396 | |
| No. of protein-coding genes | 2 | |
| GC content (%) | 47.25 | |
| Plasmid coverage (×) | 329 | |
The databases used (and the numbers of searched BUSCOs) were as follows: MM221, pseudomonadales_odb10 (782 BUSCOs); MM222, micrococcales_odb10 (537 BUSCOs).
The genome of the presented Pseudomonas sp. strain MM221 has Pseudomonas putida NBRC 14164 (GenBank accession number NC_021505.1) (13) as the closest relative (Fig. 1A). A plasmid was coisolated and contains two open reading frames, encoding hypothetical proteins with unknown functions. According to CARD (12), the genome of Pseudomonas sp. strain MM221 contains genes conferring antibiotic resistance.
FIG 1.
Genome BLAST distance phylogeny (GBDP) trees. Phylogenetic trees for Pseudomonas sp. strain MM221 (A) and Arthrobacter sp. strain MM222 (B) were constructed from genome sequences with TYGS (10). (A) The closest relative for MM221 is Pseudomonas putida NBRC 14164, with 51.2% digital DNA-DNA hybridization (dDDH) (formula d4) similarity (10). (B) With a dDDH (formula d4) value of 26.7%, Pseudoarthrobacter albicanus NJ-Z5 is the closest relative for Pseudoarthrobacter sp. strain MM222. The presented isolates MM221 and MM222 are highlighted in bold type. The pseudo-bootstrap support values from 100 replications are indicated at each branch point. A., Arthrobacter; P., Pseudomonas; Ps., Pseudoarthrobacter.
The other presented genome belongs to Pseudoarthrobacter sp. strain MM222, with Pseudoarthrobacter albicanus NJ-Z5 (GenBank accession number GCA_018224015.1) as the closest relative (Fig. 1B). A KAAS analysis (11) indicated that Pseudoarthrobacter sp. strain MM222 is likely auxotrophic for biotin.
Data availability.
The MM221 and MM222 assemblies, gene annotations, and reads are available in GenBank/ENA under BioProject accession number PRJEB56332. The SRA accession number for the MM221 raw reads is ERS13525428, and the BioSample accession number for the MM221 assembly and annotation is ERS13579683. The SRA accession number for the MM222 raw reads is ERS13525429, and the BioSample accession number for the MM222 assembly and annotation is ERS13579684.
ACKNOWLEDGMENTS
We thank Arno Krieger for preparation of growth media, Levin J. Klages for assistance during library preparation, and Jörn Kalinowski and Tobias Busche for provision of the sequencing platform. Sequences were generated, assembled, and analyzed as part of the practical course Fundamentals of Molecular Microbiology at Bielefeld University.
We acknowledge support for the publication costs by the Open Access Publication Fund of Bielefeld University and the Deutsche Forschungsgemeinschaft (DFG). This work was supported by the BMBF-funded de.NBI Cloud within the German Network for Bioinformatics Infrastructure (de.NBI).
Contributor Information
Bianca Frommer, Email: frommer@cebitec.uni-bielefeld.de.
David A. Baltrus, University of Arizona
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Associated Data
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Data Availability Statement
The MM221 and MM222 assemblies, gene annotations, and reads are available in GenBank/ENA under BioProject accession number PRJEB56332. The SRA accession number for the MM221 raw reads is ERS13525428, and the BioSample accession number for the MM221 assembly and annotation is ERS13579683. The SRA accession number for the MM222 raw reads is ERS13525429, and the BioSample accession number for the MM222 assembly and annotation is ERS13579684.