Draft Genome Sequence of Pseudomonas sp. Strain FEN, Isolated from the Fe- and Organic Matter-Rich Schlöppnerbrunnen Fen

Pseudomonas spp. are Gram-negative bacteria belonging to the Pseudomonadaceae family within the class Gammaproteobacteria and are characterized by metabolic versatility and colonization of a wide range of diverse habitats (1–3). A single nonfluorescent, siderophore-producing Pseudomonas sp. strain was isolated from soil samples collected from the moderately acidic (pH 4.5 to 6.0), Fe-rich minerotrophic Schlöppnerbrunnen fen located in the Fichtelgebirge (northern Bavaria, Germany; 50°7′54″N, 11°52′51″E). Enrichments were grown by inoculating Fe gradient tubes, prepared as described previously (4, 5), with 25 μl soil slurry. Briefly, the gradient tubes contained an FeS bottom layer (1:1 FeS mixed with modified Wolfe’s mineral medium [MWMM] [ATCC medium 2672] and 3% [wt/vol] agarose) overlaid with 0.2% (wt/vol) agarose-stabilized MWMM supplemented with Wolfe’s vitamin solution, 1 mM ferulic acid, and 1 mM syringic acid. Active enrichment cultures were transferred to new gradient tubes every 4 to 6 weeks, and the dilution-to-extinction method was used to obtain the Pseudomonas sp. strain FEN isolate.

Pseudomonas sp. FEN stock cultures were cultivated at room temperature on solid agar plates containing PS medium (ATCC medium 3). Overnight shaking cultures grown in 100 ml PS medium at room temperature were inoculated from a single colony picked from the PS plate. Overnight cultures were used for genomic DNA extraction with the GenElute bacterial genomic DNA kit (Sigma-Aldrich, Taufkirchen, Germany). Whole-genome sequencing was performed on the RS II platform (Pacific Biosciences [PacBio], Menlo Park, CA) according to the standard manufacturer's protocol. Briefly, a 10- to 20-kb library was prepared and sequenced on a PacBio RS II sequencer using C4-P6 chemistry on single-molecule real-time (SMRT) cells, with a 180-min collection protocol. Sequencing and subsequent filtering with Hierarchical Genome Assembly Process 4 (HGAP4) resulted in 153,019 reads with an average read length of 5,607 bp. The maximum read length was 74,202 bp. The genome was assembled de novo with HGAP4 using default parameters except for the estimated genome size (6.0 Mbp) (6). Genome sequence annotation and gene identification were performed with RASTtk v2.0 using default parameters (7–9).

The draft genome of Pseudomonas sp. FEN was assembled into 36 contigs totaling 6.79 Mbp, with a G+C content of 61.1%, an N₅₀ value of 576,490 bp, and an L₅₀ value of 4. Mapping of filtered raw reads onto the assembly revealed 90-fold coverage. The draft genome contains 6,294 coding sequences and 98 non-protein-coding genes. Initial cloning and 16S rRNA gene-based sequencing approaches indicated that the isolate was most closely related to Pseudomonas fluorescens. However, recent advances in genomics and taxonomic classifications, specifically implementation of average nucleotide identity (ANI)-based comparisons, standardized taxonomic classifications (Genome Taxonomy Database [GTDB] taxonomy) (10, 11), and the automated genome-based taxonomic analysis tool Type Strain Genome Server (TYGS) (12), revealed that the isolate is more closely related to the proposed type strain Pseudomonas batumici UCM B-321 (3, 13), rather than to any of the publicly available P. fluorescens genome sequences (Fig. 1). According to GTDB-Tk v0.3.2 analysis (10, 11, 14–19), the ANI between P. batumici and Pseudomonas sp. FEN is 90.56%, based on an aligned fraction of 0.73 (proportion of regions displaying significant similarity). This taxonomic classification clarifies why fluorescent siderophores, such as pyoverdine (20), were not detected (21).

Analysis of the Pseudomonas sp. FEN genome revealed putative genes encoding pathways for siderophore biosynthesis, Fe-transport systems, Fe-siderophore sensor proteins, siderophore receptor proteins, and ferrichrome (hydroxamate siderophore) uptake systems and receptors. Several genes encoding nonspecific siderophore uptake systems were identified, suggesting that these systems play a role in the uptake of exogenous siderophores, including pyoverdines. Homologs of the ferric Fe-ABC transport system (pitADC) and a ferrous Fe-transport system (efuUOB) were also identified. The draft genome of Pseudomonas sp. FEN provides supporting evidence that this microorganism is capable of siderophore production, import, and export, as well as detection of exogenous siderophores. Further investigations may reveal additional genes or regulatory mechanisms involved in siderophore production and uptake in environments characterized by high concentrations of dissolved organic matter (DOM) and Fe-DOM complexes, such as the Schlöppnerbrunnen fen.

This study was part of the Collaborative Research Centre Chemical Mediators in Complex Biosystems (SFB 1127 ChemBioSys) of the Friedrich Schiller University Jena, funded by the Deutsche Forschungsgemeinschaft.

We thank Ulrike Litzba (Friedrich Schiller University Jena, Aquatic Geomicrobiology Group) for isolating this strain.