Complete Genome Sequence of Kinneretia sp. Strain DAIF2, Isolated from a Freshwater Pond

The Gram-negative Kinneretia sp. strain DAIF2 was isolated from a eutrophic pond in Göttingen, Germany. The sample (51°33′29″N, 9°56′41″E) was collected on 24 September 2018. The strain was enriched and isolated as described previously (1). DAIF2 was chosen for sequencing, since it was most similar at the 16S rRNA gene level to the genus Kinneretia, which was until now only represented by the type strain, Kinneretia asaccharophila DSM 25082 (2). For DNA isolation, DAIF2 was cultivated in PCa medium (peptone medium supplemented with 0.015% CaC1₂ [3]) at 25°C. DNA was extracted using the MasterPure complete DNA and RNA purification kit (Epicentre, Madison, WI, USA) as described previously (1). Illumina sequencing libraries were constructed using the Nextera XT DNA sample preparation kit (Illumina, San Diego, CA, USA) and sequenced using a MiSeq instrument and reagent kit v3 (600 cycles), as recommended by the manufacturer (Illumina). For Nanopore sequencing, a separate batch of 1.5 μg high-molecular-weight DNA was used for library preparation by employing the ligation sequencing kit 1D (SQK-LSK109) and the native barcode expansion kit (EXP-NBD114; barcode 19) as described by the manufacturer (Oxford Nanopore Technologies, Oxford, UK). The MinION device Mk1B, the SpotON flow cell R9.4.1, and MinKNOW software v19.06.8 were used for sequencing (72 h) as recommended by the manufacturer (Oxford Nanopore Technologies). For demultiplexing and base calling, Guppy v3.0.7 (Oxford Nanopore Technologies) was applied. Default parameters were used for all software unless otherwise specified. Sequencing resulted in 3,208,102 300-bp Illumina reads and 5,612,523 Nanopore reads with a mean length of 1,631 bp. The Illumina reads were quality filtered using Trimmomatic v0.36 (4), and paired reads were joined with FLASH (5). The Nanopore reads were adapter and quality trimmed with a length cutoff of 10 kb using fastp v0.20.0 (6), resulting in 75,898 Nanopore reads with an N₅₀ value of 31,759 bp. Together with the Illumina reads, a de novo hybrid assembly was performed using Unicycler v0.4.8 (7) in normal mode. The assembly revealed a single circular chromosome (6,010,585 bp) with a GC content of 69.28%. Coverages calculated with Qualimap v2.2.1 (8) using Bowtie 2 v2.3.5 (9) and minimap2 v2.17-r941 (10) were 127-fold (Illumina) and 204-fold (Nanopore). The Prokaryotic Genome Annotation Pipeline (PGAP) v4.11 (11) was used for automatic DAIF2 genome annotation. Annotation revealed 5,538 putative genes, 5,398 of which were protein coding. Moreover, 64 tRNA genes, 15 rRNA genes, 1 transfer-messenger (tmRNA) gene, and 3 noncoding RNA (ncRNA) genes were identified.

Whole-genome-based phylogeny of the DAIF2 genome was performed with the Type (Strain) Genome Server (TYGS [12], accessed 12 November 2020). In general, close relationships of DAIF2 to the genera Kinneretia, Paucibacter, Mitsuaria, and Roseateles, which belong to the family Comamonadaceae, were detected (Fig. 1). The closest relative was the type strain Kinneretia asaccharophila DSM 25082 (GenBank accession number NZ_SNXE00000000.1) of the genus Kinneretia, which was announced in 2010 as a new genus in the Rubrivivax branch (2), with a calculated digital DNA-DNA hybridization (dDDH) of 34.5%. This result indicates that strain DAIF2 may be a new species (Fig. 1).

We thank Sarah Teresa Schüßler for technical assistance.

We also acknowledge support by the Open Access Publication Funds of the University of Göttingen.