High quality draft genome sequence of Janthinobacterium psychrotolerans sp. nov., isolated from a frozen freshwater pond

Strain S3-2T, isolated from sediment of a frozen freshwater pond, shares 99% 16S rRNA gene sequence identity with strains of the genus Janthinobacterium. Strain S3-2T is a facultative anaerobe that lacks the ability to produce violacein but shows antibiotic resistance, psychrotolerance, incomplete denitrification, and fermentation. The draft genome of strain S3-2T has a size of ~5.8 Mbp and contains 5,297 genes, including 115 RNA genes. Based on the phenotypic properties of the strain, the low in silico DNA-DNA hybridization (DDH) values with related genomes (<35%), and the low whole genome-based average nucleotide identity (ANI) (<86%) with other strains within the genus Janthinobacterium, we propose that strain S3-2T is the type strain (= DSM 102223 = LMG 29653) of a new species within this genus. We propose the name Janthinobacterium psychrotolerans sp. nov. to emphasize the capability of the strain to grow at low temperatures. Electronic supplementary material The online version of this article (doi:10.1186/s40793-017-0230-x) contains supplementary material, which is available to authorized users.


Introduction
The genus Janthinobacterium includes Gram-negative, motile, aerobic rod-shaped bacteria, which were isolated from soil and aquatic environments. Production of violacein, a purple, water-insoluble, secondary metabolite, is a feature commonly found in this genus [1,2]. Violacein has anti-bacterial, anti-viral, and anti-fungal properties [3], and has even been reported to protect frogs against fungal infection, when produced by the frog skin microbiota [4].
Strain S3-2 T , which is affiliated with the genus Janthinobacterium was isolated from freshwater sediment while screening for denitrifying bacteria. However, strain S3-2 T has traits that unambiguously distinguish it from the other strains of the genus [2,5,6]. Among these traits is the ability of strain S3-2 T to grow at −3°C, and to ferment different sugars. In contrast to the other strains, strain S3-2 T does not produce the violet pigment violacein, not even when grown on glycerol medium (20 g L −1 ) that induces violacein synthesis in other members of the genus Janthinobacterium. Here we present the genome of strain S3-2 T as well as its classification and phenotypic features. Taken together, these characteristics support the circumscription of S3-2 T as novel species, Janthinobacterium psychrotolerans sp. nov.

Classification and features
Sediment was obtained from a small fresh water pond near Aarhus, Denmark (coordinates 56.182804 N, 10.176294 E); the pond was covered with a thick layer of ice at the time of sampling. Strain S3-2 T was isolated at room temperature under oxic conditions from a diluted sediment sample (3 g in 10 mL sterile water) by direct plating on TSB agar, containing 3 g tryptic soy broth (Scharlau Chemie S.A., Spain) L −1 , 15 g agar L −1 .
Cells of strain S3-2 T are rod-shaped, and stain Gramnegative. Cells in stationary growth phase on TSB agar were motile, and had a mean length of 1.9 ± 0.3 μm, and a mean width of 0.7 ± 0.1 μm under a phase contrast microscope (n = 27) (Fig. 2).
The generation time of strain S3-2 T was approx. 160 min in TSB (10 g L −1 ) with 5 mM nitrate when grown aerobically at 20°C. Overall, strain S3-2 T has traits that unambiguously distinguish it from other strains of the genus [2,5,6]. Among these traits is the ability of strain S3-2 T to grow at −3°C. In contrast to the other strains, strain S3-2 T does not produce the pigment violacein, not even when grown on glycerol medium (20 g L −1 ), which induces violacein synthesis in other members of the genus Janthinobacterium [10]. Strain S3-2 T is available from the Belgian Co-ordinated Collection of Micro-organisms -BCCM/LMG Bacteria Collection as strain LMG 29653 and the Leibniz Institute DSMZ -German Collection of Microorganisms and Cell Cultures as strain DSM 102223; its general properties are summarized in Table 1.

Genome project history
The draft genome sequence of strain S3-2 T was completed on December 21, 2015. The genome project is deposited in the Genomes OnLine Database (GOLD) as project Gp0124039. This Whole Genome Shotgun project has been deposited at GenBank under the accession LOCQ00000000. The version described in this paper is version LOCQ01000000. The summarized information of this project is shown in Table 2.

Growth conditions and genomic DNA preparation
Strain S3-2 T was grown at 25°C in TSB (10 g L −1 ) supplemented with 5 mM nitrate. The cells were harvested by centrifugation and DNA was extracted from the pellet using the PowerLyser® PowerSoil® DNA extraction kit (MoBio, Carlsbad, CA, USA) according to the manufacturer's protocol.

Genome sequencing and assembly
The genome of strain S3-2 T was sequenced with the Illumina MiSeq Reagent Kit V3 (Illumina Inc. San Diego, CA, USA). Sequencing libraries were prepared using the Nextera XT Library Preparation Kit (Illumina). The sequencing library produced 3,761,645 paired end reads to-talling~2.11 Gbp. In total, 2,868,634 reads remained after quality trimming and adapter removal with Trimmomatic-0.33 [12] and the following trimming parameters: CROP:235 HEADCROP:25 SLIDINGWINDOW:4:20. Read quality before and after trimming was assessed by FastQC version 0.11.4 [13]. The trimmed reads (~1.04 Gbp) represented an average genome coverage of~178-fold based on , not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). These evidence codes are from the Gene Ontology project [41] the size of the assembled draft genome of strain S3-2 T . Reads were assembled using SPAdes 3.6.1 [14]. Contigs shorter than 1,000 bp were removed after the assembly.

Genome properties
The properties of the draft genome of strain S3-2 T are summarized in Table 3, and the assignment of genes into COG functional categories is shown in

Insights from the genome sequence
There is a high 16S rRNA sequence identity of 99% between strain S3-2 T and all other strains with validly published names within the genus Janthinobacterium (Table 5). This value is higher than the 98.7% identity threshold recommended by Meier-Kolthoff et al. [29] to propose a new species within the phylum Proteobacteria. Therefore, in silico DNA-DNA hybridization (DDH) against reference genomes was performed using the online genome-togenome calculator with the GGDC 2.0 BLAST+ model [27]. DDH values calculated according to formula 2 (to confidently predict DDH values of incomplete genomes [27]) were always <35% (Table 5, and Additional file 1: Table S1), and thus clearly below the 70% threshold to differentiate bacterial species [27]. Whole-genome-based average nucleotide identities (ANI) to other Janthinobacterium genomes were calculated by the online tool [30,31]. ANI was always < 86% (Table 5, and Additional file 1: Table S2) [30], and thus also well below the threshold for species delineation (95%; [31]). Hence, both results support the phenotypic distinction of strain S3-2 T as novel species within the genus Janthinobacterium. Violacein production, a common feature in Janthinobacterium, was never observed in growth studies with strain  S3-2 T . This observation is consistent with the absence of the vioABCDE operon, which encodes the genes required for the synthesis of this pigment; neither the automated annotation nor manual BLAST searches of the S3-2 T genome for known components of the vioABCDE operon (Additional file 1: Table S3) [2,6] identified any genes encoding violacein synthesis. The genome of strain S3-2 T features all necessary genes for nitrate reduction to N 2 O but lacks genes encoding the nitrous oxide reductase (Additional file 1: Table S4), which is consistent with N 2 O as end-product of denitrification. Genes affiliated with aerobic respiration were identified, including terminal oxidases with both high-and lowaffinity for oxygen (Additional file 1: Table S5). Another characteristic of strain S3-2 T is its capability to ferment different sugars, a trait which has not been reported for other strains in the genus Janthinobacterium [1,5,11]. The genes that encode these properties were summarized (Additional file 1: Table S6, and Figure S1).

Conclusions
Based on the phenotypic properties, phylogenetic position, and whole genome comparison, we formally propose strain S3-2 T as novel species of the genus Janthinobacterium, for which we propose the name Janthinobacterium psychrotolerans sp. nov. with strain S3-2 T (=DSM 102223 = LMG 29653) as the type strain.
Janthinobacterium psychrotolerans is a facultative anaerobic, Gram-negative bacterium. Cells are rod-shaped, motile, and have a size of 1.9 ± 0.3 × 0.7 ± 0.1 μm. Colonies are pale yellow and mucoid on TSB agar. Growth occurs between −3 and 30°C, with optimal growth observed at 25°C. Strain S3-2 T tolerates salinity between 0.17% and 2.17% NaCl, and grows within the pH range of 6 to 8 with optimal growth observed at pH 7.
The G + C content of the genome is 63.04 mol%. The genome project is deposited in the Genomes OnLine Database (GOLD) as project Gp0124039. This Whole Genome Shotgun project is deposited at GenBank under the accession LOCQ00000000. The type strain S3-2 T (= LMG 29653 = DSM 102223) was isolated from sediment of a small, frozen pond in Hasle, Aarhus, Denmark (coordinates 56.182804 N, 10.176294 E) in January, 2015.