Pseudomonas atagosis sp. nov., and Pseudomonas akappagea sp. nov., New Soil Bacteria Isolated from Samples on the Volcanic Island Izu Oshima, Tokyo

During the exploration of microbial natural resources, two strains of Pseudomonas, PS14T and PS24T, were isolated from samples taken from Izu Oshima, a volcanic island located 120 km southwest of central Tokyo. Phylogenetic analysis based on 16S rRNA gene sequences showed that PS14T was most similar to Pseudomonas baetica a390T (99.6%) and Pseudomonas helmanticensis OHA11T (99.5%), and that PS24T was most similar to Pseudomonas qingdaonensis JJ3T (98.8%) and Pseudomonas lutea OK2T (98.7%). The major fatty acids of these two strains were C16:0 and C17:0 cyclo, summed feature 3 (C16:1 ω6c and/or C16:1 ω7c), and summed feature 8 (C18:1 ω7c and/or 18:1 ω6c). The phylogenetic analyses, DNA-DNA hybridization results and phenotypic traits indicated that PS14T and PS24T constitute two novel species, Pseudomonas atagosis sp. nov. (type strain PS14T = CECT 9940T, = LMG 31496T) and Pseudomonas akappagea sp. nov. (type strain PS24T = CECT 9941T, = LMG 31497T), respectively. The sequence data of the draft genomes of PS14T and PS24T were deposited in the GenBank database under accession numbers VXCA00000000 and VXCP00000000, respectively, and the sequence data of their 16S rRNA genes were deposited in the GenBank database under accession numbers MN396717 and MN382268, respectively. Electronic supplementary material The online version of this article (10.1007/s00284-020-01943-2) contains supplementary material, which is available to authorized users.


Introduction
The genus Pseudomonas was first described at the end of the nineteenth century [1]. Pseudomonas strains are Gramnegative, rod-shaped, motile, catalase-positive and oxidasepositive bacterial cells. These bacteria have been isolated from various environments worldwide, including soil, animals, plants, and water [2]. To date, the List of Prokaryotic Names with Standing in Nomenclature (https ://www.bacte rio.net) includes 255 species of Pseudomonas, including 18 subspecies.
During the exploration of microbial natural resources, we collected soil samples from Izu Oshima, in January 2017. PS14 T was isolated from soil collected at Mt. Atago, which is located in the northwest part of the island. Mt. Atago, where Castanopsis sieboldii trees grow, is the transitional final stage, known as a climax community. PS24 T was isolated from a red scoria cone located on the west coast of the island, which the local inhabitants call Akappage. The present study describes the phenotypic and phylogenic characteristics of these two strains. These characteristics indicate that these two strains represent novel species of the genus Pseudomonas.

Morphological, Physiological and Biochemical Studies
Cell morphology was examined by scanning electron microscopy (Hitachi S-4800). Colony morphology was assessed on tryptic soy agar (TSA, Becton Dickinson) plates after culture for 24 h at 28 °C. Growth at various temperatures was tested by culturing in Luria-Bertani broth (LB, Becton Dickinson) [3]. Briefly, overnight cultures of tested strains were adjusted to OD 600nm = 0.225, and 20 μl of each sample were inoculated into 10 ml of LB. These strains were incubated at 5, 8, 12, 24, 28, 32, 36 and 40 °C while shaking at 25 rpm in a photorecording incubator (TN-2612; ADVANTEC, Tokyo, Japan). Gram-staining was performed by a staining kit (Muto Pure Chemicals co. ltd, Tokyo Japan). Motility was directly assessed using a Bacteria Self-Checker mil-kin® (https :// www.mil-kin.com/). Fluorescent pigmentation was assessed on King B medium (Eiken, Tokyo Japan), as described previously [4]. Oxidase activity as assessed using Cytochrome Oxidase Test Strips (Nissui, Tokyo, Japan). Catalase activity was analyzed by dropping 3% hydrogen peroxide solution onto the cells and monitoring the production of bubbles. Growth at different NaCl concentrations was assessed in nutrient broth (Becton Dickinson) [3] containing 0, 1, 2, 3, 4, 5, 6 and 7% NaCl. Growth at different pH levels (5, 6, 7, 8, 9, and 10) was investigated by adding hydrochloric acid or sodium hydroxide to 7.5 ml of twofold-higher TSB and 3 ml of buffer agent (MOPS for pH 5 to pH 7, HEPES for pH 8 to pH 9, and CAPS for pH 9 to pH 10). The broth was diluted with sterile water to adjust the TSB concentration to onefold. API 20 NE strips (bioMérieux) and Biolog GN3 MicroPlates were used according to the manufacturers' instructions. API 20 NE and GN3 tests for Pseudomonas granadensis DSM 28040 T was performed by German Collection of Microorganisms and Cell Cultures GmbH (DSMZ).

Chemotaxonomic Characterization
Fatty acid methyl ester analysis was performed at Techno Suruga Laboratory Co., Ltd (Shizuoka, Japan). Fatty acids were prepared as described by MIDI Microbial Identification System [5] and analyzed using the Sherlock Microbial Identification (MIDI) system (version 6.0).

Genomic DNA Preparation, Sequencing, and Assembly
Genomic DNA was extracted from PS14 T and PS24 T using QIAamp DNA Mini Kits (Qiagen), and genomic libraries of both strains were prepared using Nextera XT DNA Library Preparation Kits (Illumina). Paired-end sequencing was performed using MiSeq Reagent Kits v3 (600-cycles) through the Illumina MiSeq platform. De novo assembly was performed using CLC Genomics Workbench v7 (Qiagen). The DNA sequences of the 16S rRNA genes were analyzed using BigDye® Terminator v3.1 Cycle Sequencing Kits and an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, Life Technologies, Carlsbad, CA), along with the primers 8F (5′-AGA GTT TGA TCC TGG CTC AG-3′) and 1541R (5′-AAG GAG GTG ATC CAG CCG CA-3′) [6].

Phylogenetic Analysis
Sequences were aligned using CLUSTAL W software and phylogenetic trees were constructed using MEGA 7.0 software [7]. Evolutionary distances were calculated using Tamura's 3-parameter model [8]. To account for heterogeneity of substitution rate among nucleotide sites, the discrete gamma model with 5 categories was used. Phylogenetic trees were reconstructed using maximum-likelihood (ML) methods [9]. The sequences of all Pseudomonas type strains used for the analysis except Pseudomonas helmanticensis LMG 28168 T were retrieved from the National Center for Biotechnology Information (NCBI) GenBank database and EzBioCloud (https ://www.ezbio cloud .net/). Pseudomonas helmanticensis LMG 28168 T (GOLD ID Gp0112928) was retrieved from Department of Energy Joint Genome Institute (https ://www.jgi.doe.gov) under Genomes Online Database IMG.

Genome Analysis
The similarity of the sequenced genomes to genomes of other type strains was determined based on the Average Nucleotide Identity with OrthoANIu algorithm [10] and Genome-to-Genome-Distance (GGDC) version 2.1 software [11]. The GGDC results were based on formula 2, which is independent of genome length and is therefore recommended to use for incomplete draft genomes.

Results and Discussion
Phylogenetic trees were constructed based on the 16S rRNA sequences (1459 bp) of PS14 T and PS24 T and of representative Pseudomonas strains (Fig. 1). GenBank accession numbers are listed in Table S1. The highest interspecific sequence similarities that were found between strain PS14 T and its phylogenetic neighbors were Pseudomonas baetica a390 T (99.6%) and P. helmanticensis OHA11 T (99.5%), and that of PS24 T were P. qingdaonensis JJ3 T (98.8%) and P. lutea OK2 T (98.7%). Figure 2 is a phylogenetic tree constructed based on concatenated sequences of 16S rRNA and three housekeeping genes linked in the order 16S rRNA (1459 bp)-gyrB (801 bp)-rpoD (718 bp) -rpoB (915 bp) (Fig. 2). These sequences were retrieved from the genome sequences, and GenBank accession numbers of these genes are listed in Tables S1 and S2. Strain PS14 T clusters in a separate branch that is related to a group including P. baetica, P. helmanticensis and P. koreensis. PS24 T was placed near P. qingdaonensis and P. rhizosphaerae. These results indicate that both of these Izu Oshima strains belong to the P. fluorescens lineage, but they are distinct from other species in that lineage.  Table S1 Genomic Analysis The DNA G+C contents of PS14 T and PS24 T were found to be 59.6% and 60.2%, respectively. Assessments of ANI scores and dDDH values of PS14 T , PS24 T and closely related strains are listed in Table S3. The highest correlations were between PS14 T and P. helmanticensis, with an ANI score of 88.3% and a dDDH score of 35.7%, and between PS24 T and P. qingdaonensis, with an ANI score of 80.8% and a dDDH score of 24.5%. These ANI and dDDH scores were lower than the cutoff values for species delineation (> 95% for ANI and > 70% for dDDH) [12], indicating that PS14 T and PS24 T are likely novel species of the genus Pseudomonas.

Growth Conditions, Physiology, Morphology, and Biochemical Characteristics
The phenotypic features of PS14 T and PS24 T are presented in Table 2. The phenotypic features of PS14 T were similar to those of P. koreensis, although they differed in utilization of gelatin hydrolysis, d-fucose, d-arabitol, l-histidine, glucuronamide, and α-keto-glutaric acid. PS24 T was found to be more restricted than PS14 T , with d-glucose being the only sugar source found to be utilized by PS24 T .

Compliance with Ethical Standards
Conflict of interest The authors declare that they have no conflicts of interest.
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