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ORIGINAL RESEARCH
published: 08 February 2022
doi: 10.3389/fmicb.2021.809431
Edited by:
Damien Paul Devos,
Andalusian Center for Development
Biology, Spanish National Research
Council (CSIC), Spain
Reviewed by:
Stefanie P. Glaeser,
University of Giessen, Germany
Geeta Chhetri,
Dongguk University, South Korea
*Correspondence:
Olga I. Nedashkovkaya
olganedashkovska@piboc.dvo.ru;
oned2004@mail.ru
Larissa A. Balabanova
balaban@piboc.dvo.ru;
lbalabanova1@gmail.com
Specialty section:
This article was submitted to
Evolutionary and Genomic
Microbiology,
a section of the journal
Frontiers in Microbiology
Received: 08 November 2021
Accepted: 23 December 2021
Published: 08 February 2022
Citation:
Nedashkovkaya OI, Kim S-G,
Balabanova LA, Zhukova NV,
Son OM, Tekutyeva LA and
Mikhailov VV (2022) Genome-Based
Classification of Strain 16-SW-7,
a Marine Bacterium Capable
of Converting B Red Blood Cells, as
Pseudoalteromonas distincta
and Proposal to Reclassify
Pseudoalteromonas paragorgicola as
a Later Heterotypic Synonym
of Pseudoalteromonas distincta.
Front. Microbiol. 12:809431.
doi: 10.3389/fmicb.2021.809431
Genome-Based Classification of
Strain 16-SW-7, a Marine Bacterium
Capable of Converting B Red Blood
Cells, as Pseudoalteromonas
distincta and Proposal to Reclassify
Pseudoalteromonas paragorgicola
as a Later Heterotypic Synonym of
Pseudoalteromonas distincta
Olga I. Nedashkovkaya1*, Song-Gun Kim2, Larissa A. Balabanova1*, Natalia V. Zhukova3,
Oksana M. Son4, Liudmila A. Tekutyeva4and Valery V. Mikhailov1
1G.B. Elyakov Pacific Institute of Bioorganic Chemistry, Far Eastern Branch of the Russian Academy of Sciences,
Vladivostok, Russia, 2Korean Collection for Type Cultures, Biological Resource Center, Korea Research Institute
of Bioscience and Biotechnology, Daejeon, South Korea, 3A.V. Zhirmunsky National Scientific Center of Marine Biology, Far
Eastern Branch of the Russian Academy of Sciences, Vladivostok, Russia, 4Department of Bioeconomy and Food Security,
School of Economics and Management, Far Eastern Federal University, Vladivostok, Russia
A strictly aerobic, Gram-stain-negative, rod-shaped, and motile bacterium, designated
strain 16-SW-7, isolated from a seawater sample, was investigated in detail due
to its ability to produce a unique α-galactosidase converting B red blood cells into
the universal type blood cells. The phylogenetic analysis based on 16S rRNA gene
sequences revealed that the strain 16-SW-7 is a member of the Gammaproteobacteria
genus Pseudoalteromonas. The closest relatives of the environmental isolate were
Pseudoalteromonas distincta KMM 638Tand Pseudoalteromonas paragorgicola KMM
3548T, with the plural paralogous 16S rRNA genes of 99.87–100% similarity. The
strain 16-SW-7 grew with 1–10% NaCl and at 4–34◦C, and hydrolyzed casein, gelatin,
tyrosine, and DNA. The genomic DNA G+C content was 39.3 mol%. The prevalent
fatty acids were C16:1ω7c, C16:0, C17:1ω8c, C18:1ω7c, C17:0, and C12:03-OH.
The polar lipid profile was characterized by the presence of phosphatidylethanolamine,
phosphatidylglycerol, two unidentified amino lipids, and three unidentified lipids. The
major respiratory quinone was Q-8. The finished genome of the strain 16-SW-7
(GenBank assembly accession number: GCA_005877035.1) has a size of 4,531,445 bp
and comprises two circular chromosomes L1 and S1, deposited in the GenBank
under the accession numbers CP040558 and CP040559, respectively. The strain
16-SW-7 has the ANI values of 98.2% with KMM 638Tand KMM 3548Tand the
DDH values of 84.4 and 83.5%, respectively, indicating clearly that the three strains
belonged to a single species. According to phylogenetic evidence and similarity for the
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Nedashkovkaya et al. Genome-Based Reclassification of Pseudoalteromonas distincta
chemotaxonomic and genotypic properties, the strain 16-SW-7 (= KCTC 52772 = KMM
701) represents a novel member of the species Pseudoalteromonas distincta. Also, we
have proposed to reclassify Pseudoalteromonas paragorgicola as a later heterotypic
synonym of P. distincta based on the rules of priority with the emendation of the species.
Keywords: marine bacteria, taxonogenomics, phenotype, emended description of Pseudoalteromonas distincta,
Pseudoalteromonas paragorgicola
INTRODUCTION
The genus Pseudoalteromonas was proposed by Gauthier et al.
(1995) by splitting the genus Alteromonas into two genera
due to its high level of heterogeneity. At the time of writing,
the genus Pseudoalteromonas comprises 49 validly published
species, including Pseudoalteromonas haloplanktis as the type
species. Cells of the genus were described as Gram-stain-negative,
aerobic, chemoorganotrophic, non-spore-forming, straight and
curved rods or ovoid, those are motile by means of a single
polar flagellum. All species of the genus were oxidase-positive,
required a seawater base for growth, and did not accumulate
poly-p-hydroxybutyrate. Subsequently, the genus was emended
due to the newly obtained data, including the presence of polar,
bipolar, or lateral flagella, gelatin and Tween 80 hydrolysis,
glucose fermentation, and the ability of some strains to produce
buds and prosthecae (Ivanova et al., 2002a;Hwang et al., 2016;
Beurmann et al., 2017). In addition, the G+C content of DNA
was extended up to 37–55 mol% (Park et al., 2016). Currently,
the G+C content of the genomic DNA ranges from 34.8 mol%
for Pseudoalteromonas denitrificans DSM 6059T(NCBI RefSeq:
NZ_FOLO00000000.1) to 54.9 mol% for Pseudoalteromonas
aestuariivivens DB-2T(Park et al., 2016). Members of the genus
Pseudoalteromonas are often isolated from different marine
environments, including surface and deep seawater, sediments
and sea ice samples, ascidians, coral, a surface slime of a
puffer fish, mussels, brown and green algae, diatoms, and
the halophyte plants (Bowman, 1998;Sawabe et al., 2000;
Egan et al., 2001;Ivanova et al., 2002b,c, 2004;Romanenko
et al., 2003a,b;Park et al., 2005;Matsuyama et al., 2013;
Wu et al., 2017;Navarro-Torre et al., 2020). In this study,
we characterized a non-pigmented strain 16-SW-7, isolated
from seawater of the Okhotsk Sea. This strain is attracting
the attention of researchers for its ability to convert B red
blood cells into the universal type of blood cells (Bakunina
et al., 1998;Balabanova et al., 2010). A preliminary study of
the taxonomic position of the strain by Sanger sequencing
of the 16S rRNA gene indicated its closest relationship with
the type strains of the recognized species of the genus
Pseudoalteromonas,P. distincta, and P. paragorgicola, with a
similarity of 99.9% by the taxonomic EzBioCloud 16S rRNA
database (Yoon et al., 2017). The type and single strain of
P. distincta KMM 638T(formerly Alteromonas distincta) was
originally isolated from a marine sponge collected at a depth
of 350 m near the Komandorskie Islands, Russia (Romanenko
et al., 1995). This strain formed non-pigmented colonies and
was shown to produce the dark-gray diffusible melanin-like
pigments. The type of strain of P. paragorgicola KMM 3548T
was originally isolated from a gorgonian, Paragorgia arborea,
collected from the Pacific Ocean, and formed lightly orange-
pigmented colonies (Ivanova et al., 2002a). Further investigation
presented here on the whole-genome sequences has shown
the affiliation of the strain 16-SW-7 and the above-mentioned
species with validly published names to the single species.
In the present study, we clarify the taxonomic position of
the strain 16-SW-7, reclassify the species P. paragorgicola
as a later heterotypic synonym of P. distincta, and specify
the description of the species P. distincta based on the
results of phylogenetic analysis, and genotypic and phenotypic
characterization.
MATERIALS AND METHODS
Strain Isolation and Cultivation
The strain 16-SW-7 was isolated from a seawater sample
collected near Island Paramushir (Kuril Islands), the Okhotsk
Sea, during the 16th cruise of the Research Vessel Academician
Oparin by plating 0.1 ml of seawater directly onto nutrient
medium as described previously (Nedashkovskaya et al.,
2007). After primary isolation and purification, the bacterium
was cultivated at 28◦C on the same medium or marine agar
2216 (Difco, bioMérieux, Pacific, Biosciences) and stored
at −80◦C in artificial seawater or marine broth (Difco,
bioMérieux, Pacific, Biosciences) supplemented with 20%
(v/v) glycerol. The strain 16-SW-7 was deposited in the
collection of marine microorganisms (KMM) at the G.B.
Elyakov Pacific Institute of Bioorganic Chemistry FEB
RAS (Vladivostok, Russia), Korean Collection for the type
cultures (KCTC) and VKM under deposit numbers KMM
701, KCTC 52772, and VKM B-2135 D, respectively. The
type strains P. distincta KMM 638T(=ATCC 700518T)
and P. paragorgicola KMM 3548T(=DSM 26439T) were
obtained from the collection of marine microorganisms
(KMM) and used as the reference strains for comparative
taxonomic analysis.
Morphological, Biochemical, and
Physiological Characterization
The physiological, morphological, and biochemical properties of
the strain 16-SW-7 were studied using the standard methods.
The novel isolate was also examined in the API 20E, API 20NE,
API 50 CH, API 32 ID GN, and API ZYM galleries (bioMérieux,
France) according to the manufacturer’s instructions, except that
the inoculum was prepared using ASW (Bruns et al., 2001)
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Nedashkovkaya et al. Genome-Based Reclassification of Pseudoalteromonas distincta
and the galleries were incubated at 28◦C. Gram-staining was
performed as recommended by Gerhardt et al. (1994). Oxidative
or fermentative utilization of glucose was determined on Hugh
and Leifson’s medium modified for marine bacteria (Lemos
et al., 1985). Catalase activity was tested by the addition of
3% (v/v) H2O2solution to a bacterial colony and observation
for the appearance of gas. Oxidase activity was determined
by using tetramethyl-p-phenylenediamine. Degradation of agar,
starch, casein, gelatin, chitin, DNA and urea and production
of acid from carbohydrates, hydrolysis of Tween 80, nitrate
reduction, production of hydrogen sulfide, acetoin (Voges–
Proskauer reaction), and indole were tested according to standard
methods (Gerhardt et al., 1994). The temperature range for
growth was assessed on MA. Tolerance to NaCl was assessed
in medium containing 5 g Bacto Peptone (Difco), 2 g Bacto
Yeast Extract (Difco), 1 g glucose, 0.02 g KH2PO4, and 0.05 g
MgSO4.7H2O per liter of distilled water with 0, 0.5, 1.0, 1.5,
2.0, 2.5, 3, 4, 5, 6, 8, 10, 12. 15, 17, 19, and 20% (w/v) of
NaCl. Susceptibility to antibiotics was examined on MA plates
at 28◦C by the routine disk diffusion plate method. Disks were
impregnated with the following antibiotics: ampicillin (10 µg),
benzylpenicillin (10U), carbenicillin (100 µg), cefalexin (30 µg),
cefazolin (30 µg), chloramphenicol (30 µg), erythromycin
(15 µg), gentamicin (10 µg), kanamycin (30 µg), lincomycin
(15 µg), nalidixic acid (30 µg), neomycin (30 µg), ofloxacin
(5 µg), oleandomycin (15 µg), oxacillin (10 µg), polymyxin B
(300 U), rifampicin (5 µg), streptomycin (30 µg), tetracycline
(5 µg), and vancomycin (30 µg).
Chemotaxonomic Characterization
For whole-cell fatty acid and polar lipid analysis, the strains 16-
SW-7, P. distincta KMM 638Tand P. paragorgicola KMM 3548T
were grown under optimal physiological conditions for all strains
(at 28◦C for 24 h on MA). Cellular fatty acid methyl esters
(FAMEs) were prepared according to the methods described by
Sasser (1990), using the standard protocol of Sherlock Microbial
Identification System (version 6.0, MIDI), and analyzed with the
use of a GC-21A chromatograph (Shimadzu) equipped with a
fused-silica capillary column (30 m ×0.25 mm) coated with
Supelcowax-10 and SPB-5 phases (Supelco) at 210◦C. FAMEs
were identified by using equivalent chain-length measurements
and comparing the retention times to those of authentic
standards. The polar lipids of the strains studied were extracted
using the chloroform/methanol extraction method of Bligh and
Dyer (1959). Two-dimensional TLC of polar lipids was carried
out on silica gel 60 F254 (10 cm ×10 cm; Merck) using
chloroform/methanol/water (65: 25: 4, by vol.) in the first
dimension, and chloroform/methanol/acetic acid/water (80: 12:
15: 4, by vol.) in the second dimension (Collins and Shah,
1984). For detection of the lipids, 10% sulfuric acid in methanol,
molybdenum blue, ninhydrin, and a-naphthol were applied.
Isoprenoid quinones were extracted with chloroform/methanol
(2:1, v/v) and purified by TLC, using a mixture of n-hexane
and diethyl ether (85:15, v/v) as the solvent. Isoprenoid quinone
composition of the strain 16-SW-7 was characterized by HPLC
(Shimadzu LC-10A) using a reversed-phase type Supelcosil LC-
18 column (15 cm ×4.6 mm) and acetonitrile/2-propanol (65:35,
v/v) as a mobile phase at a flow rate of 0.5 ml min−1as described
previously (Komagata and Suzuki, 1988).
Whole-Genome Sequencing and
Phylogenetic Analysis
The genomic DNA of the strain Pseudoalteromonas sp. 16-SW-
7 (=KMM 701 = KCTC 52772) was extracted from the cells
grown on marine agar (25◦C, 72 h), using a NucleoSpin microbial
DNA kit (Macherey-Nagel, 54 Germany), and sequenced at
Macrogen, Inc. (Seoul, South Korea). To construct libraries,
the high-molecular-weight DNA (15 µg) was fragmented to
generate 20-kb SMRTbellTM templates, and then, the fragments
were annealed using a PacBio DNA polymerase binding kit and
sequenced by the PacBio RS II platform (Pacific Biosciences,
United States), with the use of PacBio version 4.0 sequencing
kit with single-molecule real-time cells. Hierarchical Genome
Assembly Process 3 (HGAP3) was used to perform de novo
assembly of the PacBio reads for Pseudoalteromonas sp. 16-SW-
7. The circular shape of the contigs was formed by testing the
overlap ability of the contig ends. Because of the mapping reads
against the assembled contigs and error correction using Quiver,
the final sequence with the highest quality was generated.
The 16S rRNA gene and genome phylogenetic analyses
were performed by the Type (Strain) Genome Server (TYGS),
an automated high-throughput platform for state-of-the-art
genome-based taxonomy (Meier-Kolthoff and Göker, 2019). The
genome of the strain 16-SW-7 (GenBank assembly accession:
GCA_005877035.1) was compared against all types of strain
genomes available in the TYGS database via the MASH
algorithm, a fast approximation of intergenomic relatedness
(Ondov et al., 2016), and the 10 types of strains with the
smallest MASH distances were chosen. In addition, the set
of 10 closely related type strains was determined via the
16S rRNA gene sequences, extracted from the genomes using
RNAmmer (Lagesen and Hallin, 2007), and BLASTed (Camacho
et al., 2009) against the 16S rRNA gene of each of 14,309
type strains. This was used as a proxy to find the best 50
matching type strains, according to the bit score for the 16-
SW-7 genome and to subsequently calculate precise distances
using the Genome BLAST Distance Phylogeny approach (GBDP)
under the algorithm “coverage” and distance formula d5(Meier-
Kolthoff et al., 2013). These distances were finally used to
determine the top 10 closest genomes of the type strains. For
the phylogenomic inference, all pairwise comparisons among
the set of genomes were conducted using GBDP and accurate
intergenomic distances inferred under the algorithm “trimming”
and distance formula d5. One hundred distance replicates were
calculated each. Digital DNA-DNA hybridization (DDH) values
and confidence intervals were calculated using the recommended
settings of the GGDC 2.1 (Meier-Kolthoff et al., 2013). The
resulting intergenomic distances were used to infer a balanced
minimum evolution tree with branch support via FastME 2.1.6.1
including SPR post-processing (Lefort et al., 2015). Branch
support was inferred from 100 pseudo-bootstrap replicates each.
The trees were rooted at the midpoint (Farris, 1972) and
visualized with PhyD3 (Kreft et al., 2017). The type-based species
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Nedashkovkaya et al. Genome-Based Reclassification of Pseudoalteromonas distincta
clustering, using a 70% dDDH radius around each of the 13 type
strains, was done as previously described (Meier-Kolthoff and
Göker, 2019). Subspecies clustering was done using a 79% dDDH
threshold as previously introduced (Meier-Kolthoff et al., 2014).
The whole-genome average nucleotide identity (ANI) values
were calculated with the use of ChunLab’s ANI calculator (Yoon
et al., 2017). The average amino acid identity (AAI) values
were calculated by an AAI-profiler available at http://ekhidna2.
biocenter.helsinki.fi/AAI (Medlar et al., 2018). Biosynthetic gene
clusters (BGCs) were identified with antiSMASH version 5.1.1
(Medema et al., 2011). The whole-genome sequence analyses
and comparative genomics of Pseudoalteromonas sp. 16-SW-
7, P. paragorgicola KMM 3548T(GenBank assembly accession:
GCA_014918315.1), and P. distincta ATCC 700518T(GenBank
assembly accession: GCA_000814675.1) were additionally carried
out using the high-performance computing servers Rapid
Annotation of microbial genomes using Subsystems Technology
(RAST; Overbeek et al., 2014), EzBioCloud (Yoon et al., 2017),
and Integrated Microbial Genomes and Microbiomes (IMG/M)
system (Chen et al., 2021).
RESULTS AND DISCUSSION
Morphological, Biochemical, and
Physiological Characterization
The strain 16-SW-7 was shown to be a strictly aerobic,
heterotrophic, Gram-stain-negative, and motile bacterium,
which formed non-pigmented colonies on marine agar and
required NaCl or seawater for growth. It was positive for
cytochrome oxidase and catalase and hydrolyzed aesculin, casein,
gelatin, Tweens 20, 40, and 80, DNA, and tyrosine (Table 1).
The strains 16-SW-7, P. distincta KMM 638T, and
P. paragorgicola KMM 3548Tshared many common phenotypic
features, such as respiratory type of metabolism, motility by
means of flagella, the presence of catalase, alkaline phosphatase,
esterase lipase (C8), leucine arylamidase, valine arylamidase,
acid phosphatase, and naphthol-AS-BI-phosphohydrolase
activities (Table 1). They could not synthesize lipase (C14),
N-acetyl-β-glucosaminidase, β-glucosidase, α-galactosidase,
β-glucuronidase, α-mannosidase and α-fucosidase, hydrolyse
agar, chitin, and urea and reduce nitrate to nitrite. However, the
strain 16-SW-7 can be distinguished from its closest phylogenetic
relatives by the several phenotypic traits, including the ability
to form acid from D-raffinose, to produce hydrogen sulfide and
cysteine arylamidase, trypsin and α-chymotrypsin, and to be
resistant to ampicillin and vancomycin (Table 1). The above
findings can extend the phenotypic characteristics those were
reported for the species P. distincta (Romanenko et al., 1995;
Ivanova et al., 2000, 2004) after justification of the placement
of the strains 16-SW-7 and P. paragorgicola KMM 3548Tin the
species P. distincta.
Chemotaxonomic Characterization
The fatty acid profiles of the strains 16-SW-7, P. distincta KMM
638T, and P. paragorgicola KMM 3548Twere similar (Table 2).
The predominant fatty acids (>5% of the total fatty acids)
of the strain 16-SW-7 and its closest relatives were C16:1ω7c
(29–32.1%), C16:0(15.4–18.2%), C17:1ω8c(11.7–17.9%), C18:1
ω7c(5.2–11%), C17:0(5.8–10.3%), and C12:03-OH (4.8–7.5%).
The composition of other fatty acids presented in Table 2 was
also similar except that the strain 16-SW-7 contained higher
proportions of C14:0, C12:0, and iso-C16:0, and lower proportions
of C15:1ω8c, C12:03-OH, and C13:03-OH. These values were
consistent with the results of phylogenetic analysis and confirmed
the affiliation of the strains studied to the same species. The
polar lipid profile of the strain 16-SW-7 was characterized by
the presence of phosphatidylethanolamine, phosphatidylglycerol,
two unidentified amino lipids, and three unidentified lipids
(Table 2 and Supplementary Figure 1). It was similar to that
of P. paragorgicola KMM 3548Tand it can be distinguished
from another relative, P. distincta KMM 638T, by the presence
of unknown lipids L1 and L2. The nearest neighbors of the
strains under study, Pseudoalteromonas aliena LMG 22059T
and Pseudoalteromonas fuliginea KMM 216T, distinguished from
them by the presence of unknown phospholipids and unknown
aminophospholipid and two unknown glycolipids, respectively
(Machado et al., 2016;Zhang et al., 2016). The main respiratory
quinone of the strains under study was ubiquinone Q-8 that is
consistent with those reported for the members of the family
Pseudoalteromonadaceae (Ivanova et al., 2004).
16S rRNA Genes and Phylogenomic
Analysis
The analysis of the 16S rRNA gene sequence of the strain 16-
SW-7 (GenBank accession number: OL587468) in the EzTaxon
database application (Yoon et al., 2017) revealed 100% similarity
with Pseudoalteromonas arctica A 37-1-2T(CP011026) and
Pseudoalteromonas elyakovii KMM 162T(AF082562), and 99.9%
similarity with P. distincta KMM 638T(JWIG01000030) and
P. paragorgicola KMM 3548T(AY040229). However, the 16S
rRNA gene sequences obtained by the Sanger method are
recommended to be compared with the genome sequences,
as well as the use of overall genome data for the taxonomy
of prokaryotes, such as average nucleotide identity (ANI) and
digital DDH (dDDH) and relatedness between the strains and
type of strain of a species (Chun et al., 2018). The closed
genome of 16-SW-7 was found to contain nine full-length
sequences of 16S rRNA genes with 99.87–100% similarity
between each other (Table 3). The multiple 16S rRNA genes
seem to be a characteristic of the type species of the family
Pseudoalteromonadaceae, including P. distincta KMM 638Tand
P. paragorgicola KMM 3548T(Table 3). Among the nine 16S
rRNA genes found in the genome of P. distincta KMM 638T
(=ATCC 700518T), only one—the length-comparable gene in
a contig 30—was extracted for the analysis, probably due to
an incomplete genome sequencing (GenBank WGS accession:
JWIG00000000.1). In the P. paragorgicola KMM 3548Tgenome,
two 16S rRNA genes were completely sequenced among three
found (GenBank WGS accession: AQHE00000000.1).
The whole-genome sequence of the strain 16-SW-7 was
4,531,445 bp, with a G+C content of 39.3 mol% and comprised of
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TABLE 1 | Phenotypic characteristics of the strain 16-SW-7 and the closest relatives of the genus Pseudoalteromonas.
Characteristic 1 2 3
Source of isolation Seawater Sponge Gorgonian
Colony color Whitish Whitish Pale orange
Flagellation P P, L P
Temperature range for growth (◦C) 4–34 4–30 4–30
Salinity range for growth (% NaCl, optimum) 1–10 (2–6) 1–6 (1.5–3) 0.5–8 (1–8)
Requirement for seawater or artificial seawater for growth − + −
Production of melanin-like pigments − + −
H2S production − + +
Hydrolysis of:
Casein w −w
Starch − − +
Alginate + + ND
Acid production from:
D-Cellobiose, D-galactose, D-glucose, D-lactose, maltose, D-xylose + − +
D-Raffinose + − −
D-Mannitol + + −
Utilization of citrate + − w
Assimilation of (API ID 32GN gallery):
Itaconic acid, potassium-2-keto-gluconate, L-histidine + − −
D-Melibiose, D-sorbitol + − +
Inositol, sodium malonate, lactic acid, D-ribose, 3-hydroxybutyric acid − + −
Salicin + − −
Enzyme activity (API ZYM tests):
Cysteine arylamidase, trypsin, α-chymotrypsin + − −
Esterase (C4) + + −
Susceptibility to:
Ampicillin, vancomycin − + +
Oleandomycin + − +
Tetracycline − − +
DNA G+C content (mol %) 39.3 39.2 (43.8) 39.2
Strains: 1, 16-SW-7; 2, P. distincta KMM 638T; 3, P. paragorgicola KMM 3548T. All strains were positive for the following tests: respiratory type of metabolism;
motility; presence of oxidase, catalase, alkaline phosphatase, esterase lipase (C8), leucine arylamidase, valine arylamidase, acid phosphatase, and naphthol-AS-BI-
phosphohydrolase activities; hydrolysis of aesculin, gelatin, Tweens 20, 40, and 80, DNA, and tyrosine; acid production from sucrose; assimilation of D-glucose,
maltose, sucrose, D-mannitol, sodium acetate, L-alanine, L-serine, L-proline, glycogen, propionic acid, valeric acid, and capric acids; susceptibility to carbenicillin,
chloramphenicol, erythromycin, doxycycline, gentamicin, kanamycin, nalidixic acid, neomycin, ofloxacin, polymyxin, rifampicin, and streptomycin, and resistance to
benzylpenicillin, cefalexin, cefazolin, lincomycin, and oxacillin. All strains were negative for the following tests: nitrate reduction; hydrolysis of agar, chitin, CM-cellulose, and
urea; acetoin production; acid production from L-arabinose, D-fructose, D-mannose, D-melibiose, L-rhamnose, D-ribose, D-trehalose, N-acetylglucosamine, and glycerol;
assimilation of L-arabinose, L-fucose, L-rhamnose, N-acetylglucosamine, suberic acid, potassium 5-ketogluconate, 3-hydroxybenzoic acid, 4-hydroxybenzoic acid, and
salicin; lipase (C14), α-galactosidase, β-galactosidase, β-glucuronidase, α-glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase, α-mannosidase, and α-fucosidase
activities. P, polar; L, lateral; +, positive; −, negative; w, weak reaction; ND, no data available.
two circular chromosomes L1 and S1, deposited in the GenBank
under the accession numbers CP040558 and CP040559,
respectively (assembly accession: GCA_005877035.1). The
genome size of P. paragorgicola KMM 3548T(GenBank
assembly accession: GCA_014918315.1) was 4,322,351 bp,
with the G+C content 39.2 mol%. In comparison, the
genome size of P. distincta KMM 638T(GenBank assembly
accession: GCA_000814675.1) was 4,532,748 bp and the
G+C content of 39.2 mol% (Supplementary Table 1).
The GBDP phylogenomic tree is consistent with the
branching patterns, observed for only the strains 16-SW-7
and P. paragorgicola KMM 3548T(=DSM 26439T) in the
16S rRNA gene sequence-based tree, generated by TYGS,
because of the use of the P. distincta KMM 638T(=ATCC
700518T) gene with lower identity (Table 3,Figure 1, and
Supplementary Figure 2).
However, the species-specific gene clusters for the strains
16-SW-7, P. paragorgicola KMM 3548T(=DSM 26439T), and
P. distincta KMM 638T(=ATCC 700518T) were identical,
indicating that the three strains belonged to a single species
(Figure 1 and Supplementary Figure 2). The ANI calculator in
EzBioCloud, based on the use of OrthoANIu algorithm (Lee et al.,
2016), showed the similar high ANI values (98.04–98.2%) for
the strain 16-SW-7 and the reference strains P. distincta KMM
638Tand P. paragorgicola KMM 3548T(Supplementary Table 2),
which are higher than the species-level cutoff value of 95–
96% (Richter and Rosselló-Móra, 2009). According to the TYGS
results, the dDDH (d4) values between the 16-SW-7 genome
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TABLE 2 | Fatty acid composition (%) of the strain 16-SW-7 and closely related
strains of the genus Pseudoalteromonas.
Fatty acids 1 2 3
Saturated
C12:02.8 tr tr
C14:03.9 tr 1.3
C15:02.5 4.7 3.0
C16:018.2 15.4 16.0
C17:06.1 10.3 5.8
C18:01.9 1.8 1.4
Unsaturated
C15:1ω8c 2.7 4.3 4.9
C16:1ω7c 30.6 29.0 32.1
C17:1ω8c 11.7 17.9 15.3
C18:1ω7c 11.0 5.2 7.5
Branched
iso-C16:01.0 tr tr
Hydroxy
C12:03-OH 4.8 6.3 7.5
C13:03-OH tr 1.2 1.0
Strains: 1, 16-SW-7; 2, P. distincta KMM 638T; 3, P. paragorgicola KMM 3548T.
All data are from the present study. Major components (≥5.0%) are highlighted in
bold. tr, trace amount (<1.0%).
and genomes of P. distincta and P. paragorgicola were 84.4
and 83.5%, respectively (Supplementary Table 3), that are
sufficiently higher than the suggested species boundary of 70%
(Wayne et al., 1987). The proteome-wide sequence search results,
implemented by a web server AAI-profiler (Medlar et al.,
2018), showed 98.6 (87% matched proteins) and 98.8% (85.9%
matches) average amino acid identity (AAI) of the strain 16-
SW-7 with P. distincta and P. paragorgicola, which are higher
than 85% proposed as a threshold for delimitation of a species
(Goris et al., 2007).
The target count for P. paragorgicola proteins showed
98.8 and 98.5% AAI (83.5 and 83.1% matches) against the
proteins of Pseudoalteromonas sp. 16-SW-7 and P. distincta,
respectively. The calculated genetic similarities are presented in
Supplementary Tables 1–3. These values support the proposed
affiliation of the three strains to a single species of the genus
Pseudoalteromonas.
Genome Features and Comparative
Genomics
The core-gene content (about 3200 genes according to
EzBioCloud) and coding sequences (CDSs) similarity confirmed
the affiliation of the strains 16-SW-7 and P. paragorgicola KMM
3548Tto the species P. distincta (Supplementary Figures 3, 4
and Supplementary Tables 4–6). They include genes for use
of some common mechanisms to respond to high salt stress,
such as a high-affinity choline and betaine uptake system
(Supplementary Table 4, Column 1: lines 769, 898, 1257, 2699,
3969–3974), the doubled genes for the glutamate synthase
small and large subunits (Supplementary Table 4, Column
1: lines 1761, 3562, 2003, 2033), K+transporters Trk H/A
(Supplementary Table 4, Column 1: lines 3064, 3069), other
ABC transporters/permeases, and transcription factors (Fu et al.,
2014). However, analysis of the subsystem features, annotated by
RAST, indicated the presence of some individual functions for
the strains 16-SW-7, P. distincta KMM 638T(=ATCC 700518T),
and P. paragorgicola KMM 3548T(Table 4).
The RAST comparative genomics revealed 242 and 426
strain-specific CDSs (singletons) in the strain 16-SW-7
against the strains KMM 638T(=ATCC 700518T) and KMM
3548T, respectively, most of which encoded hypothetical
proteins, and only 40 and 50 genes, respectively, had a
function (Supplementary Table 4). Meanwhile, 1378 and
1229 CDSs of KMM 638T(=ATCC 700518T) and KMM
3548T, respectively, had 100% identity with the CDSs of 16-
SW-7 (Supplementary Table 5). In the genome of the strain
KMM 3548T, 1259 identical CDS and 481 singletons were
found vs. the strain KMM 638T(ATCC 700518T), and 420
singletons vs. the strain 16-SW-7 (Supplementary Table 6).
All three strains putatively have xylan and xylose degradation
specialization, but differ from each other by exopolysaccharide
and lipopolysaccharide biosynthesis pathways (Table 4
and Supplementary Tables 4, 6), which are known to be
responsible for serotypes in clinical bacterial strains (Balabanova
et al., 2020). Thus, the strain 16-SW-7 exclusively contains
the doubled genes encoding for GDP-mannose mannosyl
hydrolase, phosphomannomutase, and several sugar and
glycosyltransferases, as well as the low-identical capsular
polysaccharide synthases (up to 78–79%) of the type strain
KMM 638T(ATCC 700518T): A, B, C, D, UDP-glucose 4-
epimerase, lipid carrier: UDP-N-acetylgalactosaminyltransferase,
O-antigen acetylase, N-acetylneuraminate cytidylyltransferase
(sialic acid synthesis), polysaccharide deacetylase, dTDP-4-
dehydrorhamnose reductase, and specific lipoproteins which are
absent in KMM 3548T(Supplementary Tables 4, 6). Meanwhile,
KMM 3548Tincludes many genes for rhamnosyltransferases,
some of which are similar only to KMM 638T(ATCC 700518T),
and related hydrolases, lipoproteins, and capsular polysaccharide
synthesis and export systems (Supplementary Table 6). In
addition, 16-SW-7 and KMM 638T(ATCC 700518T) differ
from KMM 3548Tby some genes responsible for xanthine and
gluconate metabolism, carbon starvation, catechol pathway, and
phosphate metabolism (Supplementary Table 4, Column 1:
lines 3684–3691). Contrarily, the strain KMM 3548Thas almost
all genes found in the strains 16-SW-7 and ATCC 700518T,
but mostly distinguishes from them by the large number of
mobile elements, transposases, integrases, chaperone proteins
of heat shock (HtpG), components of fatty acid synthases,
enlarged pectin degradation system, and hypothetical proteins
of unknown function (Supplementary Table 6). In general,
the different level of identity and numbers of the genes for
motility functions, metal resistance, TonB-related receptors,
transporters, beta-lactamases, bactericins, signal transduction
(sensors, receptors, transporters, enzymes), mobile elements,
and DNA repair systems reflect eco-physiological diversity and
different adaptive lifestyles of the free-living 16-SW-7, and
host-associated KMM 3548Tand KMM 638T(ATCC 700518T)
(Table 4 and Supplementary Tables 4–6). Indeed, KMM 638T
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Nedashkovkaya et al. Genome-Based Reclassification of Pseudoalteromonas distincta
TABLE 3 | The 16S rRNA gene sequences content and similarity for the strains Pseudoalteromonas sp. 16-SW-7, P. distincta ATCC 700518T, and P. paragorgicola
KMM 3548T.
IMG homolog* NCBI homolog genome
locus_tag (16S rRNA gene)
Identity % Identity/length Genome ID Genome name Contig/length Coordinates/strand
2888223316 FFU37_04590 (OL587469) 100.00 1536/1536 CP040558 Pseudoalteromonas sp.
16-SW-7
1(L1)/3735685 1023946..1025481/+
2888222433 FFU37_00210 (OL587468) 100.00 1536/1536 CP040558 Pseudoalteromonas sp.
16-SW-7
1(L1)/3735685 48439..49974/+
2888226108 FFU37_18380 (OL587475) 99.94 1535/1536 CP040559 Pseudoalteromonas sp.
16-SW-7
2(S1)/795760 375599..377134/+
2888225489 FFU37_15325 (OL587473) 99.94 1535/1536 CP040558 Pseudoalteromonas sp.
16-SW-7
1(L1)/3735685 3416198..3417733/−
2888225372 FFU37_14755 (OL587472) 99.94 1535/1536 CP040558 Pseudoalteromonas sp.
16-SW-7
1(L1)/3735685 3277653..3279188/−
2888225547 FFU37_15610 (OL587474) 99.87 1534/1536 CP040558 Pseudoalteromonas sp.
16-SW-7
1(L1)/3735685 3477632..3479167/+
2888225264 FFU37_14215 (OL587471) 99.87 1534/1536 CP040558 Pseudoalteromonas sp.
16-SW-7
1(L1)/3735685 3172769..3174304/−
2888222403 FFU37_00060 (OL587467) 99.87 1534/1536 CP040558 Pseudoalteromonas sp.
16-SW-7
1(L1)/3735685 16934..18469/+
2888224778 FFU37_11820 (OL587470) 99.74 1533/1536 CP040558 Pseudoalteromonas sp.
16-SW-7
1(L1)/3735685 2652584..2654119/−
– QT16_19995 99.94 1542/1543 JWIG01000030 P. distincta strain ATCC
700518T
C30/180150 175393..176935/+
– PPAR_aR004 99.87 1522/1524 AQHE01000014 P. paragorgicola KMM
3548T
14/767276 187495..189018/+
– PPAR_aR007 100.00 1524/1524 AQHE01000021 P. paragorgicola KMM
3548T
21/300025 298305..299828/+
*From the alignment on query gene of the strain 16-SW-7 under the IMG/M database accession number 2888222433 (FFU37_00210/ OL587468), implemented by Top
IMG Isolate RNA hits or NCBI BLAST to get top RNA homologs.
FIGURE 1 | Tree inferred with FastME 2.1.6.1 from GBDP distances calculated from genome sequences by the TYGS server (Lefort et al., 2015). The branch lengths
are scaled in terms of GBDP distance formula d5. The numbers above the branches are GBDP pseudo-bootstrap support values >60% from 100 replications, with
an average branch support of 92.7%. The tree was rooted at the midpoint (Farris, 1972). The square labels colored by the same color indicate the genomes with the
same species (light lilac) and subspecies (dark lilac) gene clusters, G+C content (white), and delta statistics: Pseudoalteromonas_GCF_005877035.1 (the strain
16-SW-7), P. distincta ATCC 700518T, and P. paragorgicola DSM 26439Tform one phylogenomic clade separated from other species of the genus.
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TABLE 4 | Genome-based comparison for the presence/absence of metabolic subsystem functions in Pseudoalteromonas sp. 16-SW-7 (A), P. distincta ATCC 700518T
(B), and P. paragorgicola KMM 3548T(C).
Category Subcategory Subsystem Function A B C
Amino acids and derivatives Lysine, threonine,
methionine, and cysteine
Methionine biosynthesis
(MB)
Homoserine O-acetyltransferase (EC
2.3.1.31), MB subpathway
+ − −
Amino acids and
derivatives; protein
metabolism
Arginine; urea cycle,
polyamines
Urea decomposition; G3E
family of P-loop GTPases
(metallocenter biosynthesis)
Urea ABC transporter, ATPase protein
UrtD, UrtE, UrtB, UrtC; Urease
accessory proteins UreD, UreE, UreF,
UreG; Urease alpha, beta, and gamma
subunits (EC 3.5.1.5)
− + −
Carbohydrates Monosaccharides Mannose Metabolism GDP-mannose mannosyl hydrolase (EC
3.6.1.), Phosphomannomutase (EC
5.4.2.8)
+ − −
Cell wall and capsule Capsular and extracellular
polysaccharides
Rhamnose containing
glycans; dTDP-rhamnose
synthesis
Alpha-1,2(1,3)-L-rhamnosyltransferase
(EC 2.4.1.); polysialic acid transporter
KpsM; dTDP-4-dehydrorhamnose
3,5-epimerase (EC 5.1.3.13) and
reductase (EC 1.1.1.133);
dTDP-rhamnosyltransferase RfbF
− + −
DNA metabolism DNA repair DNA repair, bacterial
RecBCD pathway
RecD-like DNA helicase Atu2026
(exodeoxyribonuclease V)
+ − −
DNA metabolism DNA repair DNA repair, bacterial DNA-cytosine methyltransferase (EC
2.1.1.37), modulates gene expression,
a component of bacterial
restriction-modification systems
+ − −
DNA metabolism No subcategory Restriction-modification
system
Type III restriction-modification system
methylation and helicase subunits (EC
2.1.1.72), host-protective DNA
methylation
− − +
DNA metabolism No subcategory Restriction-modification
system
Putative DNA-binding protein in cluster
with Type I restriction-modification
system
−+−
Fatty acids, lipids, and
isoprenoids
No subcategory Polyhydroxybutyrate
metabolism
D-beta-hydroxybutyrate permease
(utilization of poly-HB, gluconate)
+− −
Membrane transport Protein secretion system,
Type II
Widespread colonization
island
Flp pilus assembly protein, pilin Flp − − +
Membrane transport TRAP transporters TRAP transporter collection TRAP-type C4-dicarboxylate transport
system, uptake host’s succinate,
fumarate, and malate during symbiotic
growth
− − +
Membrane transport;
virulence and defense
Cation transporters;
resistance to toxic
compounds
Copper transport system;
Cu2+homeostasis
Copper resistance proteins CopC,
CopD, CopB; multicopper oxidase
− + −
Nucleosides and
nucleotides
Detoxification Housecleaning nucleoside
triphosphate
pyrophosphatases
Deoxyuridine 50-triphosphate
nucleotidohydrolase (EC 3.6.1.23),
remove dUTP for preservation of
genetic integrity for growth and
virulence
− − +
Regulation and cell
signaling
Programmed cell death and
toxin-antitoxin systems
Phd-Doc, YdcE-YdcD
toxin-antitoxin
(programmed cell death)
systems
Death on curing protein, doc toxin
[mimicker of aminoglycoside antibiotic
hygromycin B (HygB), increase in
mRNA half-life]
− − +
Regulation and cell
signaling
No subcategory DNA-binding regulatory
proteins, strays
Aromatic hydrocarbon utilization
transcriptional regulator CatR (LysR
family)
− − +
Regulation and cell
signaling
No subcategory Orphan regulatory proteins Sensor kinase CitA, DpiB (EC 2.7.3.-),
involved in anaerobic citrate catabolism
in response to anaerobic conditions
−+−
Stress response Cold shock Cold shock, CspA family of
proteins
Cold shock protein CspD − − +
Stress response Osmotic stress Choline and betaine uptake
and betaine biosynthesis
GbcA glycine betaine demethylase,
transporter OpuD [utilization of
quaternary ammonium compounds at
high osmolalities (kidneys)]
−+−
RNA metabolism RNA processing and
modification
Ribonucleases in bacillus Metallo-beta-lactamase family protein,
RNA-specific
−+−
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Nedashkovkaya et al. Genome-Based Reclassification of Pseudoalteromonas distincta
(ATCC 700518T) has acquired the additional pathogenic-like
systems for osmotic stress response, urea decomposition, and
sensor kinase CitA (Table 4), indicating an ability to live in the
host tissues with a high osmolality and use citrate fermentation
under anaerobic conditions (Wetzel et al., 2011;Scheu et al.,
2012). KMM 3548Trather contains rhizobia-like symbiotic traits
(Table 4), such as TRAP-type C4-dicarboxylate transport system
for facultatively anaerobically fumarate and nitrate respiration
(Janausch et al., 2002), aromatic hydrocarbon utilization
regulator CatR, Type III restriction-modification system that
confers host DNA protection via methylation (Murray et al.,
2021). Meanwhile, the free-living strain 16-SW-7 is enforced
by the modification and repair systems of its own DNA and
methylation-dependent biosynthetic pathways, such as two
bacteriocin BGCs (found by antiSMASH), defending it from UV
excess, environmental competitors, and facilitating the rapid
genetic rearrangement (Table 4).
In addition, the strains KMM 638T(ATCC 700518T) and
KMM 3548Tproduce brown and lightly orange pigments,
respectively, in comparison with the colorless free-living 16-
SW-7 colonies, probably due to its active dye-decolorization
peroxidase (Supplementary Table 4, Column 1: 2637) or
inactive pigment-producing genes. P. distincta KMM 638T
(ATCC 700518T) was reported to produce diffusible dark-gray-
colored melanin-like pigment (pyomelanin) (Romanenko et al.,
1995), the responsible genes of which are found in each strain
(Supplementary Table 4, column 1: 2123, 3887). For many
pathogens, melanin production is associated with virulence
and completely suppressed at 30–35◦C (Turick et al., 2009).
According to the gene content (Supplementary Tables 4–6),
P. distincta can produce different colored polyketide secondary
metabolites from simple fatty acids (pigments, antioxidants,
toxins), which increase virulence for many pathogens by
improving its intracellular survival (Woo et al., 2012;Hug
et al., 2020). Probably, KMM 638T(ATCC 700518T) and
KMM 3548Tdeveloped and fixed their pigment-producing
phenotypic traits due to colonizing psychrophilic marine
organisms (Romanenko et al., 1995;Ivanova et al., 2002a).
Thus, the slightly orange pigments in KMM 3548Tcould belong
to antioxidant xanthomonadin-like pigments (carotenoids,
anthraquinones, zeaxanthin, flexirubins), which protect against
oxidative stress and UV exposure, establishing or maintaining
commensal relationships between bacteria and their hosts
(He et al., 2020). At least, the resorcinol and arylpolyene
biosynthetic gene clusters (66209 nucleotide region), which are
functionally related to antioxidative carotenoids, were found in
the KMM 3548Tgenome by antiSMASH (Medema et al., 2011;
Schöner et al., 2016).
In any case, all three genomes of P. distincta possess many
polyketide synthases and concomitant proteins with a high level
of identity (97–100%) (Supplementary Tables 4–6). The dark
shade of pigments in KMM 638T(ATCC 700518T) may be due
to the degree of expansion of the pyomelanin production and
weakening xanthine metabolism by some genes lost, compared
to the KMM 3548Tpathway (Supplementary Table 6).
In conclusion, based on the results of the above phylogenetic,
phenotypic, and chemotaxonomic study, we suggest that the
strain 16-SW-7 is affiliated to the species P. distincta. Moreover,
the high similarities in the genomic sequences and phenotypic
characteristics found between the species P. distincta and
P. paragorgicola places them in the same species. Therefore,
it is proposed to reclassify the species P. paragorgicola as
a later heterotrophic synonym of P. distincta in accordance
with the rules of priority of prokaryotic names, governed
by the International Code of Nomenclature of Bacteria
(Parker et al., 2019), and to emend the description of the
species P. distincta.
Emended Description of the Species
Pseudoalteromonas distincta
(Romanenko et al. 1995) Ivanova et al.
2000
The description of the species Pseudoalteromonas distincta is as
given by Romanenko et al. (1995) and Ivanova et al. (2000, 2002a)
with the following modifications and amendments. Cells are
Gram-stain-negative, non-spore-forming, strictly aerobic rods,
motile by means of a single polar or four to seven lateral flagella.
On marine agar, colonies are non-pigmented or slightly orange
colored. They can produce diffusible melanin-like pigments. Cells
are catalase- and oxidase-positive. They require Na+ions or
sea water for growth. Growth occurs in media with 0.5–10%
NaCl. Temperature for growth ranges from 4 to 34◦C. Aesculin,
gelatin, Tweens 20, 40, and 80, DNA, alginate, and tyrosine
are hydrolyzed but agar, chitin, CM-cellulose, and urea are not
hydrolyzed. Hydrolysis of casein and starch is strain dependent.
Acid is formed from sucrose but not from L-arabinose,
D-fructose, D-mannose, D-melibiose, L-rhamnose, D-ribose,
D-trehalose, N-acetylglucosamine, and glycerol. Some strains
can produce acid from D-cellobiose, D-galactose, D-glucose,
D-lactose, maltose, D-raffinose, D-xylose, and D-mannitol and
utilize citrate. In API ID 32GN gallery, they are positive for D-
glucose, maltose, sucrose, D-mannitol, sodium acetate, sodium
citrate, L-alanine, L-serine, L-proline, glycogen, propionic acid,
valeric acid, and capric acids. Assimilation of inositol, sodium
malonate, lactic acid, D-ribose, 3-hydroxybutyric acid, itaconic
acid, potassium-2-keto-gluconate, L-histidine, and salicin is
variable. In API ZYM gallery, alkaline phosphatase, esterase lipase
(C8), leucine arylamidase, valine arylamidase, acid phosphatase,
and naphthol-AS-BI-phosphohydrolase activities are present but
lipase (C14), α-galactosidase, β-galactosidase, β-glucuronidase,
α-glucosidase, β-glucosidase, N-acetyl-β-glucosaminidase, α-
mannosidase, and α-fucosidase activities are absent. Esterase
(C4), cysteine arylamidase, trypsin, and α-chymotrypsin can
be produced. Nitrate is not reduced to nitrite. Acetoin and
indole are not produced. Production of hydrogen sulfide is
strain dependent. The predominant fatty acids (>5% of the total
fatty acids) were C16:1ω7c, C16:0, C17:1ω8c, C18:1ω7c, C17:0,
and C12:03-OH. The polar lipid profile was characterized by
the presence of phosphatidylethanolamine, phosphatidylglycerol,
two unidentified amino lipids, and three unidentified lipids.
The major respiratory quinone is ubiquinone Q-8. The genomic
DNA G+C content is 39.2–39.3 mol%. The genome size is
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Nedashkovkaya et al. Genome-Based Reclassification of Pseudoalteromonas distincta
4.3–4.5 Mb. The type of strain is KMM 638T(=ATCC 700518T),
isolated from a marine sponge collected at a depth of 350 m near
the Komandorskie Islands, Russia. The GenBank/EMBL/DDBJ
assembly accession number for the genome of the type of strain
is GCA_000814675.1.
DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in
online repositories. The names of the repository/repositories
and accession number(s) can be found in the article/
Supplementary Material.
AUTHOR CONTRIBUTIONS
ON and LB contributed to conception and designed of the study.
ON, S-GK, LB, and NZ performed the experimental works.
ON, S-GK, LB, NZ, OS, LT, and VM wrote sections of the
manuscript. All authors contributed to manuscript revision, read,
and approved the submitted version.
FUNDING
This research was funded by a grant from the Ministry of Science
and Higher Education, Russian Federation 15.BRK.21.0004
(Contract No. 075-15-2021-1052).
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fmicb.
2021.809431/full#supplementary-material
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