Description and genome-wide analysis of Profundicola chukchiensis gen. nov., sp. nov., marine bacteria isolated from bottom sediments of the Chukchi Sea

Two Gram-negative, aerobic halophilic non-motile strains designated KMM 9713 and KMM 9724T were isolated from the bottom sediments sampled from the Chukchi Sea in the Arctic Ocean, Russia. The novel strains grew in 0.5−5% NaCl, at 7−42°C, and pH 5.5−10.5. Phylogenetic analyses based on 16S rRNA gene and whole genome sequences revealed that strains KMM 9713 and KMM 9724T were close to each other and shared the highest 16S rRNA gene sequence similarity of 91.28% with the type strain Ornithobacterium rhinotracheale DSM 15997T and 90.15–90.92% with the members of the genus Empedobacter in the family Weeksellaceae. Phylogenetic trees indicated that strains KMM 9713 and KMM 9724T formed a distinct line adjacent to their relative O. rhinotracheale DSM 15997T. The average nucleotide identity values between strain KMM 9724T and O. rhinotracheale DSM 15997T, Empedobacter brevis NBRC 14943T, and Moheibacter sediminis CGMCC 1.12708T were 76.73%, 75.78%, and 74.65%, respectively. The novel strains contained the predominant menaquinone MK-6 and the major fatty acids of iso-C17:0 3-OH, iso-C15:0 followed by iso-C17:1ω6. Polar lipids consisted of phosphatidylethanolamine, one an unidentified aminophospholipid, two unidentified aminolipids, and two or three unidentified lipids. The DNA G+C contents of 34.5% and 34.7% were calculated from genome sequence of the strains KMM 9713 and KMM 9724T, respectively. Based on the phylogenetic evidence and distinctive phenotypic characteristics, strains KMM 9713 and KMM 9724T are proposed to be classified as a novel genus and species Profundicola chukchiensis gen. nov., sp. nov. The type strain of Profundicola chukchiensis gen. nov., sp. nov. is strain KMM 9724T (= KACC 22806T).


Introduction
The peculiarity of the Chukchi Sea among the Arctic seas is its high-latitude location, which is reflected in the presence of ice cover most of the time during the year and low average annual water temperature. Even in summer, the temperature of water layers deeper than 10-12 m remains almost at zero values. The bottom of the Chukchi Sea is flat, the average depth of the continental shelf is 50-60 meters, and the depth of shoals is 20-30 m. Therefore, studies of microorganisms dwelling in the arctic marine sediments provide insight into their genetic capabilities to live in extreme habitat conditions. The members of the family Flavobacteriaceae [1−3] of the phylum Bacteroidota constitute one of the dominant bacterial groups which have been reported to be widespread microorganisms inhabiting marine environments [4,5]. The family Flavobacteriaceae contains a large number of species and genera while the phylogenetic relationships on the basis of the 16S rRNA gene sequences between some of them were not completely resolved until recently [6]. Phylogenomic studies based on whole genome sequencing analysis of 1000 type strains of the phylum Bacteroidota (formerly Bacteroidetes) have shown that the family Flavobacteriaceae is non-monophyletic and therefore should be divided [7]. As a result, a new family Weeksellaceae has been proposed to include the genera Algoriella, Apibacter, Bergeyella, Chishuiella, Chryseobacterium, Cloacibacterium, Cruoricaptor, Elizabethkingia, Empedobacter, Moheibacter, Ornithobacterium, Riemerella, Wautersiella, and Weeksella as the type genus that formed a separate clade from the type genus Flavobacterium of the family Flavobacteriaceae [7]. At the time of writing, the family Weeksellaceae comprises 15 genera with correct and validly published names (https://lpsn.dsmz.de/family/weeksellaceae, accessed on 15 May 2023).
During a study on the bacterial biodiversity of the bottom sediments of the Chukchi Sea in the Arctic Ocean, two Gram-negative, aerobic, yellowish-pigmented, non-motile bacteria, KMM 9713 and KMM 9724 T were recovered and investigated by using phenotypic and molecular methods; the results obtained are reported in this study. Phylogenetic analyses demonstrated that strains KMM 9713 and KMM 9724 T formed a distinct lineage within the family Weeksellaceae adjacent to the type bacterium Ornithobacterium rhinotracheale DSM 15997 T . Based on the phylogenomic analyses data and distinctive phenotypic characteristics, a novel genus and species Profundicola chukchiensis gen. nov., sp. nov. is described to accommodate the novel marine isolates KMM 9713 and KMM 9724 T .

Bacterial strains
Strains KMM 9713 and KMM 9724 T were isolated from a deep bottom sediment sampled at a depth of 29 m from the Chukchi Sea (70˚59.60 0 N, 177˚35.8 0 W, near Wrangel Island), Russia, during the expedition of R/V Academician Oparin, in September 2016, as described previously [8].

Chemotaxonomic analyses
Strains KMM 9713 and KMM 9724 T and the type strain Empedobacter tilapiae KCTC 62904 T were grown on MA 2216 at 30˚C. Lipids were extracted using the method of Folch et al. [13]. Two-dimensional thin layer chromatography of polar lipids was carried out on Silica gel 60 F 254 (10 x 10 cm, Merck, Germany) using chloroform-methanol-water (65:25:4, v/v) for the first direction, and chloroform-methanol-acetic acid-water (80:12:15:4, v/v) for the second one [14] and spraying with specific reagents [15]. Fatty acid methyl esters (FAMEs) were prepared according to the procedure of the Microbial Identification System (MIDI) [16]. The analysis of FAMEs was performed using the GC-2010 chromatograph (Shimadzu, Kyoto, Japan) equipped with capillary columns (30 m x 0.25 mm I.D.), one coated with Supecowax-10 and the other with SPB-5. Identification of FAMEs was accomplished by equivalent chain length values and comparing the retention times of the samples to those of standards. In addition, FAMEs were analyzed using a GC-MS Shimadzu QP2020 (column Shimadzu SH-Rtx-5MS, the temperature program from 160˚C to 250˚C, at a rate of 2˚C/min). Menaquinones fraction was isolated using liquid column chromatography on silica gel. The lipid extract in chloroform was applied to the column, and the neutral lipid fraction with menaquinones was washed off with three column volumes of chloroform. Analysis was performed using GC-MS with SH-Rtx-5ms column, the temperature was programed from 200˚C to 240˚C, (10˚C/min), then from 240˚C to 325˚C, (3˚C/min) and kept for 30 min at 325˚C. The injector temperature was 300˚C, mass spectrometer scan range 50-1000 m/z. The presence of flexirubin pigments was investigated as described by Fautz and Reichenbach [17].

16S rRNA gene sequencing and phylogenetic analysis
Genomic DNAs of strains KMM 9713 and KMM 9724 T were extracted using a commercial genomic DNA extraction kit (Fermentas, EU) following the manufacturer's instruction. The 16S rRNA genes were PCR-amplified and sequenced as described in a previous paper [18]. The 16S rRNA gene sequences of the strains KMM 9713 and KMM 9724 T (1393 and 1401 bp, respectively) were compared with those of the closest relatives using the BLAST (http://www. ncbi.nlm.nih.gov/blast/, accessed on 15 May 2023) and EzBioCloud service [19]. Model testing and phylogenetic analysis were conducted using Molecular Evolutionary Genetics Analysis (MEGA X, version 10.2.1) [20]. Phylogenetic trees were constructed by the neighbor-joining and the maximum-likelihood methods, and the distances were calculated according to the Kimura two-parameter model [21]. The robustness of phylogenetic trees was estimated by the bootstrap analysis of 1000 replicates.

Whole-genome sequencing, phylogenomic, and comparative analyses
The genomic DNAs were obtained from the strains KMM 9713 and KMM 9724 T using the High Pure PCR Template Preparation Kit (Roche, Basel, Switzerland). The quantity and quality of the genomic DNA was measured using DNA gel electrophoresis and the Qubit 4.0 Fluorometer (Thermo Fisher Scientific, Singapore, Singapore). The DNA sequencing libraries were prepared using Nextera DNA Flex kits (Illumina, San Diego, CA, USA) and subsequently sequenced using paired-end (2 x 150 bp) runs on an Illumina MiSeq platform. The reads were trimmed using Trimmomatic version 0.39 [22] and their quality assessed using FastQC version 0.11.8 (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/, accessed on 21 August 2021). Contigs assembled with SPAdes version 3.15.3 [23] were used to calculated genome metrics with QUAST version 5.0.2 [24]. The genome completeness and contamination were estimated by CheckM version 1.1.3 [25] based on the taxonomic-specific workflow (lineage Flavobacteriales).
The draft genome assemblies were annotated using NCBI Prokaryotic Genome Annotation Pipeline (PGAP) and Rapid Annotation using Subsystem Technology (RAST) [26,27]. Comparisons of the Average Nucleotide Identity (ANI), Average Amino Acid Identity (AAI), and digital DNA-DNA hybridization (dDDH) values of the strains KMM 9713 and KMM 9724 T and their closest neighbors were performed with the online server ANI/AAI-Matrix [28] and TYGS platform [29], respectively. The phylogenomic analysis was performed using PhyloPh-lAn software version 3.0.1 based on a set of 400 conserved bacterial protein sequences [30].

Nucleotide sequence accession number
The 16S rRNA gene sequence and genome sequence of strains KMM 9724 T and KMM 9713 were deposited in GenBank/EMBL/DDBJ under the accession numbers OP604014 and LC379507, and JANAIE010000000.1 and JANCMU010000000.1, respectively. Strain KMM 9724 T was deposited in the Korean Agricultural Culture Collection (KACC) under the number of KACC 22806 T .

Phylogenetic and phylogenomic analyses
The average nucleotide identity (96.9%) and DNA-DNA hybridization (74.4%) values obtained on the basis of whole genome sequence comparison between two novel strains KMM 9713 and KMM 9724 T confirmed their assignment to the same species. Comparative 16S rRNA gene sequence analysis showed that the novel strains KMM 9713 and KMM 9724 T belong to the family Weeksellaceae (phylum Bacteroidota) and their close phylogenetic neighbors were found to be Ornithobacterium rhinotracheale DSM 15997 T with 90. 22 (Fig 2).
The ANI and dDDH values between strain KMM 9724 T and O. rhinotracheale DSM 15997 T , Empedobacter brevis NBRC 14943 T , and Moheibacter sediminis CGMCC 1.12708 T were 76.73%, 75.78%, and 74.65%, and values of 20.5%, 18.9%, and 21.5%, respectively ( Fig  3A, 3C). These values obtained were significantly lower than the ANI and dDDH values of 95 −96% and 70%, respectively, which have been accepted for bacterial species discrimination [34]. The AAI values between genomes of both novel strains and related bacteria O. rhinotracheale DSM 15997 T , E. brevis NBRC 14943 T , and M. sediminis CGMCC 1.12708 T ranged from 60.28% to 52.73% (Fig 3B). These values fall into the range of AAI values between 45% and 65% proposed by Konstantinidis et al. [35] to delineate bacterial genera. The phylogenomic analysis data evidence that the strains KMM 9713 and KMM 9724 T do not belong to any of recognized genera and could be classified as an individual genus and species of the family Weeksellaceae.

Genomic characteristics and comparative analysis
The whole genome sequences of strains KMM 9724 T and KMM 9713 were determined using Illumina MiSeq platform. Both genomes were obtained at high completeness (99.01%) without contamination according to the CheckM evaluation. The 16S rRNA sequences extracted from the genomes were identical to those obtained by PCR amplification. The draft genomes were de novo assembled into 108 and 47 contigs, with a N50 values of 440,336 and 448,720 bp, a L50 values of 3 and 2, respectively. The genome sizes were estimated at 2.62 and 2.67 Mbp in length with coverage of 180 X and 140 X, respectively. The genome sequences were in accordance with the proposed minimal standards for the bacterial taxonomy [34]. The genome sequences contain a total of 2,366 and 2,348 genes, 37 and 38 tRNAs, and 3 rRNA genes (one each of 5S, 16S, and 23S).
Based on RAST annotation, the genome of type strain KMM 9724 T showed the presence of 220 functional subsystems, of which the largest number of annotated genes, about 49%, were assigned to three subsystems: "Protein Metabolism" (149 genes), "Amino Acids and Derivatives" (135 genes), and "Cofactors, Vitamins, Prosthetic Groups, Pigments" (119 genes). Among peptidases for protein degradation were predicted aminopeptidases (EC 3.

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Annotation of bacterial protein secretion systems in the genomes was conducted with Mac-SyFinder (TXSScan) program. It was shown that strains KMM 9724 T and KMM 9713 have all mandatory and accessory genes for the type IX secretion system (T9SS), such as gldK, gldL, gldM, gldN, porV, sprA, sprE, sprT, and gldJ, porU, porQ, respectively. Interestingly, the T9SS is

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exclusively present in the majority of families of the phylum Bacteroidota and also required for gliding motility of bacterial cells, though it was first detected in the non-motile human pathogen Porphyromonas gingivalis [36,37]. As known, T9SS substrates are cell-associated proteins that contain a C-terminal domain (CTD) and are involved in virulence, gliding motility, and the degradation of complex biopolymers [38]. Among predicted substrates of the T9SS strains KMM 9724 T and KMM 9713 possess a set of endonucleases, peptidases S8 and M1, reprolysins, adhesins, Cu-binding proteins, and other Por_Secre_tail (pfam: PF18962.3) containing proteins. This may imply that the strains are able to consume complex biopolymers of sediments in the Chukchi Sea as a specific metabolic strategy. Another secretion system identified in the genomes was type I secretion system (T1SS), which is widespread in Gram-negative bacteria. It provides a one-step secretion of substrates across two membranes without any periplasmic intermediate into the extracellular space [39]. The genomes of the KMM 9724 T and KMM 9713 encode from two to five copies of each mandatory gene abc, mfp, and omf.
Whole-genome comparative analysis of novel strains and representatives of six related genera from the same clade on the phylogenomic tree (Fig 2) was performed using orthologous clustering with OrthoVenn2 server. According to the pairwise genome comparisons (Fig 4A), the strains KMM 9724 T and KMM 9713 share the most of orthologous protein clusters of 3167 with each other, while those values between studied strains with type strains of genera Algoriella, Chishuiella, Moheibacter, Weeksella, Empedobacter, and Ornithobacterium were from 2705 to 2711. However, despite on an almost equal distribution of shared clusters among strains KMM 9724 T and KMM 9713, and representatives of related genera, their phylogenetically closest relative genus is Ornithobacterium.
To clarify genus-related features, the orthologous clustering analysis of strains KMM 9724 T , KMM 9713, and O. rhinotracheale DSM 15997 T was conducted (Fig 4B). The analysis revealed

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that strains form 2218 orthologous gene clusters (gene families), of which 1011 orthologous clusters (at least contains two strains) and 1207 single-copy gene clusters. It was shown that presumably genus-specific genes of the strain O. rhinotracheale DSM 15997 T are represented by 64 clusters of orthologs formed by 178 paralogous genes and 665 single-copy genes. The strains KMM 9724 T and KMM 9713 share 901 clusters of 1808 genes, including paralogs, which are likely genus-and species-specific. A close inspection of these genes revealed that most of genes were assigned to following biological processes: biological processes (GO:0008150, 11.3%), metabolic processes (GO:0008152, 10.9%), cellular metabolic processes (GO:0044237, 8.8%), and nitrogen compound metabolic processes (GO:0006807, 6%). Among gene ontology annotations of molecular function activities of transferases, transporters, oxidoreductases, and hydrolases were enriched. In addition to shared species-specific genes, interstrain differences between KMM 9724 T and KMM 9713 were also identified. As such, the type strain KMM 9724 T has 9 unique clusters of 22 paralogous genes and 220 single-copy genes, and strain KMM 9713 has 8 unique clusters of 19 paralogous genes and 211 single-copy genes.

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To elucidate intra-species differences the unique single-copy genes of the strains were functional annotated via eggNOG-mapper v2 server (Fig 4C). Genes were assigned to the following COG categories: C, energy production and conversion; D, cell cycle control and mitosis; E, amino acid metabolism and transport; F, nucleotide metabolism and transport; G, carbohydrate metabolism and transport; H, coenzyme metabolism, I, lipid metabolism; J, translation; K, transcription; L, replication and repair; M, cell wall/membrane/envelop biogenesis; N, cell motility; O, post-translational modification, protein turnover, chaperone functions; P, inorganic ion transport and metabolism; Q, secondary structure; T, signal transduction; V, defense mechanisms; S, function unknown. According to the COG classes annotation of these unique genes, the most abundant functional classes in both strains were "transcription", "replication and repair", and "cell wall/membrane/envelop biogenesis".
Apparently, these genes may be responsible for adaptation to extreme conditions and let this species to inhabit the Chukchi Sea in the Arctic Ocean. It can be concluded that metabolism is mainly carried out by consumption protein-containing substrates rather than carbohydrates, since both strains KMM 9724 T and KMM 9713 encode a few periplasmic glycoside hydrolases ( Fig 4D). Nevertheless, strains of novel species of the novel genus may be a source of biotechnological relevant pullulanases (GH13), peptidases and proteases.

Phenotypic characterization and chemotaxonomy
Bacteria KMM 9713 and KMM 9724 T were observed to be Gram-negative, aerobic, catalaseand oxidase-positive, non-motile. They formed yellowish colonies with regular edges of 1−3 mm in diameter on MA 2216. Electron microscopy images of the cells depicted ovoid or rodshaped morphology (Fig 5). The cell dimensions were 0.5−0.75 μm in width and 1.2−3.5 μm in length. Capsular material around cells can be produced.
The novel strains required sodium ions for growth and grew in the narrow salinity range of 0.5 −5% (w/v) NaCl and at a temperature between 7˚C and 42˚C. The novel bacteria were not able to hydrolyse a broad number of polymeric substrates (Table 1) and assimilate most carbon sources in API 32GN, API 20E, and API 20NE tests. Cultural, physiological, and biochemical characteristics of the novel bacteria are given in Table 1 and in the genus and species descriptions.
The phylogenetic relationships observed on the basis of 16S rRNA gene and whole genome sequences, and genetic distinctness as revealed by ANI and dDDH analyses were supported by phenotypic differences of the novel isolates KMM 9713 and KMM 9724 T in their growth temperature and salinity ranges, enzymes activity and substrate hydrolysis. Differential phenotypic  Table 1. Based on the combined phylogenetic evidence, phenotypic and biochemical characteristics, it is proposed to classify strains KMM 9713 and KMM 9724 T as a novel genus and species, Profundicola chukchiensis gen. nov., sp. nov. with the type strain of the type species KMM 9724 T (= KACC 22806 T ).
Cells are Gram-negative, non-motile, non-spore-forming and rod-shaped. Aerobic. The major fatty acids are iso-C 17:0 3-OH, iso-C 15:0 . The major polar lipid is phosphatidylethanolamine. The predominant menaquinone is MK-6. Isolated from the marine environment. Phylogenetically the genus Profundicola belongs to the family Weeksellaceae in the phylum Bacteroidota. The type species is Profundicola chukchiensis with the type strain KMM 9724 T .
The major menaquinone is MK-6. Major fatty acids are iso-C 17:0 3-OH, iso-C 15:0 followed by iso-C 17:1 ω6. Polar lipids consisted of phosphatidylethanolamine, one an unidentified aminophospholipid, two unidentified aminolipids, and two or three unidentified lipids. The DNA G+C content of 34.5−34.7% is calculated from the genome sequence.
The type strain of Profundicola chukchiensis gen. nov. sp. nov. is strain KMM 9724 T (= KACC 22806 T ). Isolated from a bottom sediment sampled from the Chukchi Sea in the Arctic Ocean, Russia. The DDBJ/ENA/GenBank accession numbers for the 16S rRNA gene and the whole-genome shotgun sequences of strains KMM 9724 T and KMM 9713 are OP604014 and LC379507, and JANAIE010000000.1 and JANCMU010000000.1, respectively.