Photobacterium halophilum sp. nov. and a Salt-Loving Bacterium Isolated from Marine Sediment

: A Gram-stain-negative, rod-shaped, and facultatively anaerobic bacterium named strain GJ3 T was isolated from coastal sediment of Jeju Island, South Korea. Catalase and oxidase activity were detected in the cell of strain GJ3 T , as well as white pigmented colony and motility with polar ﬂagellum. The cell grew optimally at 30 ◦ C, pH 7.0, in the presence of 4% ( w / v ) sodium chloride. Phylogenetic analysis using the 16S rRNA gene sequence indicated that strain GJ3 T was classiﬁed to the genus Photobacterium , with high sequence similarity to Photobacterium galatheae S2753 T (98.30%), Photobacterium halotolerans MACL01 T (97.90%), and Photobacterium panuliri LBS5 T (96.55%). Strain GJ3 T possessed only ubiquinone-8 (Q-8) as a respiratory quinone and summed feature 8 as the major fatty acid (38.18%). Additionally, the dominant polar lipids phosphatidylglycerol and phosphatidylethanolamine were identiﬁed. The complete genome size and G + C content of strain GJ3 T was estimated to be 3,603,274 bp in length and 50.70%, respectively. Polyphasic approach and genomic analyses (e.g., ANI and digital DDH) revealed that strain GJ3 T (=KCTC 72816 T = KMM 6822 T ) represented a novel species within the genus Photobacterium , and the name Photobacterium halophilum sp. nov., is proposed for this novel bacterium.


Introduction
The taxonomy study is a definition for nomenclature and classification for (microbial) organisms in nature ecosystem based on their shared properties, including 16S rRNA gene sequence [1,2]. To classify the organisms, the phenotypical properties were considered as a key distinguishing feature; however, it was dramatically included with genotypic characterizations (e.g., DNA-DNA relatedness) [3,4]. Then, the bacterial systematics (i.e., taxonomy study) is supposed as a consensually polyphasic approach [1]. Nonetheless, this polyphasic approach has a critical limitation that this research can apply to the already isolated (i.e., cultivated) microorganisms in the laboratory [5] or technical handling concerns (e.g., DNA-DNA hybridization) [6].
In the sequencing era, the taxonomic assignment for bacterial organisms has been challenged, resulting in reclassification of the incorrectly identified taxa and revisiting their ecological implications [7][8][9][10][11]. At the moment, as a genomic gold standard, genomes are dramatically applied into the definition for prokaryotic species. The average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) have been most frequently utilized in these sorts of studies [12][13][14]. Additionally, the value for the average amino acid identity (AAI) is employed for novel species validation [15].
During a culturable-bacterial diversity investigation of marine environments, including coastal sediments on Jeju Island, South Korea, we isolated a novel bacterium belonging to the genus Photobacterium, designated as GJ3 T . The genus Photobacterium belongs to the family Vibrionaceae and the phylum Proteobacteria. It was first proposed by Beijerink et al. [16] with the description of Photobacterium phosphoreum as the type species. At the time of this writing, the genus Photobacterium has 42 species with valid names (https://lpsn.dsmz.de/genus/photobacterium, accessed on 10 January 2022). The species of the genus Photobacterium have been mostly isolated from marine and aquatic environments, including samples associated with marine animals [17][18][19][20][21][22][23][24][25]. Members of this genus share common characteristics of being Gram-stain negative, facultative anaerobic, motile, and rod shaped with cells containing ubiquinone 8 (Q-8) as a major or sole respiratory quinone [16]. Additionally, some Photobacterium species are luminous due to the presence of the lux gene (luxCDABEG), and many marine fish species form bioluminescent through these Photobacterium species [26]. Therefore, in this study, we describe its taxonomic properties, including biochemical and genomics traits. Finally, we proposed the strain GJ3 T as a representative novel species of the genus Photobacterium.

Isolation and Culture Conditions
Strain GJ3 T was isolated from a coastal sediment sample on Jeju Island (March 2019), South Korea (GPS; 33 • 27 15 N, 126 • 18 26 E). The sample was collected into a falcon tube, stored in a container maintained at 4 • C by icepack, and transported to the laboratory. To isolate bacteria, a sediment sample was placed in a sterile 50-mL conical tube and slurried with artificial seawater (ASW) [27]. After serial dilution to 10 −5 , the diluted slurry was spread onto marine agar 2216 (MA; BD Difco) plates. For two weeks, these plates were incubated at 30 • C. Single colonies were transferred to new MA plates. Each colony was subcultured on MA plates at least five times to obtain a pure colony. Cells were subcultured and grown on MA at 30 • C for 3 days before being stored at −80 • C as a suspension in marine broth (MB; BD Difco) supplemented with 30% (w/v) glycerol for subsequent analyses. Based on 16S rRNA gene sequence similarity and phylogenetic position, Photobacterium galatheae S2753 T (LMG 28894 T ) and Photobacterium halotolerans MACL01 T (LMG 22194 T ) were selected and obtained from Belgian Coordinated Collections of Microorganisms (BCCM/LMG) as experimental reference strains for comparison with strain GJ3 T . Strain GJ3 T was deposited in the Korean Collection for Type Cultures (KCTC) and Collection of Marine Microorganisms (KMM) as KCTC 72816 T and KMM 6822 T , respectively. Unless otherwise stated, all strains were grown on MA medium under their optimal culture conditions. However, during paper writing after polyphasic experiments, Photobacterium salinisoil LAM9072 T has been validly published by Li et al. [28]. We conducted whole-genome-based taxonomic analyses (such as digital DNA-DNA hybridization (dDDH), average nucleotide identity (ANI), and average amino acid identity (AAI)) for genomes of P. salinisoli and the isolated strain GJ3 T (see DDH results and discussion on genomics relatedness).

Phylogenetic Analysis
To determine the phylogenetic position of strain GJ3 T , genomic DNA (gDNA) was extracted using a commercial gDNA extraction kit (GeneAll Biotechnology Co., Ltd., Seoul, Korea) according to the manufacturer's instructions. The 16S rRNA gene was amplified by polymerase chain reaction (PCR) using gDNA as template and universal bacterial primer set 27F (5 -AGAGTTTGATCMTGGCTCAG-3 ) and 1492R (5 -TACGGYTACCTTGTTACGACTT-3 ) as previously described [29]. For sequencing the amplified 16S rRNA gene, the PCR product was purified using a PCR purification kit (GeneAll Biotechnology Co., Ltd.) according to the manufacturer's instructions. Sequencing of the 16S rRNA gene was performed using primers 27F, 518F, 800R, and 1492R as described previously [29,30]. Finally, the 16S rRNA gene sequence of strain GJ3 T (1474bp) was obtained using SeqMan software (DNASTAR) and compared with sequences of related taxa obtained from the GenBank database (www.ncbi.nlm.nih.gov, accessed on 12 January 2022) and the EzBioCloud server (https://www.ezbiocloud.net, accessed on 12 January 2022). Sequences were edited and aligned using Clustal_X of BioEdit program [31]. Evolutionary distances were calculated using the Kimura two-parameter model [32]. The neighbor-joining [33], maximum parsi-mony [34], and maximum likelihood methods [35] were used to reconstruct phylogenetic trees in MEGA X software [36], with bootstrap values based on 1000 replications.

Chemotaxonomic Analyses
Whole-cell fatty acid methyl ester compositions of GJ3 T and two reference strains were determined using a 6890 N gas chromatograph (Agilent Technologies, Santa Clara, CA, USA). Cultivation of strains, harvesting of cells, fatty acid extraction, and analysis of fatty acid methyl esters were performed according to recommendations of the Sherlock Microbial Identification System (MIDI). For fatty acid analysis, all strains were grown on MA for 3 days at 30 • C under aerobic conditions. They were then identified using the TSBA identification library version 5.0. For quinone and polar lipid analyses, GJ3 T , P. galatheae LMG 28894 T , and P. halotolerans LMG 22194 T cultures first underwent lyophilization. Quinones were extracted as described by Hu et al. [39]. Quinone component was separated and identified by reversed-phase high-performance liquid chromatography as previously described [30]. Polar lipids were extracted according to procedures described by Minnikin et al. [40] and separated by two-dimensional thin-layer chromatography as described by Minnikin et al. [41].

Whole-genome Sequencing and Genome Analyses
For whole-genome sequencing, 20-kb SMRTbell TM template libraries were constructed using the extracted gDNA. Whole-genome sequencing of GJ3 T was performed using a PacBio RSII platform (Pacific Biosciences, Menlo Park, CA, USA) at DNA Link, Inc. (Seoul, Korea). Sequences were obtained from high-quality reads with about 244-fold coverage and assembled using a hierarchical genome assembly process (HGAP, v3.0) with assembly polishing using Quiver [42]. To examine species boundaries, ANI [43] values, AAI [44], and dDDH were calculated using an ANI calculator (http://enve-omics.ce.gatech.edu/ani/, accessed on 13 January 2022), AAI (http://enveomics.ce.gatech.edu/aai/, accessed on 13 January 2022), and a genome-to-genome comparison (GGDC) [45], respectively. Before genomic trait analysis, the genome of GJ3 T was annotated using the Prokaryotic Genome Annotation Pipeline (PGAP) [46]. There was no difference between the 16S rRNA gene sequence identified from the whole-genome sequence and that obtained by the PCR-Sanger sequencing method (see phylogenetic analysis).
The genome sequence data were uploaded to the Type (Strain) Genome Server (TYGS), a public bioinformatics platform available at https://tygs.dsmz.de (accessed on 13 January 2022) for whole genome-based taxonomic analysis [47]. Information on nomenclature, synonymy, and associated taxonomic literature was obtained from TYGS's sister database, the List of Prokaryotic names with Standing in Nomenclature (LPSN, available at https://lpsn.dsmz.de, accessed on 13 January 2022) [48]. For 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 [45]. One hundred distance replicates each were calculated. Digital DDH values and confidence intervals were calculated using recommended settings of the GGDC 3.0 [45,48]. Resulting intergenomic distances were used to infer a balanced minimum evolution tree with branch support via FASTME 2.1.6.1, including SPR postprocessing [49]. Branch support was inferred from 100 pseudo-bootstrap replicates each. Trees were rooted at the midpoint [50] and visualized with PhyD3 [51].

Phylogenetic, Phylogenomic, and Genome Features
The 16S rRNA gene sequence revealed that strain GJ3 T was clearly clustered to the genus Photobacterium (Figure 1). Based on the sequence similarity in EzBioCloud server, GJ3 T was most closely related to Photobacterium galatheae S2753 T (98.30% 16S rRNA gene sequence similarity), followed by Photobacterium halotolerans MACL01 T (97.90%), and Photobacterium panuliri LBS5 T (96.55%). The sequence similarity between GJ3 T and the additional-validated strain P. salinisoil LAM 9072 T was 98.55%. Additionally, the resultant phylogenomics tree provided strong evidence that strain GJ3 T belonged to the genus Photobacterium ( Figure 2).
From genome, the branch lengths are scaled in terms of GBDP distance formula d5. Numbers above branches are GBDP pseudo-bootstrap support values > 60 assemble, a complete circular genome 3,603,274 bp length with a G+C content of 50.70% and two plasmids. The values for ANI, AAI, and dDDH between GJ3 T and two selected reference strains were calculated by 79.24%, 81.94%, and 23.50% (P. galatheae LMG 28894 T ), and 77.90%, 81.83%, and 22.30% (P. halotolerans LMG 22194 T ), respectively. In addition, those values between GJ3 T and P. salinisoli LAM 9072 T (GenBank access. No. GCA_003614885) were 81.11%, 81.99%, and 22.30%, respectively. Despite its 16S rRNA gene sequence similarity with P. salinisoli (98.55%) was slightly higher than P. galatheae (98.30%), values of ANI, AAI, and dDDH from whole-genome-based genomic analyses indicated that GJ3 T was clearly distinguished from P. salinisoli as a novel species. Strain GJ3 T clearly represented a novel species in the genus Photobacterium based on ANI and dDDH criterion values [43,47].  Whole-genome sequence-based tree inferred with FastME 2.1.6.1 [49] from GBDP distances calculated from strain GJ3 T and relative genome sequences. The branch lengths are scaled in terms of GBDP distance formula d 5 . Numbers above branches are GBDP pseudo-bootstrap support values > 60% from 100 replications, with average branch support of 55.6%. The tree was rooted at the midpoint [51]. Strain GJ3 T genome was annotated by PGAP predicted 4719 coding sequences (CDSs) and 164 RNAs, including 125 transfer RNAs and 6 non-coding RNA genes. A total of 2259 CDSs were matched in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (47.87% of total CDSs). Based on KEGG results, quinone electron carrier (ArcBA) enzymes for anaerobic respiration and low nitrogen availability (GlnDBLGA) were found in the genome. Anaerobic respiration enzymes (FrdABCD) for converting fumarate to succinate and regulating the expression of anaerobic respiration gene (NarQL) and dissimilatory nitrate reduction (NapAB and NirBD) were also found in the genome, indicating that strain GJ3 T can grow under a low oxygen environment [55,56].
Strain GJ3 T genome encoded enzymes for phosphate limitation (PhoRB), misfolded proteins (CpxAR, HtrA), and Mg 2+ starvation and antimicrobial peptide (PhoQP). In addition, the genome contained gene contents entirely for bacterial chemotaxis, including flagella assembly (cheWYARYVB). The genome of strain GJ3 T included entire genes for the biosynthesis of ectoine and glycine betaine as a compatible solute. These have been implicated in the maintenance of osmotic equilibrium in hypersaline environments [57]. The resultant comparative genomic analysis for the genus Photobacterium showed that biosynthetic gene cluster for ectoine has been well presented in a number of species of the genus [58]. These results indicate that strain GJ3 T might have an ecological advantage for surviving in various environments, including nutrient starvation. Additionally, using antiSMASH analysis, the genome encoded the genes for the biosynthesis for non-ribosomal peptide synthetase cluster, althiomycin, and type I PKS (polyketide synthase) with antagonistic properties, including antibiotics [59]. Unexpectedly, the gene cluster for butyrolactone synthesis has firstly been identified in the Photobacterium spp. genomes. The butyrolactone is related to a quorum sensing molecule, regulating for antibiotic production and cell differentiation in streptomyces [60]. Taken together, although natural secondary-metabolic compounds are commonly produced from various organisms, including plant, this result indicates that strain GJ3 T may have a competitive benefit against other organisms [61].
Interestingly, the gene encoding the biosynthesis of siderophores aerobactin (iuc) was found in the genome of strain GJ3 T . This gene has been widely reported in members of pathogenic genera, including Vibrio [62]. The gene involved in siderophores synthesis is known as related to virulence (e.g., Klebseilla [63]). However, for non-pathogenic species, the gene might have been acquired via horizontal gene transfer (HGT) [64]. It was deduced that this gene might have a strategic advantage for iron uptake from the environment. To the best of our knowledge, in the genus Photobacterium, except strain GJ3 T , the iuc gene encoded in the P. halotolerans MELD1 genome was reported [65]. Also, we only identified three incomplete prophage regions (score < 70 estimated by PHASTER) in the strain GJ3 T genome with slightly lower GC range (47.09-47.86%) than its genome GC (50.70%). In these prophage sequences, genes involved in transposase, integrase, hypothetical protein, exonuclease, recombination, and tail assembly were identified. However, we found no CRISPRs in the genome. Taken together, these results indicate that strain GJ3 T might have a little stress for genetic exchange of foreign DNA (i.e., HGT via phage), and there might be no driving force for evolution opportunity (e.g., genome conservation) in their habits [66,67].

Physiology and Chemotaxonomy
The isolated strain GJ3 T was Gram-stain-negative, catalase-and oxidase-positive, facultatively anaerobic, white pigmented, motile, and rod shaped ( Figure S1). Cells grew at 15-37 • C (optimum: 30 • C), pH 5.5-8.5 (optimum: 7.0), and with 0.5-9.0% (w/v) NaCl (optimum: 4%) under aerobic conditions. Cells were sensitive to ampicillin, kanamycin, tetracycline, gentamicin, neomycin, and sulfamethoxazole/trimethoprim, but resistant to penicillin, oxacillin, clindamycin, and streptomycin. Degradation activity for starch, skim milk, DNase, cellulose, and Tween 60 was not observed. In addition, strain GJ3 T was negative for reduction of nitrates to nitrite, reduction of nitrates to nitrogen, indole production, and glucose acidification. Numerous additional phenotypic characteristics, as listed in Table 1, can be used to differentiate strain GJ3 T from the experimental reference strains used in this study. Table 1. Differential physiological characteristics of strain GJ3 T and reference strains of the genus Photobacterium.
Protease (gelatin hydrolysis) were Gram-stain-negative and catalase positive. All strains were positive for activities of valine arylamidase and acid phosphatase, but negative for arginine dihydrolase and β-glucosidase (esculin hydrolysis) as a sole source of carbon and energy. +, Positive; −, Negative; ND, not determined. 1, 2, 3 data from in this study; and *, data from Li et al. [28].
Based on morphological, physiological, and chemotaxonomic properties, as per the results of phylogenetic and genomics analysis (i.e., phylogenomics, dDDH relatedness, ANI, and AAI), GJ3 T belongs to the genus Photobacterium, and represents a novel species.
The type strain GJ3 T (= KCTC 72816 T = KMM 6822 T ) was isolated from the marine sediment collected from Jeju Island, South Korea.
The GenBank accession number for the 16S rRNA gene sequence is MK828357. The whole genomes, including two plasmid sequences, have been deposited in GenBank under the accession number CP073578, CP073579, and CP073580.
Supplementary Materials: The following supporting information can be downloaded at https: //www.mdpi.com/article/10.3390/d14030188/s1, Figure S1: Cell morphology, Figure S2  Data Availability Statement: The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene and the complete genome sequences of strain GJ3 T are MK828357 and CP073578-073580 (genome and two plasmids), respectively.