Whole genome sequencing and comparative genomic analyses of Planococcus alpniumensis MSAK28401T, a new species isolated from Antarctic krill

Background Extremophiles have attracted much attention in the last few decades, as they possess different properties by producing certain useful metabolites. However, the secondary metabolism of the extremophiles of Antarctic krill has received little attention. Results In this study, a new bacterial strain MSAK28401T from Antarctic krill was isolated and identified. The results of analysis on phenotypic, chemotaxonomic, and genomic characteristics showed that the strain MSAK28401T belongs to the genus Planococcus. Cells of this strain were coccoid (0.89–1.05 μm) and aerobic. The majority of the fatty acid content was C15:0 anteiso (37.67 ± 0.90%) followed by C16:1 ω7c alcohol (10.37 ± 1.22%) and C16:0 iso (9.36 ± 0.71%). The calculated average nucleotide identity and DNA–DNA hybridization values between the strain MSAK28401T and type strains P. citreus DSM 20549T and P. rifietoensis M8T were lower than 91 and 70%, respectively. The strain MSAK28401T (=KCTC 43283T and MCCC 1k05448T) represented a new member of the genus Planococcus and was named P. alpniumensis sp. nov. Moreover, genes involved in the degradation of aromatic compounds (e.g., salicylate, gentisate, and quinate) were found in the genome, implying that strain MSAK28401T has an aromatic compound as its potential metabolite. This work will help us understand the genomic characteristics and potential metabolic pathway of Planococcus from Antarctic krill. Conclusions This study reported the genomic information and phenotypic characteristics of the new strain P. alpniumensis MSAK28401T isolated from Antarctic krill, and provided the genome information of Planococcus strains for further studying the function roles in aromatic compound metabolism. Supplementary Information The online version contains supplementary material available at 10.1186/s12866-021-02347-3.

properties, G + C content in DNA, fatty acid composition, and menaquinone profiles. P. halophilus was classified under the genera Marinococcus [6,7]. These changes indicated that genus Planococcus and Planomicrobium have a close phylogenetic relationship. Usually, the main splitting points of the 16S rRNA sequence between the genera Planococcus and Planomicrobium were located at sites 183 and 190 (E. coli counting), which in the Planococcus are T and A, whereas in the Planomicrobium are C and G [8].
Planococcus has the following known features: Grampositive, multicellular morphology (cocci, short rod, or rod), aerobic, and no sporulation [8]. Representative strains of genus Planococcus usually grow in cold and/or saline-alkali soil with high salt concentrations, e.g., Arctic, Antarctic, and marine environments [9][10][11]. Planococcus has attracted much attention, because they can produce carotenoids of biotechnological significance; this metabolite has potential applications as the ingredient of cosmetics, food or feed additives, and antioxidants [12]. Planococcus can also degrade and process various contaminants, such as heavy metals and phenols, and play an important role in the bioremediation of extreme environments [13,14].
In the present study, a new strain P. alpniumensis MSAK28401 T of the genus Planococcus from Antarctic krill was isolated and identified using taxonomic, phylogenetic, chemotaxonomic, whole-genomic, and comparative genomic analysis.

Isolation, identification, and phylogenetic analysis
The single-bacterial MSAK28401 T was obtained by mixing culture on Luria-Bertani (LB) agar. The 16S rRNA sequence alignment against GenBank revealed that the strain MSAK28401 T belonged to the genus Planococcus, and it showed 98.62, 98.55, 98.43, 98.20, and 97.79% similarity with the corresponding gene sequences of P. citreus DSM20549 T , P. rifietornsis M8 T , P. maitriensis S1 T , P. dechangensis NEAU-ST10-9 T , and P. maritimus DSM17275 T , respectively (additional File 1: Table S1). The 16S rRNA phylogenetic tree showed that strain MSAK28401 T was clustered with four species of the genus Planococcus, and placed in an independent branch (Fig. 1). These results suggested that strain MSAK28401 T belongs to the genus Planococcus.

Phenotypic characterization
The transmission electron microscopy observations showed that cell coccoid and the diameter of strain MSAK28401 T was 0.89-1.05 μm with a thick cell wall ( Fig. 2A). The isolates could grow in the range of 4-50 °C, and the optimal growth temperature was 30 °C (Fig. 2B). The phenotypic characteristics of strain MSAK28401 T and related species as shown in Table 1.
Strain MSAK28401 T differed from the type strains of P. citreus DSM20549 T , P. rifietornsis M8 T , and P. maitriensis S1 T in the assimilation of β-methyl-D -glucoside, D -aspartic acid, L -arginine, quinic acid, D -glucuronic acid, and L -malic acid. Strain MSAK28401 T was distinguished from other species of the genus Planococcus by using some carbon sources and by producing acids from certain sugars. Phenotypic characteristics suggested that the strain MSAK28401 T may represent a new Planococcus species and was named P. alpniumensis sp. nov.

Fatty acid analysis
The details of the fatty acid profiles of the strain MSAK28401 T and three related species of P. citreus DSM 20549 T , P. rifietoensis M8 T , and P. maitriensis S1 T were described (Table 2). These major fatty acids (> 5%) of strain MSAK28401 T were C 15:0 anteiso (37.67 ± 0.90%), C 16:1 ω7c alcohol (10.37 ± 1.22%), and C 16:0 iso (9.36 ± 0.71%). The main fatty acid with the highest content is C 15:0 anteiso. The other major fatty acids that were the most abundant in strain MSAK28401 T , namely, C16:0 iso (9.36 ± 0.71%), C 16:1 ω7c alcohol (10.37 ± 1.22%), and C 14:0 iso (7.80 ± 0.15%), showed quantitative differences with those in the two related type species. Results of comparing fatty acid types and proportions suggested that the strain MSAK28401 T can be distinguished from the two species of a cluster in the phylogeny. Carbon source utilization:

Genome properties and mining
The genome of strain MSAK28401 T formed from 10 contigs, and the genomic length was 3,930,779 bp. The G + C content was 47.15%. We identified 3998 genes and 3897 codifying sequences (Table 3 and Fig. 3A) and assigned them to 27 subsystems with SEED viewer using the RAST pipeline ( Fig. 3B and additional File 2: Table S2). Nevertheless, only 26% (1150, genes) of this genome was annotated, and the other 74% was not assigned to the RAST subsystems. The most represented subsystem features were amino acids and derivatives (266), carbohydrates (214), protein metabolism (203), cofactors, vitamins, prosthetic group, and pigments (138). Notably, several genes involved in dormancy and sporulation were also found in strain MSAK28401 T . Carbohydrate-related enzymes and activity annotations of presumed genes showed that 24 genes encoded glycosyl transferases (GT) and 21 genes encoded glycosyl hydrolases (GH) ( Fig. 4A and additional File 3: Table S3). KofamKOALA analysis results showed that almost all of the major metabolic pathways of bacteria were found in the genome of strain MSAK28401 T ( Fig. 4B and additional File 4: Table S4). Most genes were related to amino acid and carbohydrate metabolism, suggesting that MSAK28401 T might possess the efficient nutrient uptake systems. In-depth analysis of the metabolic pathways of the strain MSAK28401Trevealed that genes related to aromatic hydrocarbon degradation pathways, such as catechol 2,3-dioxygenase (gene 0402), 4-oxalocrotonate tautomerase (gene 2794), and S-(hydroxymethyl) glutathione dehydrogenase / alcohol dehydrogenase (gene 2852) (Additional File 5: Table S5). Notably, 4-oxalocrotonate tautomerase (EC 5.3.2.-4-OT) is an enzyme that forms part of a bacterial metabolic pathway that oxidatively catabolizes toluene, o-xylene, 3-ethyltoluene, and 1,2,4-trimethylbenzene into intermediates of the citric acid cycle. In addition, we mapped the relevant pathways of aromatic hydrocarbons that the isolate may be involved in degradation (Fig. 4C). Above results indicated this isolate have a potential for application to the process of aromatic hydrocarbon metabolism.

Genetic relatedness and Pan-genome analysis
The phylogenetic tree of GBDP determined the phylogenetic position of strains, and it showed that the strain MSAK28401 T was clustered with P. citreus DSM 20549 T and P. rifietoensis M8 T (Fig. 5). The DDH and ANIb values between the strain MSAK28401 T and related species P. citreus DSM 20549 T and P. rifietoensis M8 T were less than 70 and 91%, respectively (Table 4 and Table 5), which were below the threshold for species delineation. The above results support the affiliation of the strain MSAK28401 T to a new species of the genus Planococcus.
The pan-genome analysis of strains P. alpniumensis MSAK28401 T , P. citreus DSM 20549 T , and P. rifietoensis M8 T was depicted in a Venn diagram (Fig. 6). The three strains of Planococcus possessed 3363 gene families, whereas a "core" genome comprised 2853 clusters of orthologous, accounting for 84.5% of all gene families. Most of the annotation functions of homologous clusters were involved in biological process, hydrolase activity, ion binding, molecular function, and transferase activity. A total of 63 unshared protein clusters were found in the strain MSAK28401 T , whereas 6 and 4 unshared protein clusters were found in the strains of P. citreus DSM 20549 T and P. rifietoensis M8 T , respectively. Remarkably, the number of unshared clusters of the strain MSAK28401 T was higher than those of these related species. Approximately 84% of unshared clusters involved biological processes, such as those involving nucleobase-containing compounds, cellular aromatic compounds, macromolecules, nitrogen compounds, and heterocycle metabolism, thereby indicating the unique advantages in its biological process compared with the other related strains.

Secondary metabolites
Screening the genes of secondary metabolites showed two different genes clusters, which both belong to the terpene biosynthesis-related clusters (Fig. 7A). Cluster 1 displayed orphan Biosynthetic gene clusters (BGCs), which were unable to identify the known homologous gene cluster. Cluster 2 (3,001,607-3,022,437 nucleotides) was 66% similar to the known BGC (BGC0000645), which was a gene cluster comprising carotenoids biosynthetic carotenoids. Nevertheless, the low similarity of predicted gene clusters may represent the production of new metabolites.

Islands of genome
Thirty-seven genomic islands were predicted in this new strain MSAK28401 T by IslandViewer 4, and the localization of the predicted genomic islands is shown in Fig. 7B. The 37 genomic islands were made up of 971 genes from the range of 4000-320,000 bp. Among these, 581 genes were hypothetical proteins with no function, 29 genes were mobile element protein, but genes producing secondary metabolites were not found within the genomic islands.

Discussion
The species of Planococcus are the dominant species in many marine environments, e.g., deep sea, salt marshes, and intertidal zones [5]. These aerobic heterotrophic bacteria degrade a variety of hydrocarbons, so they can make a significant contribution to the reduction of hydrocarbon contamination in the marine environment [5,15]. Thirty species of Planococcus have been characterized. Notably, six typical strains have been found in the Antarctic, namely, P. faecalis [16], P. versutus [17], P. maitriensis [18], P. antarcticus, P. psychrophilus [9], and P. mcmeekinii [19]. In this work, we isolated and identified a Defining a new species involves two consecutive steps, namely, 16S rRNA gene analysis and calculation of several parameters of the genome [20]. In conformity to this scheme, we analyzed the 16S rRNA sequence of strain MSAK28401 T and found that the similarity between the corresponding gene sequence and related stains within genus Planococcus was less than 98.7%. This finding supported the idea that this strain might be a new species, because some species recently proposed in the genus Planococcus had similar or highly similar values in the 16S rRNA gene [17,21,22]. Chun et al. proposed a minimum standard to the taxonomy of prokaryotes using genomic data [20]. The whole-genome analysis results showed that the threshold values of ANI for species differentiation were 95-96%, which were generally accepted. The calculated ANI values of the genome of related strains of Planococcus were less than 91%, thereby indicating that the strain belongs to a novel species within the genus Planococcus. Furthermore, the morphology, phenotype, and whole-genome analysis of the strain MSAK28401 T showed that it represented a new member of the genus Planococcus and was named P. alpniumensis sp. nov.
Genus Planococcus is a halophilic bacterium known for producing various secondary metabolites [23], which are often referred to as anti-inflammatory, antimicrobial, pharmaceutically significant, and chemotherapeutic [24]. Ganapathy et al. identified a new carotenoid (methyl glucosyl-3,4-dehydro-apo-8-lycopenoate) with  antioxidant activity from P. maritimus MKU009 [25]. Nevertheless, two clusters of genes that may be involved in the synthesis of terpenes were discovered by scanning potential secondary metabolites in strain MSAK2840 T . Cluster 2 had 66% similarity with the gene cluster of the carotenoid biosynthesis of Halobacillus halophilus DSM 2266, which can help the strain resist oxidative stress. Genes associated with aromatic compound metabolism, one of the most common and persistent contaminants in environments [26], were found. In general, degradation of hydrocarbons, e.g., salicylate, gentisate, and quinate degradation, was a function of Planococcus [23]. By identifying vertical genetic homologous gene clusters from unique common ancestors, comparative analysis can help clarify the relationship between different species and the evolution and adaptability of the genome [23,27]. The strain MSAK28401 T shared 2853 gene clusters with P. citreus DSM 20549 T and P. rifietoensis M8 T , and had 63 unshared protein clusters. The functional distribution of homologous gene families in core genomes showed that most homologous gene families encode the Fig. 6 The Venn diagram and the bar graph depict the comparative genomics among the genomes of P. alpniumensis MSAK28401 T , P. citreus DSM 20549 T , and P. rifietoensis M8 T , showing shared and unshared orthologous genes clusters basal metabolism of bacteria, such as protein processing, folding, and secretion and DNA and RNA metabolism [28]. Notably, the number of unshared clusters in strain MSAK28401 T was significantly higher than these related species among themselves (Fig. 6). The biological processes of unshared clusters of strain MSAK28401 T are aromatic compound, nitrogen compound, macromolecule, and heterocycle metabolic processes, indicating the unique advantages in its biological process than other related strains.

Conclusion
The analysis of genomic, chemotaxonomic, and phenotypic traits showed that the strain MSAK28401 T belongs to a new species of the genus Planococcus, named P. alpniumensis sp. nov, whose type strain is MSAK28401 T . Furthermore, genomic characterization and comparative analysis showed that the strain P. alpniumensis MSAK28401 T contained many genes related to the metabolism and transportation of amino acids and carbohydrates, thereby suggesting that MSAK28401 T might possess n efficient nutrient uptake system. Screening the secondary metabolite genes found two different types of terpene biosynthesis-related clusters. Cluster 2 was similar to carotenoids (66% of genes showed similarity), thereby indicating that these predicted gene clusters may represent the production of new metabolites. Finally, genes (catechol 2,3-dioxygenase (gene 0402), 4-oxalocrotonate tautomerase (gene 2794), and S-(hydroxymethyl) glutathione dehydrogenase / alcohol dehydrogenase (gene 2852)) involved in the degradation of aromatic compounds (e.g., salicylate, gentisate, and quinate) were identified, indicating the potential metabolism of an aromatic compound of the new species.

Bacterial isolation
Antarctic krill was collected from Antarctica (58°33.1″ W, 63°6.3″ S) in 2016. It was washed with sterile seawater thrice under aseptic conditions to remove superficial residual sediments and microbes. Three Antarctic krill samples from a collected site were ground and homogenized as one specimen. Then, the milled samples were diluted with approximately 1 ml of sterile water, collected in a 2 ml aseptic centrifuge tube, and centrifuged at 3500 rpm for approximately 5-10 min. An inoculation loop was used to obtain a small amount of supernatant liquid, which was spread on agar-mixed LB. Bacteria in inoculated dishes were allowed to multiply at 10 °C until the colonies became visible. The colonies were randomly isolated from the agar plates, picked, and sub-cultured almost thrice under the same conditions. The same strains were preserved in 20% glycerin liquid medium at − 80 °C for future use.

Phylogenetic tree construction
16S rRNA gene sequence of strain MSAK28401 T was amplified and sequenced through the sequencing DNA service of TSINGKE Biological Technology, China and then compared with the EzBioCloud database [29]. ClustalW program was used for sequencing against the closest type strains [30] to analyze phylogeny. The Neighbor-Joining phylogenetic tree was established using MEGA-X [31]. The robustness of the phylogenetic tree was evaluated through bootstrap analysis (1000 replicates) [32].

Phenotypic characterization
After incubating the MSAK28401 T strain on LB-Agar-Powder plates for 48 h at 25 °C, transmission electron microscopy confirmed the morphological characteristics. Motility was examined by stab-culture in semi-solid medium according to the method of Gerhardt et al. Oxidase activity was tested using 1% (w/v) tetramethyl-pphenylenediamine. Formation of spores was monitored by phase-contrast microscopy on cells cultured on LB agar at 30 °C for up to 7 days. Growth at different temperatures (4,10,16,25,30,37,45, and 50 °C) was determined and bacterial concentration was measured as optical density at 600 nm. Under manufacturer-indicated conditions, phenotypic characterization of this strain and two reference strains (P. citreus DSM20549 T and P. rifietornsis M8 T , which were obtained from Marine Culture Collection of China, MCCC) were identified using Biolog Gen III microstation. Strains P. citrus DSM20549 T , P. rifietornsis M8 T , and the strain MSAK28401 T were incubated together at 25 °C for 30 h and the results were tested.

Chemotaxonomic analysis
For cellular fatty acid analysis, strains MSAK28401 T , P. citreus DSM20549 T , and P. rifietornsis M8 T were incubated together on LB-Agar-Powder at 25 °C for 2 days. Culture was harvested and prepared, and fatty acid methyl esters were separated based on the method proposed by Sasser [33] and were tested by the MIDI Sherlock Microbial Identification system.

Genome sequencing and mining
Total DNA of the genome was purified from a purely cultured strain MSAK28401 T using a DNA extraction kit (TaKaRa, Japan) following the manufacturer's protocol. PacBio sequencing and analysis were conducted by OE Biotech Co., Ltd. (Shanghai, China). The total DNA obtained was subjected to quality control via agarose gel electrophoresis and quantified by Qubit. The library was constructed utilizing the SMRTbell template prep kit 1.0 from Pacific Biosciences. Single-molecule real-time (SMRT) sequencing was performed on the PacBio Sequel platform. SMRT Analysis 2.3.0 was used to filter low-quality reads [34,35]. The filtered reads were assembled into a contig without gaps. Falcon was used for the de novo assembly of these reads [36]. This draft genome sequence of MSAK28401 T was collected in GenBank and was given the accession number JAAMTH000000000.

Pan-genome and comparative genome-wide analysis
To compare genomes, the reference genome sequence of this bacteria was downloaded from the GenBank database. The pan-genome sequence comparative analysis of this strain MSAK28401 T was performed using the GBDP method [43]. Genomic homogeneous clustering analysis, including the genetic ontogeny of all predicted protein-coding genes, was performed using OrthoVenn2 [47].