Comparative Genomic Analysis of Arctic Permafrost Bacterium Nesterenkonia sp. PF2B19 to Gain Insights into Its Cold Adaptation Tactic and Diverse Biotechnological Potential

Nesterenkonia sp. PF2B19, a psychrophile was isolated from 44,800-year-old permafrost soil. This is the first report on comparative genomics of Nesterenkonia sp. isolated from Arctic. Genome of PF2B19 exhibited the presence of a vast array of genetic determinants involved in cold adaptation i.e., response to cold-associated general, osmotic, and oxidative stress. These genomic attributes proved to be valuable in unraveling the adaptive tactics employed by PF2B19 for survival in the cold permafrost soils of the Arctic. Genomic analysis of PF2B19 has given some valuable insight into the biotechnological potential of this strain, particularly as a source of cold-active enzymes, as a bioremediating agent and as plant growth-promoting bacteria.


Introduction
Permafrost defines soil, rock or sediment that is frozen for more than two consecutive years [1], covering >25% of the land surface in the northern hemisphere [2]. Harsh conditions prevail in such soils like nutrient limitation, extreme aridity and pH, low temperature, high ultraviolet irradiation, etc. [3,4]. In spite of such extreme conditions, reports suggest the presence of metabolically-active microbial life in the permafrost soil of Svalbard [5,6]. Permafrost soils are considered as chronological collections of past and present microbes [7]. These soils are characterized as extreme environments which can severely impair the cellular function by negatively affecting the cell integrity, membrane fluidity, enzyme kinetics and other interactions [8]. Therefore, for an organism to survive and grow in such extreme niches, it should harbor genes encoding enzymes involved in regulation of DNA replication, transcription, translation and membrane fluidity at low temperatures and other stress combative mechanisms. The microorganisms harboring such harsh microenvironments have evolved certain adaptive features to combat various cold environment-related stresses such as cold stress, oxidative stress, osmotic stress, low nutrient availability, etc. [9,10].
In the last few decades, there has been a growing interest in permafrost as it is known to harbor potentially novel and biotechnologically important microorganisms [11]. Psychrophiles are the most probable sources of cold-active enzymes [12]. These cold-active enzymes have high catalytic efficiency and stability at low and moderate temperatures [13]. Cold-active enzymes have huge market potential as compared to mesophilic and thermophilic enzymes as they shorten process time and cut down energy costs. These enzymes

Genomic DNA Preparation and Genome Sequencing
Genomic DNA from the strain PF2B19 was isolated using GenElute™ Bacterial Genomic DNA Isolation kit (Sigma, St. Louis, MO, USA). The PF2B19 genome was sequenced on the Ion Torrent PGM platform (Life Technologies, Carlsbad, CA, USA) using the 316™ chip and 200-bp chemistry. The obtained sequence was then de novo assembled using SPAdes assembler version 3.9.1 [21].

Comparative Genomics
Digital DNA-DNA Hybridization was executed as described by Auch et al. (2010) [22] using online tool http://ggdc.dsmz.de (accessed on 3 March 2021) with PF2B19 as query genome and Nesterenkonia JCM 19054, Nesterenkonia alba DSM 19423(T), Nesterenkonia massiliensis strain NP1, Nesterenkonia sp. AN1, Nesterenkonia sp. F and Nesterenkonia jeotgali CD08_7 as reference genomes. Genome sequence of PF2B19 further compared with the genomes of above mentioned strains in RAST tool to determine distinctive genomic determinants, i.e., gene unique in PF2B19 to prove its novelty. A circular map representing the general genome comparisons of strain PF2B19 with its close phylogenetic affiliates (Nesterenkonia JCM 19054, Nesterenkonia alba DSM 19423(T) and Nesterenkonia sp. AN1) was generated using the BRIG program. BRIG uses BLAST for genome comparisons and CGView for image generation. The circular image is generated wherein the reference genome is placed at the center and other query genomes as a set of concentric rings colored displaying similarity. The genomes of reference Nesterenkonia strains NP1, F, AN1, JCM 19054, DSM 19423 and CD08_7 were obtained from the NCBI database.

Functional Annotation
Functional annotation of PF2B19 genome was carried out by Rapid Annotation using Subsystem Technology (RAST) [23]. PF2B19 genome was mined for the presence of genes having role in cold adaptation and biotechnological potential in RAST annotation tool. Pathway elucidation was executed using Kyoto Encyclopedia of Genes and Genomes (KEGG) genes having role in cold adaptation and biotechnological potential in RAST annotation tool. Pathway elucidation was executed using Kyoto Encyclopedia of Genes and Genomes (KEGG) (http://www.genome.ad.jp) database. Virulence determinants were detected using the online tool Virulence Finder [24].

Accession Nnumber
The Nesterenkonia sp. PF2B19 whole Genome Shotgun project has been deposited at GenBank under the accession no. MDSS00000000.

Characterization and Phylogeny of PF2B19
PF2B19, a Gram positive, strictly aerobic coccoid, was identified as the affiliate of the psychrophilic genus Nesterenkonia based on 16S rRNA gene sequencing, displaying maximum 16S rRNA sequence (1312 nucleotides) homology of 99% with closest phylogenetic neighbors Nesterenkonia aethiopica DSM 17733(T), Nesterenkonia xinjiangensis strain YIM70097, Nesterenkonia sp. YIM70097 and Nesterenkonia suensis Sua-BAC020(T). PF2B19 shared 98% homology with Nesterenkonia massiliensis strain NP1. 16S rRNA gene sequences of PF2B19 were aligned with those of the publicly available Nesterenkonia 16S rRNA sequences using the Mega version 6.0 [25] Phylogenetic tree showing the taxonomic relationship of strain PF2B19 with other Nesterenkonia strains was constructed by employing the neighbor-joining algorithm ( Figure 1).

General Genome Features of Permafrost Bacterium Nesterenkonia sp. PF2B19
Sequencing of the library generated 3,698,032 bp reads, which were de novo assembled using SPAdes assembler version 3.9.1 into 135 contigs, yielding a 3.6 Mb genome with 69.5% G+C content. These results were in congruence with publicly available draft genomes of three strains of Nesterenkonia possessing sizes in the range of 2.59 to 3.01 Mb and G+C contents of 62.2 to 71.5%. Functional annotation pf PF2B19 genome by RAST revealed a total of 3763 proteins were predicted, including 3708 coding sequences and 55 total RNAs. Differentiating genome features of query genome PF2B19 along with five reference genomes are illustrated in Table 1.

General Genome Comparisons of PF2B19 with Its Closest Phylogenetic Affiliates
PF2B19 genome was compared with the available Nesterenkonia genomes, by running BLASTn in BRIG software [26]. The circular map ( Figure 2) represents the BLASTn results of each query genome (Nesterenkonia JCM 19054, Nesterenkonia alba DSM 19423(T) and Nesterenkonia sp. AN1) against the reference PF2B19. As evident from the BRIG image, gaps were more pronounced in the query genomes, emphasizing the difference between PF2B19 and the other Nesterenkonia genomes.

Comparative Genomics Identifies Unique Genes/Proteins in Nesterenkonia sp. PF2B19:
Genome annotation performed using RAST tool identified Renibacterium salmoninarum ATCC 33209 (Genome id: 288705.3, Score: 512) as the closest phylogenetic neighbor of PF2B19. On comparative analysis with ATCC 33209, 378 unique genes associated with a subsystem in PF2B19 were detected in PF2B19. These genes were scored as distinctive genomic determinants that differentiated PF2B19 from its phylogenetic associates.
PF2B19 genome was also compared with other Nesterenkonia genomes in RAST. Unique genes were detected in PF2B19 as compared to other Nesterenkonia sp., further highlighting the novelty of PF2B19 (Table 2). Glycine betaine ABC transport system permease protein 3.
Sarcosine oxidase alpha, beta and gamma subunit (EC 1.5.3.1) Counteract against cold-induced osmotic stress Figure 2. Circular map generated using BRIG program highlighting the differences between PF2B19 and publicly available Nesterenkonia genomes.

Comparative Genomics Identifies Unique Genes/Proteins in Nesterenkonia sp. PF2B19
Genome annotation performed using RAST tool identified Renibacterium salmoninarum ATCC 33209 (Genome id: 288705.3, Score: 512) as the closest phylogenetic neighbor of PF2B19. On comparative analysis with ATCC 33209, 378 unique genes associated with a subsystem in PF2B19 were detected in PF2B19. These genes were scored as distinctive genomic determinants that differentiated PF2B19 from its phylogenetic associates.
PF2B19 genome was also compared with other Nesterenkonia genomes in RAST. Unique genes were detected in PF2B19 as compared to other Nesterenkonia sp., further highlighting the novelty of PF2B19 (Table 2).

Identification of Virulence Determinants
No virulent genes were detected in the genome of PF2B19 as revealed by Virulence Finder. Thus, PF2B19 was non-pathogenic.

Genes Involved in Resistance to Antibiotics
Antibiotic-resistance genes are potentially transferable genes in specific niches such as intestinal microflora where microbial inhabitants are often exposed to an exhaustive use of antibiotics. Yet, current studies have revealed the presence of antibiotic-resistant genes and/or antibiotic-resistance bacteria in the geographically isolated natural niches which are not exploited by anthropogenic factors [27][28][29][30]. We screened the genome of PF2B19, which was isolated from Arctic for presence of antibiotic-resistance genes. Interestingly, genes conferring resistance to fluoroquinolones and Beta lactam group of antibiotics were detected in PF2B19 genome. Arctic is characterized by harsh cold conditions. The stresses encountered by bacteria in permafrost soil include limited nutrients, desiccation, oxidative stress, osmotic stress and persistent low temperatures [31,32]. A repertoire of adaptive genes associated with diverse stresses present in cold milieus have been reported in the literature [33][34][35][36][37]. PF2B19 genome was mined for the adaptive genes that may be associated with survival of PF2B19 in the permafrost soils of Svalbard. Analysis of Nesterenkonia sp. PF2B19 genome revealed a total of 128 putative stress response genes, including 16 genes linked to cold stress response, 16 genes for DNA repair, 12 genes for modulation of membrane fluidity, 39 genes for oxidative stress response, 37 genes for osmotic stress response and 4 in response to general stress (Table 3).    Cold shock proteins are vital for the cold acclimation of bacteria [38]. Cold shock proteins (Csps) serve as nucleic acid chaperons, which counteract the harmful effects of cold stress like inefficient protein folding by regulating transcription and translation at low temperatures [39,40]. Csps have also been known to contribute to various environmental stress tolerance such as osmotic, oxidative, starvation and pH stress. PF2B19 genome contains genes encoding the cold shock proteins CspA and CspC and an arsenal of chaperones like dnaJ, dnaK and grpE, which are considered pivotal for preserving the integrity and function of proteins [41]. The genome also contains genes encoding the secondary CSPs polyribonucleotide nucleotidyltransferase (PNPase), ribosome binding factor A (RbfA), transcription elongation protein (NusA), and translation initiation factor (Inf2) which are typically induced via transcription anti-termination [42].
Modulation of membrane fluidity is crucial for cell viability at lower temperatures. This is achieved by improved production of unsaturated fatty acids, alteration of fatty acid branched chains and shortening of fatty acyl chains [43][44][45]. The Nesterenkonia sp. PF2B19 genome encodes five proteins involved in fatty acid biosynthetic pathways (Table  3). These include FabG and FabH involved in fatty acid biosynthesis, the condensation of fatty acids and the synthesis of branched fatty acids [35,46]. The genome also codes for 1acyl-sn-glycerol-3-phosphate acyltransferase (PlsC), catalyzing the phospholipid synthesis, and 3-ketoacyl-(acyl-carrier-protein) reductase, involved in enhancing the production of polyunsaturated lipids [35,46]. Additionally, the pathway for unsaturated fatty acid synthesis was detected in PF2B19 using KEGG pathway tool.
At low temperatures, pigments are also known to modulate membrane fluidity [47][48][49]. The genome of PF2B19 contains three genes with putative roles in carotenoid biosynthesis (Table 3).

Oxidative Stress Response
Bacteria-harboring cold environments are more inclined to the deleterious effects of reactive oxygen species (ROS) because of better solubility of gases at low temperatures [45,50]. Nesterenkonia sp. PF2B19 encoded genes involved in detoxification of ROS such as catalase (kat), two superoxide dismutases (SodA; SodC), a thiol peroxidase (Bcp) as well as thioredoxin and thioredoxin reductase (TrxA and TrxB) [51]. Two putative dioxygenases were also detected in PF2B19 genome, known to play a key role in combating ROS damage [34].

Osmo-Protection
Accumulation of compatible solutes is an effective tactic to combat osmotic stress. These solutes are known to have dual response in stress as osmolytes and cryo-protectants [52]. Nesterenkonia sp. PF2B19 genome encodes a range of proteins involved in combating osmotic stress ( Table 2). The genome also encodes transporters for glycine/betaine and choline dehydrogenases which are well-known osmo-protectants [53]. A number of genes involved in the endogenous synthesis of compatible solutes like trehalose biosynthesis genes otsA and otsB, known to be cold-inducible and essential for low temperature survival, were also detected.

General Stress Response
In addition to cold, osmotic and oxidative stress response, the PF2B19 genome encoded a repertoire of other stress-related proteins, which was included in general stress response system ( Table 2). Ten genes involved in SOS response (cellular response to DNA damage) and DNA repair systems were detected. The genome also encoded universal stress protein, UspA, which is associated with cold acclimation [54].

Biotechnological Potential of PF2B19
Cold-active enzymes and the microbes producing them are of great biotechnological potential, with applications in detergent-, food-, textile-industry, pharmaceuticals and molecular biology. Psychrophilic enzymes are considered to be a boon to industry because of shorter process intervals, low energy budgets, low enzyme concentration requirement as well as impeding undesired chemical alterations [55]. Annotated genome sequence of PF2B19 revealed the presence of genes involved in production of cold-active enzymes, particularly of α-amylases, proteases, lipases/esterases, β-glucosidase, β-galactosidase and alkaline phosphatase (Table 4). Furthermore, genes possibly responsible for hydrocarbon degradation were detected. Genes encoding catabolism of benzoate, catechol were found in the genome. Catecholic compounds are the common inter-mediates in aerobic bacterial aromatic compound degradation pathways [56] and extradiol dioxygenases (EDOs) are known to catalyze the ring cleavage of catecholic compounds. EDOs like catechol 2,3-dioxygenase (EC 1.13.11.2), possible dioxygenase and 3-phenylpropionate dioxygenase ferredoxin subunit were detected in PF2B19. Benzoate catabolism genes 2-oxo-hepta-3-ene-1, 7-dioic acid hydratase (EC 4.2.-.-), and benzoate transport protein, 4-hydroxybenzoate transporter were also detected. Presence of these genes highlighted the bioremediation potential of PF2B19 in cold environment. Additionally, the pathway for degradation of catechol was elucidated in PF2B19 using KEGG database (Figure 3). Furthermore, genes possibly responsible for hydrocarbon degradation were detected. Genes encoding catabolism of benzoate, catechol were found in the genome. Catecholic compounds are the common inter-mediates in aerobic bacterial aromatic compound degradation pathways [56] and extradiol dioxygenases (EDOs) are known to catalyze the ring cleavage of catecholic compounds. EDOs like catechol 2,3-dioxygenase (EC 1.13.11.2), possible dioxygenase and 3-phenylpropionate dioxygenase ferredoxin subunit were detected in PF2B19. Benzoate catabolism genes 2-oxo-hepta-3-ene-1, 7-dioic acid hydratase (EC 4.2.-.-), and benzoate transport protein, 4-hydroxybenzoate transporter were also detected. Presence of these genes highlighted the bioremediation potential of PF2B19 in cold environment. Additionally, the pathway for degradation of catechol was elucidated in PF2B19 using KEGG database (Figure 3). PF2B19 also possessed the ability to promote plant growth. Genes involved in acetoin production, i.e., acetolactate synthase and zinc-containing alcohol dehydrogenase were identified in the genome. Acetoin is known to promote plant growth by stimulating root formation [57]. 1-aminocyclopropane-1-carboxylate (ACC) deaminase gene (acdS) was also detected. acdS is known to aid the degradation of a plant's ethylene precursor, thus promoting plant growth [58]. Arctic plants are challenged by various abiotic stressors in their environment, which are known to limit their growth. PF2B19 can form mutualistic relationship with plants growing in the Arctic and promote growth.
Moreover, the genes encoding proteins involved in resistance to heavy metals and toxic compounds (copper, cobalt, zinc, cadmium, mercury, chromium and arsenic) were PF2B19 also possessed the ability to promote plant growth. Genes involved in acetoin production, i.e., acetolactate synthase and zinc-containing alcohol dehydrogenase were identified in the genome. Acetoin is known to promote plant growth by stimulating root formation [57]. 1-aminocyclopropane-1-carboxylate (ACC) deaminase gene (acdS) was also detected. acdS is known to aid the degradation of a plant's ethylene precursor, thus promoting plant growth [58]. Arctic plants are challenged by various abiotic stressors in their environment, which are known to limit their growth. PF2B19 can form mutualistic relationship with plants growing in the Arctic and promote growth.
Moreover, the genes encoding proteins involved in resistance to heavy metals and toxic compounds (copper, cobalt, zinc, cadmium, mercury, chromium and arsenic) were detected in PF2B19, highlighting the potential of the PF2B19 to adapt to extreme lifestyles.

Conclusions
Based on genomic analysis, it can be concluded that Nesterenkonia sp. PF2B19 employs was found to be well-equipped with proteins involved in cold stress as well as modulation of membrane fluidity, osmotic and oxidative stress responses. Nesterenkonia sp. PF2B19 was found to be non-virulent and non-pathogenic. Genomic analysis of the PF2B19 has given valuable insight into the potential role of this strain in bioremediation in a colder environment. The genomic attributes also revealed the strategies adopted by Nesterenkonia sp. PF2B19 to survive in the extreme cold environment of permafrost.  Institutional Review Board Statement: This study did not involve humans or animals. Therefore, there are no ethical and biosafety issues.

Informed Consent Statement: Not applicable.
Data Availability Statement: The data present in this research article will be available for public interest.