sustainability Article Comparative Genomic Analysis of Arctic Permafrost Bacterium Nesterenkonia sp. PF2B19 to Gain Insights into Its Cold Adaptation Tactic and Diverse Biotechnological Potential Purnima Singh 1, Neelam Kapse 2,3, Vasudevan Gowdaman 2, Masaharu Tsuji 4, Shiv Mohan Singh 5,* and Prashant K. Dhakephalkar 2,3,* 1 Parvatibai Chowgule College of Arts and Science, Goa 403602, India; [email protected] 2 Maharashtra Association for Cultivation of Science, Agharkar Research Institute, G.G. Agarkar Road, Pune 411004, India; [email protected] (N.K.); [email protected] (V.G.) 3 Savitribai Phule Pune University, Ganeshkhind Rd., Pune 411007, India 4 Department of Materials Chemistry, National Institute of Technology, Asahikawa College, Hokkaido 071-8142, Japan; [email protected] 5 Department of Botany, Institute of Science, Banaras Hindu University, Varanasi 221005, India * Correspondence: [email protected] (S.M.S.); [email protected] (P.K.D.) Abstract: 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 Citation: Singh, P.; Kapse, N.; Gowdaman, V.; Tsuji, M.; Singh, S.M.; cold permafrost soils of the Arctic. Genomic analysis of PF2B19 has given some valuable insight Dhakephalkar, P.K. Comparative into the biotechnological potential of this strain, particularly as a source of cold-active enzymes, as a Genomic Analysis of Arctic bioremediating agent and as plant growth-promoting bacteria. Permafrost Bacterium Nesterenkonia sp. PF2B19 to Gain Insights into Its Keywords: Nesterenkonia sp.; permafrost; comparative genomics; cold adaptation Cold Adaptation Tactic and Diverse Biotechnological Potential. Sustainability 2021, 13, 4590. https:// doi.org/10.3390/su13084590 1. Introduction Permafrost defines soil, rock or sediment that is frozen for more than two consecutive Academic Editor: Silvia Tabacchioni years [1], covering >25% of the land surface in the northern hemisphere [2]. Harsh condi- tions prevail in such soils like nutrient limitation, extreme aridity and pH, low temperature, Received: 3 March 2021 high ultraviolet irradiation, etc. [3,4]. In spite of such extreme conditions, reports suggest Accepted: 15 April 2021 Published: 20 April 2021 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]. Publisher’s Note: MDPI stays neutral These soils are characterized as extreme environments which can severely impair the cellu- with regard to jurisdictional claims in lar function by negatively affecting the cell integrity, membrane fluidity, enzyme kinetics published maps and institutional affil- and other interactions [8]. Therefore, for an organism to survive and grow in such extreme iations. niches, it should harbor genes encoding enzymes involved in regulation of DNA replica- tion, 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]. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. In the last few decades, there has been a growing interest in permafrost as it is known This article is an open access article to harbor potentially novel and biotechnologically important microorganisms [11]. Psy- distributed under the terms and chrophiles are the most probable sources of cold-active enzymes [12]. These cold-active conditions of the Creative Commons enzymes have high catalytic efficiency and stability at low and moderate temperatures [13]. Attribution (CC BY) license (https:// Cold-active enzymes have huge market potential as compared to mesophilic and ther- creativecommons.org/licenses/by/ mophilic enzymes as they shorten process time and cut down energy costs. These enzymes 4.0/). Sustainability 2021, 13, 4590. https://doi.org/10.3390/su13084590 https://www.mdpi.com/journal/sustainability Sustainability 2021, 13, 4590 2 of 17 find wide applications in biotechnological and industrial usage, especially in detergents, cosmetics, textiles, etc. Although permafrosts are known to cover 27% of the Earth [14], there are very few reports on bacterial community composition of permafrost soil from Svalbard (78 ◦N) [15,16]. Additionally, genomes sequenced from cold environments are relatively few [17]. The molecular strategy employed by bacteria for cold-adaption in such harsh environments remains poorly understood. Genus Nesterenkonia belongs to the family Micrococcaceae, within the phylum Actinobacteria [18]. Nesterenkonia sp. is coccoid, aerobic and non-spore forming bacteria [18,19]. At present, only nine genomes of Nesterenkonia, sp. are available publicly. Reports suggest that some of the Nesterenkonia strains are associated with extreme environments underlining their importance as sources of industrially important cold active enzymes [20]. In this study, a psychrophilic bacterium, Nesterenkonia sp. strain PF2B19 was isolated from permafrost soil. Here, we attempted, by means of genome sequencing of this strain, to unravel the molecular machineries associated with cold adaptation and to identify industrially important cold-active enzymes. 2. Materials and Methods 2.1. Sampling Site, Bacterial Strain and Growth Conditions Nesterenkonia sp. PF2B19 (PF2-B6) was isolated from permafrost soil gathered from Svalbard, Arctic (78◦55.1650 N, 11◦52.6600 E) on 20 August 2007. This strain was cultured routinely at 15 ◦C on Zobell Marine Agar. The pure culture of Nesterenkonia sp. PF2B19 has been deposited with accession number MCC 3408 at Microbial Culture Collection (MCC), India. 2.2. Genomic DNA Preparation and Genome Sequencing Genomic DNA from the strain PF2B19 was isolated using GenElute™ Bacterial Ge- nomic 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]. 2.3. 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. 2.4. 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. Path- way elucidation was executed using Kyoto Encyclopedia of Genes and Genomes (KEGG) Sustainability 2021, 13, x FOR PEER REVIEW 3 of 15 Sustainability 2021, 13, 4590 genes having role in cold adaptation and biotechnological potential in RAST annotation3 of 17 tool. Pathway elucidation was executed using Kyoto Encyclopedia of Genes and Ge- nomes (KEGG) (http://www.genome.ad.jp) database. Virulence determinants were de- tected using the online tool Virulence Finder [24]. (http://www.genome.ad.jp accessed on 3 March 2021) database. Virulence determinants were2.5. Accession detected Nnumber using the online tool Virulence Finder [24]. 2.5. AccessionThe Nesterenkonia Nnumber sp. PF2B19 whole Genome Shotgun project has been deposited at GenBank under the accession no. MDSS00000000. The Nesterenkonia sp. PF2B19 whole Genome Shotgun project has been deposited at GenBank under the accession no. MDSS00000000. 3. Results and Discussion 3.1.3. Results Characterization and Discussion and Phylogeny of PF2B19 3.1. CharacterizationPF2B19, a Gram and positive, Phylogeny strictly of PF2B19 aerobic coccoid, was identified as the affiliate of the psychrophilicPF2B19, agenus Gram positive,Nesterenkonia strictly based aerobic on 16S coccoid, rRNA was gene identified sequencing, as the displaying affiliate of maximumthe psychrophilic 16S rRNA genus sequenceNesterenkonia (1312 nucleotidebased ons) 16S homology