The genus Kocuria was proposed by Stackebrandt et al.²⁴ and at the time of writing the genus includes 26 validly published species names.²⁵ Among Kocuria species, K. polaris was isolated from the Antarctic cyanobacterial mat, and as mentioned above it was reported to share high 16S rRNA gene sequence similarity with the type strain of K. rosea,⁸ hence the present study evaluates its taxonomic position. We further evaluated the metabolic potentials and survival strategy of K. polaris in a cold environment.

The genome size of K. polaris CMS 76or^T was 3779800 (bp) with 72.8% G+C content while the genome size of K. rosea ATCC 186^T was 3946651 (bp) with 72.7% G+C content. The genome completeness of K. polaris CMS 76or^T and K. rosea ATCC 186^T was 99.1 and 98.6%, respectively with zero contamination. A total of 48 tRNAs were predicted in both K. polaris CMS 76or^T and K. rosea ATCC 186^T. The graphical representation of the genome’s comparison is mentioned in Figure 1.

Metabolic potential and survival strategy of K. polaris
K. polaris CMS 76or^T encodes genes for glycolysis, citrate cycle, and pentose phosphate pathway. Nitrate stands as the most highly oxidized variant among fixed nitrogen compounds, constituting a vital nutrient crucial for the sustenance of microbial and plant life.²⁶ In prokaryotes, dissimilatory nitrate reduction mechanisms have been extensively explored.^26-28 Reduction of nitrate to nitrite by respiratory membrane-bound NarG or periplasmic nitrate reductase NapA is the first step in dissimilatory nitrate reduction. Nitrite is next reduced to ammonia by cytoplasmic nitrite reductase NirB or periplasmic nitrite reductase NrfA.²⁷ In the present study, the genes encoding dissimilatory nitrate reduction (NarGHI and NirBD) were noticed in K. polaris CMS 76or^T. In addition, genes related to nitrate assimilation were also noticed in K. polaris CMS 76or^T.

Microorganisms use assimilatory sulfate reduction to convert inorganic sulfate to sulfide.²⁹ In the present study, it was noticed that K. polaris CMS 76or^T encodes genes (CysND, CysH and Sir) for assimilatory sulfate reduction. K. polaris CMS 76or^T encodes genes for various amino acid metabolism (like betaine, methionine, lysine, ornithine, arginine biosynthesis, etc). A detailed list of metabolic potentials of K. polaris CMS 76or^T is mentioned in Table S1.

Low temperatures significantly limit cellular function by impacting cell structure, water thickness, solute movement, membrane flexibility, enzyme activity, and large molecule interactions.³⁰ Microorganisms that survive in cold environments rely on adaptive strategies to keep their fundamental cellular processes intact.³⁰ In reaction to a quick temperature drop, many bacteria produce small cold shock proteins.³¹ The cold-shock protein, CspA, was found to be significantly upregulated during the cold-shock response.³² In the present study, CspA was also noticed in K. polaris CMS 76or^T. Universal stress proteins (USP) are key regulatory stress proteins that help bacteria survive in stressful environments.³³ In the present study, USP (ABCDEFG) was noticed in K. polaris CMS 76or^T. Trehalose production was reported to play a significant role in resistance to freezing in cold environments.³⁴ In the present study, The genes encoding trehalose biosynthesis were noticed in K. polaris CMS 76or^T.

A sudden drop in temperature can cause phase separation of cell membrane phospholipids, resulting in decreased membrane fluidity and increased permeability.³⁵ Palmitoleate has been shown to increase the flexibility of cell membranes while decreasing the temperature at which phase transition occurs. This helps to mitigate the adverse effects of cold temperatures. When the temperature drops, in certain bacteria, the cold-induced acyltransferase LpxP induces the attachment of palmitoleate to lipid A.³⁶ The adaptation of membrane fluidity also involves the fast desaturation of fatty acids in pre-existing phospholipids. This is accomplished by the activation of fatty acid desaturase (Des), which is regulated by the sensor kinase DesK and the response regulator DesR.³⁷ Genome analysis of K. polaris CMS 76or^T revealed the presence of LpxP, DesK and DesR.

K. polaris CMS 76or^T was also reported to tolerate NaCl up to 2.9%.⁸ Salt-in and salt-out mechanisms help microorganisms regulate osmoregulation.^38,39 Microorganisms employ the salt-in strategy to maintain osmotic equilibrium through the accumulation of large amounts of inorganic salts or ions within their cytosol.^38,39 The salt-out strategy entails the removal of salt ions from the cytoplasm while concurrently accumulating large amounts of compatible solutes.^38-40 Genome analysis of K. polaris CMS 76or^T revealed the presence of genes related to potassium uptake protein (Trk system), and multicomponent Na⁺:H⁺ antiporter. In addition, it was also noticed that K. polaris CMS 76or^T encodes genes for compatible solutes like betaine, proline, and trehalose.

Taxonomic position re-evaluation
In the present study, the 16S rRNA gene sequence extracted from K. polaris CMS 76or^T and K. rosea ATCC 186^T genome showed 99.7% similarity to each other. Even in the original article K. polaris CMS 76or^T was reported to share 99.8 and 71% 16S rRNA gene sequence and DNA–DNA hybridization (DDH) similarity with K. rosea ATCC 186^T, respectively.⁸ The 16S rRNA gene sequence similarity was above the threshold value (98.7%), while the DDH value was close to the threshold value (70%) for species delineation.⁴¹ In the phylogenomic tree (Figure 2), K. polaris CMS 76or^T and K. rosea ATCC 186^T clustered together.

The proposed cut-off values for ANI and dDDH values for species delineation were 95-96% and 70%, respectively.^14,42,43 The ANI value and dDDH value between K. polaris CMS 76or^T and K. rosea ATCC 186^T were 98.7 and 87.6%, respectively which were above the cut-off value indicating they are the same species. Based on the above results we propose to reclassify Kocuria polaris as a later heterotypic synonym of Kocuria rosea.