**1. Introduction**

Bacteria of the genus *Cobetia* are Gram-negative, aerobic and halotolerant and belong to the *Halomonadaceae* family. For the first time, a bacterium of the genus *Cobetia* was described in 1970 by Cobet et al. [1] and was originally assigned to the species *Arthrobacter marinus* sp. nov. In further studies, it was assigned to various genera, such as *Pseudomonas* [2], *Deleya* [3], and *Halomonas* [4]. Then, based on an analysis of the 23S and 16S rRNA sequences and amending the description of the species *Halomonas marina*, including new features, the authors [5] proposed assigning it to a new genus, *Cobetia* gen. nov., within the *Halomonadaceae* family. The type species is *Cobetia marina*, with the type strain LMG 2217 (= CIP 104,765 = IAM 14,107 = CECT 4278 = DSM 4741 = NCIMB 1877 = CCUG 49,558 = NBRC 102,605 = JCM 21,022 = ATCC 25374).

At present, a few species are characterized for marine bacteria of the genus *Cobetia*, namely: *C. marina* [5], *C. crustatorum* [6], *C. amphilecti* [7]*, C. pacifica* [7], and *C. litoralis* [7]. The whole genome sequence analysis is presented only for three strains of the genus *Cobetia*, including a type strain *C. marina* JCM 21022T [8], and the non-type strains *C. amphilecti* KMM 296 (formerly *C. marina* KMM 296) [9] and *Cobetia* sp. cqz5-12 [10]. At the same time, more and more information appears about *Cobetia* isolates, which are promising sources of unique enzymes and secondary metabolites degrading oil [11], bacterial biofilms [12], alginate [10,13], and phenol [14]. However, their species identification are complicated due to the high level of identity for their 16S rRNA genes and the absence of whole genome sequences for the type strains of known species, excluding from *C. marina* JCM 21022<sup>T</sup> [8]. The 16S rRNA gene usually is highly specific to each bacterial species that makes it a good target for identification of both environmental and clinical bacterial isolates [15–17]. However, the 16S rRNA sequences were found to be indistinguishable for a few species [18].

**Citation:** Noskova, Y.; Seitkalieva, A.; Nedashkovskaya, O.; Shevchenko, L.; Tekutyeva, L.; Son, O.; Balabanova, L. Are the Closely Related *Cobetia* Strains of Different Species? *Molecules* **2021**, *26*, 690. https://doi.org/ 10.3390/molecules26030690

Academic Editors: Pavel S. Dmitrenok and Francesco Epifano Received: 18 December 2020 Accepted: 26 January 2021 Published: 28 January 2021

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The usage of 16S rRNA gene as a marker is limited for the closely related species, which have a high percentage of the sequence similarity and lack enough variations. Thus, a conventional method of 16S rRNA phylogeny has often failed to correctly identify *Vibrio* species [19]. Similarly, we found that the *Cobetia* species have 99–100% identity of their compared 16S rRNA sequences. In this regard, searching for the additional molecular markers and/or methods for the species identification of the *Cobetia* isolates is especially relevant.

In this work, we have applied the genome-based found species-specific coding sequences for the essential enzymes from each functional category and different structural families, such as nucleases, proteases, phosphatases, and phospholipase, as the additional molecular markers in the polymerase chain reaction (PCR)-based method, to differentiate the species of the closely related isolates of the marine bacteria *Cobetia,* collected in the Pacific Ocean from various habitats. In general, we have raised the issue of the possible species reclassification, due to their highly homologous 16S rRNA sequences, PCR-patterns with the use of the additional molecular markers, which were suggested here, and a high similarity between two whole genome sequences of the closely related non-type strains.
