*3.2. Isolation and Analysis of DNA from the Strains*

All strains of marine bacteria were cultivated in Petri dishes on sterile agar LB medium of the following composition (g/L): bacto-tryptone—10; yeast extract—5; NaCl—5; agaragar—15; distilled water—0.98 l; the pH of the medium is 7.7. Five passages were made for each type of bacterium. Then, DNA was isolated from the grown colonies using the PureLink Genomic DNA Mini Kit protocol (Invitrogen). The isolated bacterial DNA was used to carry out a polymerase chain reaction (PCR), with universal primers to 16S rRNA sequences (BF/20: 5- -AGAGTTTGATCMTGGCTCA -3- ; BR2/22: 5- - TACGGTTACCTTGT-TACGACTT -3- ) or the primers corresponding to the coding DNA sequences (CDSs) for housekeeping enzymes used in this work as molecular markers for the *Cobetia* species identification (Table 2).


**Table 2.** Oligonucleotides for molecular differentiation of *Cobetia* species.

\* The molecular markers are the enzymes, predicted according to the structural classification of the National Center for Biotechnology Information (NCBI) Conserved Domain Database [23]; \*\* the reference genes' IDs are from the whole genome sequence annotation of the *C. amphilecti* KMM 296 (GenBank ID: JQJA00000000.1).

> Reaction conditions for PCR of 16S rRNA sequence in DNA amplifier C1000TM Thermal Cycler (Bio-Rad Laboratories, Inc., California, USA) or Mastercycler gradient (Eppendorf, Hamburg, Germany): 10× buffer for polymerase, 50× mixture of Encyclo polymerases ("Encyclo PCR kit", Evrogen, Moscow), 50× mixture of dNTP (10 mM each),

mixture of forward and reverse primers (5 μM each), and 20 ng DNA of a bacterial clone. The amplification process consists of the following stages: 30 PCR cycles × (15 s—95 ◦C; 30 s—55 ◦C; 1 min 30 s—72 ◦C), then incubation at 72 ◦C for 5 min. After amplification, PCR products were used for sequencing. The PCR products from each clone of each strain (four replicates) were sequenced and verified with the use of an ABI Prism 3130xl sequencer and Chromas program (version 2.5.1), respectively. Homology searches were performed against EzBioCloud 16S database using the Blast program to find sequences that provide significant alignment [22].

Reaction conditions for marker genes (Table 2) in DNA amplifier C1000TM Thermal Cycler (Bio-Rad Laboratories, Inc., California, USA) or Mastercycler gradient (Eppendorf, Hamburg, Germany): 10× Encyclo buffer, 50× Encyclo polymerase mixture (Encyclo PCR kit, Evrogen, Moscow), 50× dNTP mixture (10 mM each), a mixture of forward and reverse primers (5 μM each), and 20 ng DNA of a bacterial clone. The amplification process consisted of the following stages: 38 PCR cycles x (2 min—95 ◦C; 15 s—95 ◦C; 1 min 40 s—72 ◦C). After amplification, the PCR products from each clone of each strain (four replicates) were separated by gel electrophoresis in a 1% agarose gel stained with ethidium bromide. The PCR product visualization and documentation were performed with Herolab imaging system (Herolab GmbH Lab., Wiesloch, Germany). The PCR products for the species-specific coding sequencing regions of *C. amphilecti* and *C. litoralis* were confirmed by sequencing as described above.

Comparative analysis of the marker genes and whole genome sequences of *Cobetia* sp. 2AS1 (GenBank: JADAZN000000000.1) and *C. amphilecti* KMM 296 (GenBank: JQJA-00000000.1) was carried out by the SEED Viewer at the RAST server [31].
