Halophilic bacteria of salt lakes and saline soils of the Peri-Caspian lowland (Republic of Daghestan) and their biotechnological potential

The article presents the results of studying the biodiversity and biotechnological potential of halophilic microorganisms from the thermal highly mineralized Berikey Lake, the salty Lake Tarumovskoye and saline soils of the Peri-Caspian Lowland (Republic of Daghestan). Denitrifying halophilic bacteria of the genus Halomonas and Virgibacillus were identified using microbiological methods and 16S rRNA gene analysis. A new species Halomonas sp. G2 (MW386470) with a similarity of the nucleotide sequences of the 16S rRNA genes is 95 %. Strain G2 is an extreme halophile capable of growing in the range of 5–25 % NaCl (optimum 25 %) and forming a carotenoid pigment. Mesophil, 30–37 °С (optimum 30 °С); neutrophil, pH 6–8 (optimum 7.2–7.4). Strain G2 chemolithotroph; reduces nitrate or nitrite as electron donors; catalase-, amylase-, protease- and β-galactosidase-positive; lipase-, oxidase- and urease-negative. Not able to hydrolyze inositol, indole; produces lysine, gelatin, ectoine; uses citrate and sodium malate as a source of carbon and energy; does not produce ornitin, H2S or acid from d-mannose, sucrose, glycerol, cellobiose, except for lactose and d-glucose. Susceptible to trimethoprim, ciprofloxacin, ofloxacin, kanamycin, vancomycin, rifampicin, cefuroxime, ampicillin, ceftazidime, fosfomycin, clarithromycin, cefepime, cefaclor. The G+C content in DNA is 67.3 %. A distinctive characteristic of the isolate was the production of industrially significant hydrolytic enzymes such as amylase, protease, β-galactosidase, and oxidoreductase (catalase) at a NaCl concentration of 25 % in the medium. Habitat: saline soils on the territory of the Tersko-Kumskaya lowland (Republic of Daghestan, Russia). The rest of the halophilic isolates of H. ventosae G1 (MW386469), H. elongata G3 (MW386471), V. salinarius B2 (MW386472), and V. salinarius B3 (MW386473) had a high degree of similarity (100 %) with the type strains of H. elongata DSM 2581Т and V. salarius DSM 18441Т ; the content of G+C in DNA was 65.8, 66.5, 42.8 and 37.3 %, respectively. The strains had a high biotechnological potential at NaCl concentrations of 5 and 25 % in the medium. The data obtained expanded the understanding of the diversity and ecological significance of denitrifying bacteria in the functioning of arid ecosystems and make it possible to identify strains producing enzymes of industrial importance.


Introduction
The interest in extremophilic microorganisms is relatively high due to their biological uniqueness. A great contribution to the study of natural microbial communities was made by the school of Russian scientists (Zavarzin, 2004;Namsa raev et al., 2010;Bonch-Osmolovskaya, Atomi, 2015). Archaea and highly-specialized bacteria of the genera Alcaligenes, Bacillus, Halobacillus, Virgibacillus, Micrococcus, and Pseudomonas (Wang et al., 2019;Banciu et al., 2020;Begmatov et al., 2020) occupy a dominant place in ecological niches with a high salt content (solar salt works, oceans, seas, hypersaline lakes, saline soils, deserts, plants, saline products) and anthropogenic ecosystems with an increased level of mineralization.
The largest ecosystems on the planet are saline and hypersaline environments (Ghosh et al., 2019). Daghestan is a unique natural province of Russia that has a variety of natural landscapes due to the influence of tectonic processes, the erosive activity of flowing waters, transgressive and regressive dynamics of the Caspian Sea, and arid climate. There are a number of works on the study of microbial communities of various ecological niches of the region: lithotrophic sulfur-oxidizing representatives of sulfide sources, hydrocarbon-oxidizing bacteria of the geothermal source of the Kizlyar field (Chernousova et al., 2008;Gridneva et al., 2009;Khalilova et al., 2014).
The highly mineralized lakes of the Tersko-Kumskaya lowland with high salinity form conditions for the existence of halophilic bacteria. Microorganisms from extreme habitats are the producers of valuable industrially important enzymes, antibiotics; they can participate in soil biodegradation, and are highly resistant to contamination by foreign microflora (Corral et al., 2020).
The study considers the spatial distribution of halophilic microbial communities of halophyte plants, saline soils, and highly mineralized lakes in arid regions of the Peri-Caspian Lowland (Khalilova et al., 2017(Khalilova et al., , 2020. Chemoorganoheterotrophic bacteria of the genera V irgibacillus, Bacillus, Halomonas and Salimicrobium from the phylum Firmi cutes and Proteobacteria have proved to be the main components of the microbial flora of the Tersko-Kumskaya and Tersko-Sulakskaya provinces. A major correlation was revealed between isolated microbial communities and concentrations of chemicals Na, K, Ca, Mg, Cl, Cu, Sr, SO 4 , Cl, and HCO 3 , as one of the main regulators of microbiological activity in soils and lakes.
The aim of the paper is a molecular taxonomic study of isolated halophilic bacteria and their biotechnological potential.

Materials and methods
The objects of research are natural microbial communities of saline reservoirs and soils in the territory of the Peri-Caspian Lowland of the Republic of Daghestan (Khalilova et al., 2020) (Table 1). Samples were taken in July-September 2014.
Morphology of bacterial cells (cell morphology, motility, the presence of spore formation) was studied using Halophilic bacteria of salt lakes and saline soils of the Peri-Caspian lowland (Republic of Daghestan) a light microscope CX21 FS1 (Olympus, Japan) and the PowerShot A640 digital camera (Canon, Japan) at a working magnification of ×600. Ecological and physiological characteristics of growth (temperature, pH, salinity). Effect of NaCl concentration (0, 5, 10, 15, 25 %, weight/volume) in an amount of 2 % of the medium volume for cell growth in liquid and solid media was determined at 30-37 °C in the Binder-115 incubator (USA). The growth was monitored at 24-hour intervals for 7 days by measuring turbidity using a Genesys-20 spectrophotometer (Thermo Spectronic, USA). The effect of temperature (30 and 37 °C) on the growth rate was determined by cultivation under the same conditions. The use of growth substrates (assimilation of organic acids, the formation of acid from carbohydrates, reduction of nitrates to nitrites) was studied using standard methods (Gordon, Smith, 1953;Holt et al., 1997;Netrusov et al., 2005).
The use of electron acceptors. The ability to use nitrate as an electron acceptor was determined using the BD BBL Taxo Differentiation Discs Nitrate (Becton Dickinson and Company, Australia), in compliance with the manufacturer's instructions. The discs were impregnated with a solution containing 40 % potassium nitrate and 0.1 % sodium molybdate. The reduction of nitrate to nitrite was detected by the addition of sulfanilic acid and N,N-dimethyl-α-naphthylamine, which reacts with nitrite to form a red-colored substance-n-sulfobenzenazo-α-naphthylamine (positive result).
In the absence of a change in color after the addition of reagents (negative result), zinc dust was added to determine the presence of unrecoverable nitrate or products other than nitrite.
Determination of oxidoreductases: catalase -using 3 % H 2 O 2 as a substrate in the medium for 24-48 hours, oxidase -by Kovac's method (Steel, 1961). All screening tests for enzymatic activity were performed in three repetitions. The bacteria were incubated at 37 °C for seven days.
G+C content and phylogenetic analysis. Genomic DNA was isolated according to Marmur (1961) and Thomas (Thomas et al., 1997) methods. The DNA nucleotide composition was determined by thermal denaturation (0.5 °C • min -1 ) using a Cary-100 Bio UV-VIS spectrophotometer (Varian, Australia). The GC content in the composition of DNAaccording to the method (Owen et al., 1969). Escherichia coli K-12 DNA (51.7 %) was used as the standard.
For phylogenetic analysis, the DNA was isolated from samples using the modified Birnboim-Doly alkaline DNA
A total of 18 sequences with a minimum length of 1381 nucleotides were used. Bar corresponds to two substitutions per 100 nucleotides. The Boots trap values (500 repeats) are shown next to the tree branches. method (Birnboim, Doly, 1979) and the Wizard technology of Promega (USA) (Bulygina et al., 2002). The concentration of the resulting DNA sample when using this method was 30-50 μg /ml. RNA in the resulting preparation is present in trace amounts (less than 1 %, according to the data of electrophoretic analysis, which are not presented). For polymerase chain reaction (PCR) and further sequencing of PCR fragments of the 16S rRNA gene for each of the studied samples, universal primer systems were used to detect both eubacteria (11f-1492r) (Lane, 1991) and archaea (8fa-A915R) . The volume of the amplification mixture was 50 μl with the following composition: 1× buffer of BioTaq DNA polymerase (17 mM (NH 4 ) 2 SO 4 , 67 mM Tris-HCl, pH 8.8, 2 mM MgCl 2 ); 12.5 nmol of each dNTP, 50 ng of the DNA matrix; 5 pmol of the corresponding primers and 3 units of BioTaq DNA polymerase (Dialat LTD, Russia). The temperature-time profile of the PCR was as follows: the first cycle -94 °C × 9 min, 55 °C × 1 min, 72 °C × 2 min; the next 30 cycles -94 °C × 1 min, 55 °C × 1 min, 72 °C × 2 min; the final cycle -72 °C × 7 min. The PCR products were analyzed by electrophoresis in 2 % agarose gel at an electric field strength of 6 V/cm. Isolation and purification of PCR products were carried out from low-melting agarose using a set of reagents by WizardPCRPreps (Promega, USA), according to the manufacturer's recommendations.
Sequencing of PCR products was carried out at the Center of "Bioengineering" of the Russian Academy of Sciences, Moscow, by the method of (Sanger et al., 1977) using a set of BigDyeTerminatorv.3.1 reagents on the ABIPRIZM 3730 genetic analyzer (Applied Biosystems, Inc., USA). Standard primers were used for sequencing (Camacho et al., 2009).
Analysis of 16S rRNA sequences. The primary analysis of the similarity of the nucleotide sequences of the 16S rRNA genes of the studied strains was performed using the BLAST program on the following web-site: https://blast.ncbi.nlm. nih.gov (Van de Peer, De Wachter, 1994).

Results and discussion
Strains of halophilic bacteria G1, G2, G3, B2 and B3, isolated from salt lakes and salt marshes of the Tersko-Kumskaya and Tersko-Sulakskaya lowlands, grew at a temperature of 30-37 °C and pH 6.4-7.4. The cultures showed steady growth in the agarized elective medium in the presence of 5-25 % NaCl with an optimum of 5, 10, 25 %, which indicated that they belonged to moderate and extreme halophiles following the known classification (Kushner, Kamekura, 1988).
The analysis of the 16S rRNA gene sequence allowed us to determine their phylogenetic position. The 16S rRNA gene sequences of the new halophilic strains were analyzed and compared with the 16S rRNA sequences of the validly described bacterial species. The analysis has shown that the new isolates belong to two genera of bacteria containing halophilic microorganisms Halomonas and Virgibacillus (Table 2, Fig. 1).

Morphology of cells and colonies.
Rod-shaped, gramnegative, mobile bacillus of the size of 0.8-1.0 × 1.5-3.0 µm. The cells were observed singly, in pairs, or in short chains (Fig. 2, d ). Cell mobility was ensured by one or two lateral flagella located on one side of the cell, forming endospores. On an elective solid medium, the strain formed an active growth of colonies of a round shape with a wavy edge, yellow and pale-yellow color with a gloss. With an increase in the concentration of NaCl in the culture medium, a bright carotenoid pigment appeared in the beige-colored colonies. On meat-peptone agar (MPA) -small colonies located close to each other in a chain, rounded shape with a wavy edge, turning into a solid growth; smooth, shiny, light beige with a pinkish tinge. In all variants, a smearing consistency was observed (Fig. 3, c).
Physiology of strain growth (temperature, pH, salinity). When determining the optimal growth parameters, the G2 strain was assigned to mesophiles (30 to 37 °C, 30 °C op-timum) and moderate alkalophiles (pH 6-8, 7.2-7.4 optimum). As a representative of the genus Halomonas, it can grow in a wide range of NaCl concentrations from 10 to 25 % with an optimum of 25 %; an extreme halophile.
The substrates and electron acceptors used. Oxygen ratio. The G2 strain is capable of denitrification by using nitrates as an electron acceptor, reducing them to nitrites.
The differentiating characteristics of the G2 strain are presented in Table 3. The strain had a positive reaction to lysine, gelatin, ectoine, lactose, and d-glucose; utilized citrate and Na malonate. Tests for β-galactosidase, amylase, protease, and catalase are positive; for oxidase, lipase, and urease -negative. Growth does not occur under anaerobic conditions. Antibiotic sensitivity. The G2 culture differed in sensitivity to trimethoprim of the sulfanilamide group; fluoroquinolones of the 1st and 2nd generations (ciprofloxacin, ofloxacin, kanamycin); vancomycin of the macrolide group; rifamycins of the rifampin group; cefuroxime and ampicillin of the penicillin group; antibiotics of the 3rd generation cephalosporins of the macrolides (ceftazidime, fosfomycin and clarithromycin); antibiotics of the 4th generation cephalosporins (cefepime, cefaclor).
The results of phylogenetic analysis of 16S rRNA gene sequences indicated that the closest type strains (100 %) for G1 and G3 are the type strain of H. elongata DSM 2581 T , and for B2 and B3 -V. salarius DSM 18441 T . On the dendrogram, the cultures formed a common cluster with the typical strains, making it possible to classify isolated   (Vreeland et al., 1980;Schwibbert et al., 2011;Kindzierski et al., 2017)  Notе. "+" -positive; "-" -negative; "n" -not studied. The distinctive characteristics differentiating the cultures of H. ventosae G1 and H. elongata G3 were: optimum growth at 5-25 % NaCl vs. 32 %; pH 7.2-7.4 vs. 7-9; no utilization of sucrose, glycerol, d-mannose, cellobiose, lactose, and production of urease, oxidase, and protease (except G3) at a concentration of 5-25 % NaCl in the medium (see Table 3). At the same time, such traits for V. salarius B2 and B3 strains in comparison with the typical V. salarius DSM 18441 T were: no need for d-mannose, the ability to produce amylase, protease, and β-galactosidase enzymes at a concentration of 5-10 % NaCl in the medium ( Table 4).

Characteristics of genera Halomonas and Virgibacillus
Thus, based on phenotypic and genetic studies, we provide a brief description of the strain of the new species Halomo nas sp. G2 and halophilic strains of H. ventosa G1, H. elon gata G3, V. salinarius B2, V. salinarius B3.
Currently, the genus Halomonas includes 91 species, where H. elongata acts as the type species (http://www. bacterio.cict.fr/h/halomonas.html). The Halomonadaceae family was first described in 1988 by combining moderately halophilic and marine bacteria of the genera Deleya and Halomonas (Franzmann et al., 1988). Over the past three decades, many species have been assigned to the genus Halomonas, the domain Bacteria, the phylum Proteobacte ria, the class Gammaproteobacteria, the order Oceanospiril lales, and the family Halomonadaceae; however, at the time of writing, 7 species have been reclassified. Representatives of the genus -gram-negative, facultative anaerobes, aerobes, prototrophs, mesophiles, denitrifying, produce exopolysac-Halophilic bacteria of salt lakes and saline soils of the Peri-Caspian lowland (Republic of Daghestan) Notе. "+" -positive; "-" -negative; "n" -not studied.
charides; they mainly utilize oxygen, nitrate or nitrite as an electron acceptor; under conditions of saline stress, they synthesize ectoine, which protects cells from adverse environmental influences (Schwibbert et al., 2011).
The genus Virgibacillus was created as a result of the reclassification of Bacillus pantothenticus, Virgibacillus pantothenticus and, subsequently, established as a species of Virgibacillus (Heyndrickx et al., 1998;Heyrman et al., 2003). At the moment, the genus consists of 27 species, representatives of which are gram-positive, obligate aerobes or facultative anaerobes, moderate halophiles, chemotaxonomic; the main fatty acid is C 15:0 (Lee et al., 2012).
Based on its physiological, biochemical, and phylogenetic properties, the G2 strain represents a new species, called Halomonas sp. G2. A distinctive characteristic of the isolate is the production of hydrolytic enzymes protease, amylase, β-galactosidase, and oxy-reductase-catalase at a concentration of 25 % NaCl in the medium.
Habitat: saline soils (Tarumovsky district, Kochubey bio sphere station) and Lake Tarumovskoe on the territory of the Tersko-Kumskaya lowland (Republic of Daghestan, Russia). The type strain of H. elongata DSM 2581 Т was isolated from equipment for extraction of salt from brine (Netherlands Antilles, southern island of Bonaire).
Description of V. salinarius B2 (MW386472) and B3 (MW386473) strains. The strains are gram-positive; mesophiles, neutrophils, chemolithotrophs, moderate halophiles (optimum 5, 10 % NaCl). The cultures are unable to hydrolyze inositol, sodium malonate; they do not produce lysine (except for B3), indole, H 2 S and acid from d-mannose, sucrose, except for d-glucose, reduce nitrate to nitrite and are capable of utilizing the polypeptide substrate gelatin and sodium citrate as a carbon source. The GC content in the DNA of strains B2 and B3 was 42.8 and 37.3 %, respectively. Based on the phenotypic and genotypic characteristics, the isolated cultures have been classified as V. salinarius B2 (MW386472) and V. salinarius B3 (MW386473) strains.

Biotechnological potential of halophilic microorganisms
Halophilic bacteria are increasingly being studied for their biotechnological potential for the production of biochemically active and resistant enzymes to alkaline pH, high temperature and salt concentration (Di Donato et al., 2019;Liu et al., 2019). These multifaceted properties are useful for a variety of industries (Delgado-Garcia et al., 2012), such as fermented food, textiles, pharmaceuticals, cosmetics, and leather production (De Lourdes Moreno et al., 2013). Most producers of extracellular hydrolytic enzymes lipase, amylase, protease, inulinase, xylanase, cellulase, DNase, and pectinase are halophilic bacteria, including strains of the genera Halomonas and Virgibacillus (Cira-Chávez et al., 2018;Liu et al., 2019;Kaitouni et al., 2020;Varrella et al., 2020).

Conclusion
The study confirms the biotechnological and scientific importance of halophilic denitrifying bacteria inhabiting the extremophilic ecological niches of the Peri-Caspian Lowland in the Republic of Daghestan. The strains of bacteria of the genera Halomonas and Virgibacillus isolated proved to be not strictly confined to the salt lakes and soils of the Peri-Caspian Lowland (Republic of Daghestan, Russia), having a wide distribution area, including the ecological niches of Bonaire Island (Netherlands Antilles) and Tunisia. Isolation and study of natural strains have revealed a new species Halomonas sp. G2 that complement the collection of the already known strains -producers of industrially useful enzymes such as amylase, protease, lactase, lipase, urease, β-galactosidase, catalase, and oxidase.