Bacteria of the family Halomonadaceae (class Gammaproteobacteria) are gram-negative, weakly or moderately halophilic, non-sporulating aerobic microorganisms. Members of this family, which comprises 14 genera, are widely present in saline environments, such as salty lakes, saline soils, solonchaks, marine ice, seafood, marine invertebrates, waste waters, seawater, and hydrothermal springs (de la Haba et al., 2014). Bacteria of the family Halomona-daceae have been attracting researchers’ attention due to their ability to synthesize osmoprotectors (ectoine, glycine betaine, and hydroxyectoine), biopolymers (exopolysaccharides and polyhydroxyalkanoates), and biosurfactants, as well as to mediate degradation of aromatic compounds (García et al., 2005; de la Haba et al., 2014; Monzón et al., 2018). These bacteria are considered promising agents for biotechnological purposes, for instance, for remediation of saline soils and water bodies from toxic organic pollutants. A large number of studies have reported degradation of aromatic compounds by members of the family Halomonadaceae, either individually or in bacterial associations (Le Borgne et al., 2008; Fathepure, 2014). An important intermediate produced by bacterial oxidative catabolism of many aromatic compounds, including environmental pollutants, such as phenol, toluene, biphenyl, or phthalates, is benzoate (Moreno et al., 2011; Li et al., 2013). Benzoate accumulation in the environment is also related to metabolic activity of plants, as well as to anthropogenic activity, since benzoic acid and its derivatives serve as a source for production of a broad range of chemical compounds; they are also used in food preservation, medicine, and perfumery industry (García et al., 2005; Le Borgne, 2008; Li et al., 2013). Among Halomonadaceae, the ability to degrade benzoate has been described in H. halodurans ATCC 29686T (Rosenberg, 1983), H. organivorans CECT 5995T (Moreno et al., 2011), H. elongata A15.6, H. eurihalina A17.6, A25.2 (Garcia et al., 2005), H. campisalis ATCC 700597T (Oie et al., 2007), Halomonas sp. KHS3 (Monzón et al., 2018), Chromohalobacter salexigens DSM 3043T (Csonka et al., 2005), and Chromohalobacter sp. HS-2 (Kim et al., 2008). However, the currently available information on the structure and functioning of the genetic systems and the metabolic pathways that mediate the degradation of aromatic compounds under conditions of saline environments in this microbial group is extremely limited (Fathepure, 2014).

The metabolic pathway of benzoate degradation most commonly occurring in prokaryotes begins with incorporation of hydroxy groups into the chemically stable aromatic ring of the molecule with production of dihydroxybenzoate (catechol); this step is mediated by benzoate 1,2-dioxygenase (benzoate 1,2-DO; EC 1.14.12.10) and dihydroxybenzoate dehydrogenase (EC 1.3.1.25) (https://www.genome.jp/pathway/ map00362+C00180). It was shown that the substrate specificity of benzoate 1,2-DO is determined by its α‑subunit (Parales and Resnick, 2006); therefore, the benA gene encoding this subunit is frequently employed as a genetic marker indicating the possibility of benzoate 1,2-DO induction in bacterial cells.

Previously, individual bacterial strains of the family Halomonadaceae and bacterial consortia that included members of this family were isolated at the mining site of the Upper Kama deposit of potassium and magnesium salts, which is characterized by a high salinity level and the presence of various organic pollutants, including mono- and polyaromatic compounds (Anan’ina et al., 2005; Bachurin and Odintsova, 2009; Korsakova et al., 2013; Olsson et al., 2017; Pyankova et al., 2020). Several strains of the genus Halomonas were capable of growing on naphthalene, ortho-phthalic, protocatechuic, gentisic, and benzoic acids as sole sources of carbon and energy (Yastrebova et al., 2019).

The goal of the present work was to characterize benzoate-degrading bacteria of the family Halomonadaceae isolated from various ecotopes of the salt mining area of the Upper Kama deposit and to describe the diversity of key genes of benzoate degradation (benA) in these halophilic strains.

MATERIALS AND METHODS

Specimens. The study was conducted in bacteria of the family Halomonadaceae (genera Halomonas, Chromohalobacter, Kushneria, Salinicola) from the working collection of the Laboratory of Microbiology of Technogenic Ecosystems (Institute of Ecology and Genetics of Microorganisms). These bacterial strains were isolated from various samples collected in the salt mining area of the Upper Kama deposit (near the towns of Solikamsk and Bereziniki, Perm krai): plant rhizosphere, soil, ground, clay bottom sediments of mine brine tanks, production waste (slurry storages, salt tailing piles), and brine ponds (Anan’ina et al., 2005; Korsakova et al., 2013; Olsson et al., 2017; Yastrebova et al., 2019; Pyankova et al., 2020). The study also involved the type strains Halomonas taeanensis DSM 16463T, Chromohalobacter canadensis DSM 6769T, Chromohalobacter beijerinckii DSM7218T, Chromohalobacter japonicus CECT7219T, Chromohalobacter salarius CECT5903T, Salinicola socius SMB35T, and Salinicola salarius DSM18044T.

Benzoate-degrading capacity was assessed by culturing the bacteria in liquid Raymond’s mineral medium (RMM) (Raymond, 1961) with different concentrations of NaCl (30‒150 g/L), as well as without salt addition. Benzoate (in the form of 10% sodium benzoate solution) was added to the final concentration of 1 g/L. Cultures were grown for 14 days in a thermostated shaker at 28°C and 140 rpm. Growth was evaluated by measuring the optical density (OD) of the culture liquid at the wavelength of 600 nm using a UV-Visible BioSpec-mini spectrophotometer (Shimadzu, Japan) and a cell with the optical pathway length of 1 cm.

To assess benzoate utilization, bacterial cells were removed from the culture liquid by centrifugation at 9660 g for 3 min in a miniSpin centrifuge (Eppendorf, Germany). The presence of benzoate in the supernatant was determined using HPLC analysis in the acetonitrile–0.1% H3PO4 (60 : 40) system on an LC-20AD Prominence chromatograph (Shimadzu) with a C-18 column 150 × 4.6 mm (Sigma-Aldrich, United States) and a SPD-20A UV detector (at 205 nm). The substrate amounts were evaluated based on the height of the chromatogram peaks and the area under the peaks in comparison to the standard curve obtained for 0.01% benzoate solution in water. Experiments were performed in three replicates.

Identification of benzoate-degrading bacteria. DNA was isolated from pure bacterial cultures using the conventional technique (Short Protocols in Molecular Biology, 1995). Bacteria were identified based on analysis of the nucleotide sequences of the 16S rRNA genes (fragment length, 915‒1418 bp). Fragments of the 16S rRNA gene were amplified by PCR with the universal bacterial primers 27F (5'-AGAGTTTGATC(A/C)TGGCTCAG-3') and 1492R (5'-АCGG(C/T)TACCTTGTTACGACTT-3') (Lane, 1991) using a C1000 TouchTM Thermal Cycler (Bio-Rad Laboratories, United States). Electrophoresis, sequencing, and analysis of the obtained 16S rRNA gene sequences were performed as described below.

The key genes of benzoate degradation (benA) were studied by amplifying a 521-bp-long fragment of the gene encoding the α-subunit of benzoate 1,2-DO with the primers benA-F (5'-GCCCACGAGAGCCAGATTCCC-3') and benA-R (5'-GGTGGCGGCGTAGTTCCAGTG-3') as described (Baggi et al., 2008) with subsequent sequencing of the amplicons and analysis of their sequences.

PCR products were detected by horizontal electrophoresis in a 1% agarose gel in 1× TBE buffer (Tris, 10.8 g/L, borate, 5.5 g/L, 0.5 M EDTA, 4 mL, distilled water, 79.7 mL/L) at 5‒15 V/cm and room temperature for 20‒40 min. The gels were stained with 0.5 μg/mL ethidium bromide solution for 10‒15 min and photographed in UV light using the BioDocAnalyze gel documentation system (Bio-Rad Laboratories). The size of the amplified fragments was determined using the 100+ bp DNA Ladder (Eurogene, Russia).

Nucleotide sequences of 16S rRNA and benA genes were determined on an automated Genetic Analyzer 3500XL system (Applied Biosystems, United States) using the Big Dye Terminator Cycle Sequencing Kit v3.1 (Applied Biosystems) according to the manufacturer’s protocol. Phylogenetic analysis of the 16S rRNA and benA gene sequences was conducted using the programs Sequence Scanner v. 2.0. and MEGA 7.0 (http://www.megasoftware.net), as well as the BLAST service (http://www.ncbi.nlm.nih.gov). The search for homologous sequences was performed in the international databases EzBioCloud (http://www.ezbiocloud.net) and GenBank (http://www.ncbi. nlm.nih.gov). Phylogenetic trees were constructed using the Neighbor-Joining algorithm of MEGA 7.0. Statistical significance of the tree branching was assessed by bootstrap analysis based on 1000 alternative trees.

The nucleotide sequences of the 16S rRNA and benA genes of the strains studied were deposited in the GenBank database under the accession numbers MW757272‒MW757286, MZ359852‒MZ359857 (16S rRNA gene), and MW862486‒MW862498 (ben A).

RESULTS AND DISCUSSION

Screening for the ability to utilize benzoate as growth substrate. One hundred and twenty four strains of the family Halomonadaceae (genera Halomonas, Chromohalobacter, Salinicola, and Kushneria) isolated previously from various ecotopes of the salt mining area of the Upper Kama deposit, as well as seven type strains of the genera Halomonas, Chromohalobacter, and Salinicola, were tested for their ability to grow on benzoate as the sole carbon and energy source. Among the 63 analyzed strains representing different species of the genus Halomonas, 29 strains (including H. taeanensis DSM 16463T) exhibited growth on RMM with benzoate in the presence of 30 g/L NaCl. Growth on benzoate was observed in bacteria closely related to the type strains of the species H. taeanensis, H. olivaria, H. ventosae, H. titanicae, H. alkaliantarctica, H. neptunia, H. radicis, and H. sulfidaeris, but not in members of H. venusta, H. hydrothermalis, H. utahensis, H. alimentaria, H. meridiana, H. piezotolerans, and H. songnenensis. Twenty-two active benzoate degraders of the genus Halomonas were next investigated in greater detail (Table 1). Among 52 analyzed strains of the genus Chromohalobacter (closely related to C. beijerinckii, C. japonicus, C. salarius, and C. canadensis) and four type strains of the genus, three strains of C. canadensis (including the type strain C. canadensis DSM 6769T) were capable of degrading (transforming) benzoate, as indicated by substrate depletion and changing color of the medium in the course of cultivation (Table 1). No benzoate-degrading activity was detected in six members of the genus Salinicola closely related to the species S. halophyticus, S. socius, and S. salarius, the type strains S. socius SMB35T and C. salarius CECT 5903T, as well as in three strains of the genus Kushneria closely related to K. phyllosphaerae EAod3T.

Table 1. Growth of bacterial strains of the family Halomonadaceae in the presence of various NaCl concentrations
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Characterization of benzoate-degrading bacteria of the genus Halomonas. Bacteria of the genus Halomonas are moderately halophilic microorganisms capable of growing both in the absence of salt and in the presence of high sodium chloride concentrations of up to 325 g/L (de la Haba et al., 2014). We found that all the strains isolated from various ecotopes of the salt mining area could grow on Raymond’s rich medium (Yastrebova et al., 2019) with 30 to 150 g/L NaCl. Several strains (Halomonas spp. PD13-5, PMK3, D2, 610-2, 61g, and H. taeanensis DSM 16463T) did not grow in the absence of salt, and the optimal NaCl concentration in the medium was 70‒150 g/L. Furthermore, the strains NDT27, D2, NDT28, NDT31, and H. taeanensis DSM 16463T were able to grow at higher NaCl concentrations (200‒300 g/L). The strains were tested for their ability to grow on RMM with benzoate in the presence of various NaCl concentrations (Table 1). For most strains, the highest biomass yield as determined by the optical density of the culture was observed in the medium containing 30 g/L NaCl. A the same time, a number of strains (NDT27, D2, BNL26, BBL18, BBL22, M56-2, M135-4Nt, 61g, and 610-2) exhibited active growth at higher salt concentrations: 50 and 70 g/L NaCl. None of the strains studied could grow on benzoate at salt concentrations of 100 g/L and higher.

In this study, we determined and analyzed nearly complete 16S rRNA gene sequences (1350‒1418 bp) of 14 benzoate-degrading strains: BBL18, BNL26, BNL3-2, BFL1, PD13-5, NDT13, BBL22, NDT27, D2, 610-2, 61g, NDT21, NDT31, and NDT28. The strains SMB56, M56-2, M135-4Nt, SMB61, TC193, and TC195 were for the first time identified based on analysis of their 16S rRNA genes. Comparison of the nucleotide sequences of the 16S rRNA genes of the bacteria studied (915‒1418 bp long) and the type strains of the genus Halomonas (http://www.ezbiocloud.net) showed that active benzoate degraders were phylogenetically close to the species H. alkaliantarctica, H. alkaliphila, H. neptunia, H. olivaria, H. sulfidaeris, H. taeanensis, H. titanicae, H. ventosae, and H. radices. Based on the 16S rRNA gene sequences, six strains had 99.24‒100% identity to the marine strain H. titanicae BH1T (Sanchez-Porro et al., 2010), four strains were closely related (99.29‒100% identity) to H. taeanensis DSM 16463T (Lee et al., 2005), and five strains had 99.62‒99.85% identity to H. alkaliantarctica CRSST isolated from an Antarctic salt lake (Poli et al., 2007). Other strains exhibited high (99.02‒100%) similarity to the species H. olivaria (strains NDT21 and NDT31, 99.93‒100%), H. ventosae (strains 610-2 and PMK3, 99.06‒99.42%), H. neptunia (NDT28, 99.57%), H. sulfidaeris (M135-4Nt, 99.02%), and H. alkaliphila (M56-2, 99.79%). It should be mentioned that the 16S rRNA gene sequence (1408 bp) of the strain 61g isolated from the bottom sediment of the brine collector at the mining site had low similarity (98.41%) to the closest type strain H. radicis EAR18T (Navarro-Torre et al., 2020), suggesting that this strain may represent a novel taxon of the family Halomonodaceae. The positions of the strains studied and the closely related type strains of the genus Halomonas in the phylogenetic tree constructed using the neighbor-joining method are shown in Fig. 1.

Fig. 1.
figure 1

Positions of the studied strains of the genus Halomonas in the phylogenetic tree constructed using the neighbor-joining approach based on comparison of the nucleotide sequences of their 16S rRNA genes. The evolutionary distances were calculated using the Jukes-Cantor method. Numbers indicate the statistical significance of the branching order determined by bootstrap analysis of 1000 alternative trees (only the values higher than 50% are shown). The scale bar corresponds to 5 substitutions per 1000 nucleotides. The GenBank accession numbers are given in parentheses.

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Diversity of benA genes in bacteria of the genus Halomonas. PCR amplification of the benA genes encoding the α-subunit of benzoate 1,2-DO showed that all 22 benzoate-degrading strains of the genus Halomonas, including H. taeanensis DSM 16463T, possessed benA genes. Twelve strains of different species affiliation (Table 2) were selected for further sequencing and sequence analysis of their benA gene amplicons. The sequences of these benA amplicons were compared to homologous sequences from the GenBank database, and it was found that the sequences in question had most similarity (no higher than 97.28%) to genes encoding the α-subunits of benzoate 1,2-DO, 2-chlorobenzoate 1,2-DO, and a Rieske cluster-containing protein of bacteria of the genus Halomonas (Table 2). The sequences of the benA genes of the strains BBL18, M135-4Nt, and H. taeanensis DSM 16463T exhibited similarity to genes of a strain representing the genus Chromohalobacter (C. salexigens 40a_TX): benzoate/toluate 1,2-DO (83.82‒82.04%) and benzoate 1,2-DO (80.87 and 82.25% for BBL18 and H. taeanensis DSM 16463T, respectively). The benA and homologous genes of other members of Gammaproteobacteria, as well as of strains representing Betaproteobacteria, Alphaproteobacteria, and Actinobacteria, had lower level of identity to the benA genes studied: the highest value of 80.54% was observed for Marinobacter hydrocarbonoclasticus strain ATCC 49840T, class Gammaproteobacteria (Table 3).

Table 2. Comparison of the benA gene sequences of strains of the genus Halomonas to homologous sequences from the GenBank database
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Table 3. Comparison of the nucleotide sequences of the benA genes of strains of the genus Halomonas (family Halomona-daceae, class Gammaproteobacteria) to the closest homologous sequences of bacteria of other taxa (BLAST analysis)*
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The phylogenetic tree in Fig. 2 was constructed for the translated amino acid sequences (TASs) encoded by the benA genes of the benzoate-degrading strains of the genus Halomonas, as well as by the closest homologous genes of Halomonas members and of species representing other taxa of proteobacteria (classes Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria) and actinobacteria. In this tree, benA TASs of Halomonadaceae strains (genera Halomonas and Chromohalobacter) formed a separate phylogenetic group clearly divided in several clusters (Fig. 2). In particular, the largest cluster included TASs of eight strains: NDT28, M56-2, M125-1, M135-4Nt, PD13-5, 8CN1-1, BBL18, and BBL22, which are phylogenetically close to different species of the genus Halomonas (H. alkaliantarctica, H. alkaliphila, H. neptunia, H. sulfidaeris, H. titanicae, and H. utahensis) based on their 16S rRNA gene sequences. A BLAST search in the genomes of the type strains of these species showed that they lacked benA genes. At the same time, TASs closely related to this group of genes were detected in the genomes of H. sulfidaeris SST4 (QNTU01000040) and Halomonas sp. HL-93 (LJST01000014) (Fig. 2). The benA sequences of this cluster had the highest level of identity to the genes of the large subunit of benzoate/2-halobenzoate 1,2-DO and a Rieske cluster-containing protein of Halomonas strains isolated from marine ecosystems (Table 2, Fig. 2). The amino acid sequences encoded by the benA genes of the strains BBL18 and BBL22 formed a separate branch together with the sequences of 2‑halo(chloro)benzoate 1,2-DO of two Halomonas strains (Fig. 2).

Fig. 2.
figure 2

Positions of the benA genes of the Halomonas strains studied in the phylogenetic tree constructed using the neighbor-joining approach based on comparison of the amino acid sequences translated from these benA genes. The evolutionary distances were calculated using the p-distance method. Numbers indicate the statistical significance of the branching order determined by bootstrap analysis of 1000 alternative trees (only the values higher than 50% are shown). The scale bar corresponds to 2 amino acid substitutions per 100 amino acids. The GenBank accession numbers are given in parentheses.

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Based on the 16S rRNA gene sequences, the strains SMB56, NDT27, and D2 are phylogenetically close to H. taeanensis DSM 16463T, and the strain 610-2 is close to H. ventosae Al12T. Comparison of the benA sequences of these strains and of H. taeanensis DSM 16463T to homologous sequences from the GenBank database showed that their similarity was the highest to the genes of the α-subunit of benzoate 1,2-DO of the strain H. aestuarii Hb3 isolated from a solar saltern in South Korea and the phenol-degrading strain H. organivorans CECT 5995T isolated from saline soil in the south of Spain, as well as to a gene encoding a Rieske cluster-containing protein in a Halomonas sp. strain BM-2019 isolated from lake water in Tanzania (Table 2). In the phylogenetic tree, the benA TASs of the strains SMB56, NDT27, and D2 (closely related to H. taeanensis) and that of the strain 610-2 (closely related to H. ventosae) formed two separate clusters (Fig. 2). Interestingly, analysis of the genomes of the Halomonas strains present in the GenBank database (https://www.ncbi.nlm.nih.gov/) revealed that the type strain H. ventosae CECT 5797T (SNZJ01000041) and the strain H. ventosae USBA 854 (PVTM01000013) possess genes of the α-subunit of benzoate/toluate 1,2-DO. The level of identity between the benA sequences of the strain 610-2 and the homologous sequences detected in the genomes of H. ventosae strains was 89.66‒90.44% (Table 2). In the phylogenetic tree, the corresponding TASs belonged to the same cluster as the benA TAS of strain 610-2 (Fig. 2). It was also found that the benA TASs of the strains SMB56, NDT27, D2, and the type strain H. taeanensis DSM 16463T (FNCI01000003) formed a separate branch in the tree. The nucleotide sequences of the benA genes of these strains had 96.13‒98.73% homology. Interestingly, these strains also formed a single cluster in the tree constructed based on the 16S rRNA gene sequences (Fig. 1). Thus, it was established that the benA genes of the strains closely related to H. taeanensis on the one hand and to H. ventosae on the other hand are phylogenetically distinct from each other, as well as from those of other species of the genus Halomonas.

Characterization of benzoate-degrading bacteria of the genus Chromohalobacter. Among the 52 Chromohalobacter strains isolated from various ecotopes of the salt mining area (Korsakova et al., 2013; Olsson et al., 2017; Pyankova et al., 2020), a majority of 35 strains were closely related to C. canadensis (99.40‒99.93% 16S rRNA gene sequence identity to C. canadensis ATCC 43984T), 16 strains were related to C. japonicus (99.54‒99.78% identity to C. japonicus 43T), and one strain was close to C. beijerinckii (99.85% identity to C. beijerinckii ATCC 19372T). The preliminary screening detected only two strains closely related to C. canadensis that were capable of degrading (transforming) benzoate (Table 1). The signs of benzoate degradation were substrate loss and appearance of dark coloration of the medium that increased in the course of strain cultivation in RMM with benzoate in the presence of 30 and 50 g/L NaCl (Table 2). For instance, for strains 55 and B201, substrate loss after 5 days of incubation in the presence of 30 g/L NaCl constituted 10 and 11% of the initial amount (1 g/L), respectively. Based on the available data concerning the metabolic pathways of benzoate degradation in bacteria (https://www.genome.jp/pathway/ map00362+ C00180), it can be supposed that benzoate was oxidized to hydroxylated aromatic metabolites (Li et al., 2013), which were not further degraded by the enzyme systems of the strains studied and accumulated in the medium. Additional experiments are required to identify the products of benzoate degradation. Our study showed that the type strain C. canadensis DSM 6769T exhibited similar activity when growing on benzoate in the presence of 30 g/L NaCl (Table 1); the substrate loss constituted 33% after 5 days. The type strains C. beijerinckii DSM 7218T, C. japonicus CECT 7219T, and C. salarius CECT 5903T did not degrade or transform benzoate.

PCR with the benA-specific primers (Baggi et al., 2008) and DNA templates of the Chromohalobacter strains studied did not detect the target nucleotide sequences (benA genes). The results of our experiments suggest that benzoate transformation in the Chromohalobacter strains studied involves enzyme systems different from the classical pathway of benzoate degradation in bacteria (Li et al., 2013). Analysis of the genome of C. canadensis DSM 6769T showed that it lacked benA genes, which confirms this notion. A search in the GenBank database identified benA genes in only two strains of the genus Chromohalobacter: a gene encoding the α‑subunit of benzoate 1,2-DO (EU155151) in Chromohalobacter sp. HS-2 and genes of the α-subunit of benzoate 1,2-DO and benzoate-toluate 1,2-DO (QGTY01000032 and QGTY01000001) in C. salexigens 40a TX (Fig. 2, Table 3).

To sum up, our study of diversity of benzoate-degrading bacteria of the family Halomonadaceae in microbial communities of the salt mining area of the Upper Kama deposit (Perm krai) showed that, among the vast variety of isolated and characterized members of the family (genera Halomonas, Chromohalobacter, Salinicola, and Kushneria), active benzoate degraders represented the genus Halomonas. The degrader strains were phylogenetically close to H. alkaliantarctica, H. neptunia, H. olivaria, H. taeanensis, H. titanicae, H. ventosae, H. radices, and H. utahensis, which have not been previously described to be capable of degrading benzoate. It was shown that the identified strains of the genus Halomonas can utilize benzoate as the only source of carbon and energy when growing in the mineral medium in the presence of 30–70 g/L NaCl. It is known that the industrial site of the salt mining area is characterized by the presence of large amounts of organic pollutants, including various mono- and polyaromatic compounds, and high levels of salinity (Bachurin and Odintsova, 2009; Korsakova et al., 2013). Our study showed that halophilic bacteria of the genus Halomonas can degrade benzoate and thus contribute to destruction of aromatic pollutants as members of the unique microbial communities developing in this industrial area. For the first time, the diversity of benA genes involved in the initial stage of benzoate oxidation has been studied in members of the genus Halomonas (isolated in the Upper Kama deposit region). Analysis of the benA nucleotide sequences and the corresponding amino acid sequences showed that the identity level was the highest for homologous sequences of the members of the genera Halomonas and Chromohalobacter (Tables 2, 3; Fig. 2). In the phylogenetic tree of amino acid sequences translated from benA genes, bacteria of the family Halomonadaceae formed a separate cluster; within this group, the level of homology among the benA nucleotide sequences was 95.50‒80.87%. Amino acid sequences translated from benA genes representing other taxa of proteobacteria and actinobacteria were separated in the tree from the group “Halomonadaceae” (Fig. 2). Recognition of the benA genes of the family Halomonadaceae as a separate group makes them a promising phylogenetic marker that can be employed to investigate the diversity and activity of benzoate degraders, in particular, members of the genera Halomonas and Chromohalobacter. Furthermore, halophilic benzoate-degrading bacteria characterized in the present work are of practical significance for development of new techniques of bioremediation and monitoring of saline soils polluted with toxic organic compounds.