Draft genome sequences of strains Salinicola socius SMB35T, Salinicola sp. MH3R3–1 and Chromohalobacter sp. SMB17 from the Verkhnekamsk potash mining region of Russia

Halomonads are moderately halophilic bacteria that are studied as models of prokaryotic osmoadaptation and sources of enzymes and chemicals for biotechnological applications. Despite the progress in understanding the diversity of these organisms, our ability to explain ecological, metabolic, and biochemical traits of halomonads at the genomic sequence level remains limited. This study addresses this gap by presenting draft genomes of Salinicola socius SMB35T, Salinicola sp. MH3R3–1 and Chromohalobacter sp. SMB17, which were isolated from potash mine tailings in the Verkhnekamsk salt deposit area of Russia. The analysis of these genomes confirmed the importance of ectoines and quaternary amines to the capacity of halomonads to tolerate osmotic stress and adapt to hypersaline environments. The study also revealed that Chromohalobacter and Salinicola share 75–90% of the predicted proteome, but also harbor a set of genus-specific genes, which in Salinicola amounted to approximately 0.5 Mbp. These genus-specific genome segments may contribute to the phenotypic diversity of the Halomonadaceae and the ability of these organisms to adapt to changing environmental conditions and colonize new ecological niches.


Introduction
The family Halomonadaceae encompasses a diverse group of moderately halophilic, aerobic, heterotrophic, Gramnegative bacteria that are ubiquitously present in ocean water, marine sediments, saline lakes, salt pans, salty foods, and saline soils [1]. These microorganisms can grow over a wide range of saline concentrations and received considerable attention as model systems to study the physiological and molecular mechanisms of prokaryotic osmoadaptation [2][3][4]. Several members of the family have been investigated because of their biotechnological potential, especially as a source of exopolysaccharides, biosurfactants, biodegradable plastics (polyhydroxyalkanoates), salt-tolerant extracellular enzymes, and compatible solutes [5]. Halomonads were used for the development of energy-efficient unsterile fermentation processes that use seawater-based growth medium [5]. Finally, certain strains of Halomonas can mobilize, degrade and neutralize environmental pollutants and may play a vital role in the process of remediation of contaminated saline environments [6].
Members of the Halomonadaceae are heterogeneous, and many currently recognized species were previously assigned to distantly related families. With the advent of molecular typing techniques in the 1990s, the group underwent a major taxonomic revision and now contains 16 validly named genera [7]. Despite the progress in understanding the diversity of these organisms, our ability to explain ecological, metabolic, and biochemical traits of halomonads at the genomic sequence level remains limited. At the time of the manuscript preparation, GenBank listed 54 genomes of bacteria of the Halomonadaceae family. However, two-thirds of these entries originated from strains of a single genus, Halomonas, while other genera within this genomically diverse group remain strongly underrepresented. Here we address this gap by presenting the description, annotation, and analysis of draft genomes of S. socius SMB35 T , Salinicola sp. MH3R3-1 and Chromohalobacter sp. SMB17, which were isolated from soils affected by potash mine tailings in the Verkhnekamsk salt deposit area of Russia. The genome sequences of strains SMB35 T and MH3R3-1 are the first analyzed Salinicola genomes, and one of these genomes represents a type strain of this genus.

Organism information
Classification and features S. socius SMB35 T (DSM 19940 T ), Salinicola sp. MH3R3-1 and Chromohalobacter sp. SMB17 are moderately halophilic Gram-negative, aerobic, catalase-positive and oxidase-negative, non-spore-forming bacteria that belong to the Halomonadaceae family in the class Gammaproteobacteria ( Fig. 1, Tables 1, 2, and 3). Strains SMB35 T and SMB17 were isolated from saline soil collected in an area affected by potash mining activity near the town of Solikamsk, Russia. Salinicola sp. MH3R3-1 was isolated from the rhizosphere of salt sand-spurrey (Spergularia salina), a halotolerant plant commonly found around salt mine tailings in the sampled area. The isolation and subsequent maintenance were carried out at 28°C in RMM medium [8] supplemented with 6% (w/v) NaCl, 0.5% (w/v) tryptone, and 0.25% (w/v) yeast extract. Cells of SMB35 T and SMB17 are straight rods (0.5-0.6 × 1.1-1.4 μm) with a single polar flagellum (Fig. 1). On RMM agar both strains form yellow, circular and convex colonies with smooth margins. Cells of the strain MH3R3-1 are straight motile rods that are 0.5-0.6 × 1.1-1.4 μm in size (Fig. 1). Colonies on spread plates are cream, circular and convex with an entire margin.
S. socius SMB35 T (DSM 19940 T ) is the type strain of this species with a well-defined status within the family Halomonadaceae [9,10], but the taxonomic placement of strains MH3R3-1 and SMB17 was not characterized in details. We compared partial (1333 bp) 16S rDNA sequences of MH3R3-1 and SMB17 to those of type strains (n = 53) representing main phylogenetic groups recognized within the family Halomonadaceae [10,11]. Results of the analysis revealed that the strain SMB17 belonged to the genus Chromohalobacter and was closely related to C. japonicus, whereas MH3R3-1 was most similar to Salinicola salarius and S. socius (Fig. 2).

Genome sequencing information
Genome project history Strains S. socius SMB35 T , Salinicola sp. MH3R3-1 and Chromohalobacter sp. SMB17 were selected for genome sequencing on the basis of their phylogenetic position and ability to produce compatible solutes ectoine and hydroxyectoine. Genome sequences provide insights into genetic diversity of halophilic bacteria associated with soils affected by potash mining activity in the Perm region of Russia. Genome sequencing was performed by the AgriLife Genomics and Bioinformatics Service at Texas A&M University (College Station, TX). Draft genome sequences of strains SMB35 T , MH3R3-1, and SMB17 are listed in the Genomes Online Database [12] of the JGI under project IDs Gp0136514, Gp0191572, and Gp0191573, respectively. The corresponding WGS projects have been deposited at GenBank under accession numbers MSDO00000000, MSDP00000000, and MSDQ00000000. A summary of the genome sequencing projects and their compliance with MIGS version 2.0 standards [13] is provided in Table 4.

Growth conditions and genomic DNA preparation
Strains SMB35 T , SMB17, and MH3R3-1 were cultured with shaking at 28°C in liquid RMM medium. The cells were pelleted by centrifugation and the genomic DNA was extracted using the UltraClean Microbial DNA Isolation Kit (MoBio Laboratories, Carlsbad, CA, USA) according to the manufacturer's recommendations. DNA concentrations were measured fluorometrically by using

Genome sequencing and assembly
Samples of purified DNA from strains SMB35 T , MH3R3-1, and SMB17 were used to construct shortinsert paired-end libraries with an average insert size of 320 bp, which were sequenced using a HiSeq 2500 instrument (Illumina, San Diego, CA, USA). Raw reads were filtered using a FastQC toolkit [14] followed by assembly with the ITMO Genome Assembler v. 0.1.3 [15]. For SMB35 T , the sequence run produced 2,530,015 × 2 reads totaling 1,265,007,500 bp of data. A k-mer length of 53 nucleotides was chosen to optimize for the highest N 50 . The resulting assembly had 164 contigs with a total length of 4.19 Mb (N 50 , 199,518 bp; N max , 370,185 bp; median coverage, 302×). Sequencing of the MH3R3-1 sample yielded 2,690,448 × 2 reads that totaled 1,345,224,000 bp of data. The maximum k-mer value of 63 was chosen for further analysis since it yielded the best combination of N 50 and genome length. The final assembly had 159 contigs with a total length of 4,121,883 bp (N 50 , 139,268 bp; N max , 370,744 bp; median coverage, 326×). The final assemblies of SMB35 T and SMB17 genomes were aligned and ordered using progressive Mauve v.2.3.1 [16]. For SMB17, a total of 2,690,448 × 2 reads were obtained that yielded 1,201,941,000 bp of data. The final 3,775,557-bp assembly was based on the k-mer value of 63 and consisted of 118 contigs (N 50 , 109,330 bp; N max , 468,741 bp; median coverage, 318×) that were aligned and ordered against Phylum: Proteobacteria TAS [42] Class: Gammaproteobacteria TAS [43] Order: Oceanospirillales TAS [44] Family: Halomonadaceae TAS [1] Genus: Salinicola TAS [1,9] Species: S. socius TAS [9] (Type) strain: SMB35 T TAS [9] Gram stain Negative TAS [9] Cell shape Rod TAS [9] Motility Motile by means of a single polar flagellum TAS [9] Sporulation Non-sporulating TAS [9] Temperature range Mesophilic TAS [9] Optimum temperature 34°C TAS [9] pH range 6.0-8.0 TAS [9] Carbon source D-glucose, D-galactose, D-xylose, D-maltose, D-trehalose, D-fructose, glycerol, mannitol, sorbitol, acetic acid Evidence codes -IDA: Inferred from Direct Assay; TAS: Traceable Author Statement (i.e., a direct report exists in the literature); NAS: Non-traceable Author Statement (i.e., not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). These evidence codes are from the Gene Ontology project the finished genome of Chromohalobacter salexigens DSM 3043 T [17].

Genome annotation
The genomes were annotated using the RAST v.2.0 pipeline [18]. Additional gene prediction analysis and functional annotation were performed using Geneious v.8.1.8 (Biomatters, Auckland, New Zealand) and tools implemented in PATRIC [19] and JGI Integrated Microbial Genomes [20] databases. Putative membrane and secreted proteins were identified using TMHMM v.2.0 [21] and SignalP 4.1 [22] servers, respectively. For SignalP analysis, the Dmaxcutoff value of 0.57 was used for sequences with transmembrane helices and 0.51 for sequences without. Secondary metabolite production clusters were identified using the anti-SMASH program [23]. The analysis of the transporter potential was conducted using the Transporter Automatic Annotation Pipeline [24]. Genomic islands were identified and visualized using the IslandViewer 3 [25]. Circular comparative maps of the genomes of the strains SMB35 T , SMB17, and MH3R3-1 were generated using the BRIG software [26].

Genome properties
The genome of S. socius SMB35 T is comprised of a 4,185,599-bp long chromosome with a 62.2% G + C content. The coding regions accounted for 85.3% of the genome and contained 3,739 protein-coding genes and 89 RNA genes. A total of 2832 genes were assigned a putative function with the remaining annotated as conserved hypothetical or hypothetical. A total of 2037 genes (54.5%) were assigned to COGs. The draft genome of Salinicola sp. MH3R3-1 is 4,121,883-bp long with a 62.0% G + C content. The genome has 3620 proteincoding genes and 130 RNA genes. The coding regions Evidence codes -IDA: Inferred from Direct Assay; TAS: Traceable Author Statement (i.e., a direct report exists in the literature); NAS: Non-traceable Author Statement (i.e., not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). These evidence codes are from the Gene Ontology project accounted for 86.0% of the whole genome and 2903 genes were assigned a putative function with the remaining annotated as hypothetical proteins. A total of 1870 genes (49.9%) were assigned to COGs. The draft genome of Chromohalobacter sp. SMB17 is 3,775,557-bp long with a 60.5% G + C content. The genome has 3486 protein-coding genes and 123 RNA genes. The coding regions accounted for 87.0% of the whole genome and 2875 genes were assigned a putative function with the remaining annotated as hypothetical proteins. A total of 1947 genes (54.0%) were assigned to COGs. The properties and the statistics of the three sequenced genomes are presented in Table 5. The distribution of genes into COG functional categories is summarized in Table 6.

Insights from the genome sequence
Phylogenetic analysis revealed that species of Salinicola and Chromohalobacter constitute two closely related yet distinct clades within the Halomonadaceae family (Fig. 2). We compared the newly sequenced Salinicola and Chromohalobacter genomes to each other and the available genome sequences of closely related strains C. japonicus CJ (NZ_CDGZ00000000), C. israelensis 6768 (NZ_JQNW00000000), and C. salexigens DSM 3043 T (CP000285). Results of these comparisons revealed that Salinicola genomes are larger than those of Chromohalobacter by an average of 0.47 Mbp, which amounts to approximately 400 additional protein-coding genes. Our analyses also showed that bacteria of Salinicola-Chromohalobacter group share a core genome containing 2817 protein-coding genes, which represents 75% to 89.8% of the predicted proteome of each strain (Figs. 3 and 4). Genes conserved among all of the genomes encode proteins contributing to many basic housekeeping functions and physiological mechanisms that allow these organisms to cope with deleterious effects of water stress and thrive in hypersaline Evidence codes -IDA: Inferred from Direct Assay; TAS: Traceable Author Statement (i.e., a direct report exists in the literature); NAS: Non-traceable Author Statement (i.e., not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). These evidence codes are from the Gene Ontology project Fig. 2 Neighbor-joining phylogeny inferred from data for 16S rRNA sequences of S. socius SMB35 T , Salinicola sp. MH3R3-1, Chromohalobacter sp. SMB17 and other species of the Halomonadaceae family. The optimal tree with the sum of branch length = 0.95863591 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Kimura 2-parameter method [39] and are in the units of the number of base substitutions per site. The analysis involved 56 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 1241 positions in the final dataset. Evolutionary analyses were conducted in MEGA7 [40] environments. The mechanisms of osmotolerance in bacteria involve alterations in the permeability or stability of cytoplasmic membrane, regulation of the concentration of intracellular solutes to adjust the cytoplasm osmolality, and production and uptake of inert metabolites collectively known as compatible solutes, osmolytes, or osmoprotectants. Such compounds help to balance the osmotic pressure across the cellular membrane without compromising protein folding or other cellular processes and include certain polyols, sugars, amino acids, amino acid derivatives, and peptides. The analysis of sequenced genomes revealed multiple pathways predicted to function in the de novo synthesis or uptake of osmoprotectants. Most of these determinants have homologs in the well-characterized strain C. salexigens DSM 3043 T [4] and collectively suggest that strains SMB35 T , SMB17, and MH3R3-1 respond to osmotic stress by accumulating the osmoprotectants ectoines, glycine betaine, and trehalose.
Each of the sequenced strains was predicted to synthesize ectoine from L-aspartate-β-semialdehyde and carried a three-gene operon encoding L-2,4-diaminobutyric acid acetyltransferase 2,4-diaminobutyric acid transaminase and ectoine synthase. Each microorganism also had a gene for the enzyme ectoine hydroxylase, which converts ectoine to  CRISPR repeats 0 -0 -0 -5-hydroxyectoine. However, in contrast to the highly conserved location of the ectABC operon, the ectD gene was present in different parts of the SMB35 T , SMB17, and MH3R3-1 genomes and not associated with ectR, which encodes the transcriptional repressor of the ectoine pathway in C. salexigens DSM 3043 T [2]. In addition to producing ectoines de novo, many bacteria will readily scavenge these metabolites from the environment, and our analyses revealed that all strains featured in this study are capable of taking up ectoines through orthologs of the ectoinehydoxyectoine ABC-type transport system of Ensifer meliloti [27]. The system consists of an ATPase EhuA, a periplasmic solute-binding protein EhuB, and permease proteins EhuC and EhuD that are encoded in genomes of SMB35 T , SMB17, and MH3R3-1 by the highly conserved ehuABCD operon. It also appears that the studied strains can utilize ectoines as sources of carbon and nitrogen. The three sequenced genomes share a cluster of ectoine catabolism genes doeABXCD that control the degradation of ectoine via hydrolysis to Nα-acetyl-L-2,4-diaminobutyric acid, deacetylation to diaminobutyric acid and ultimate conversion to aspartate.
In addition to ectoines, members of the Halomonadaceae utilize as osmoprotectants glycine betaine and other quaternary ammonium compounds, which are hypothesized to be ubiquitous and relatively abundant in the environment [28]. The three strains share six genes encoding putative BCCTtype transporters, which translocate quaternary ammonium compounds across the membrane using proton/sodiummotive force [29]. The strains also share two gene clusters encoding putative osmoprotectant-specific ATP-binding cassette transporters. The first of these clusters encodes an ortholog of ProVWX, which functions in Escherichia coli as a high-affinity uptake system for GB [30]. At lower affinities, the ProVWX complex also transports choline and many other osmoprotectants, including carnitine, proline, taurine, and ectoine. The second gene cluster encodes an ortholog of the OsmF-YehYXW system, which functions in E. coli as a low-affinity non-osmoregulatory GB-specific transporter [31]. Finally, the sequenced genomes also share at least three genes encoding proteins similar to GBspecific substrate-binding components of ABC transporters. Like most heterotrophic bacteria, SMB35 T , SMB17, and MH3R3-1 lack genes for the de novo synthesis of GB, but The total is based on the total number of protein coding genes in the genome are capable of converting choline to GB using choline oxidase BetA and betaine aldehyde dehydrogenase that are encoded by the betIBA operon. The operon also encodes BetI, a choline-sensitive transcriptional regulator of the TetR family of transcriptional repressors. Depending on the environmental conditions, GB can be accumulated as an osmoprotectant or converted to glycine and ultimately catabolized as a source of carbon and nitrogen. The catabolism of GB is a multistep process that starts with the demethylation of GB to dimethylglycine by the GbcAB oxygenase. The resultant dimethylglycine is then converted by a heterodimeric flavin-linked oxidoreductase DgcAB to sarcosine, which is further demethylated to glycine by a heterotetrameric sarcosine oxidase SoxABDG. Genes for all enzymes mentioned above were present in the genomes of SMB35 T , SMB17, and MH3R3-1, along with the AraCfamily transcriptional activator GbdR. In other species of bacteria, GbdR senses levels of GB and dimethylglycine and induces transcription of genes involved in the uptake and catabolism of GB, and detoxification of the catabolic byproducts [32]. We further analyzed the sequenced genomes for the presence of genes involved in the synthesis of trehalose, a compatible solute synthesized by many microorganisms in response to stress imposed by high osmolarity, desiccation, heat, freezing, or oxidation [33]. One of the most common ways of synthesizing trehalose by bacteria involves the TPS/TPP pathway, in which the enzyme trehalose-6-phosphate synthase catalyzes the transfer of glucose from UDP-glucose to glucose-6-phosphate, leading to trehalose-6-phosphate [34]. The latter is then dephosphorylated by TPP to release free trehalose. Our analyses revealed that otsAB genes encoding enzymes of the TPS/TPP pathway were present in genomes of strains SMB35 T , SMB17, and MH3R3-1, and other bacteria of the Salinicola-Chromohalobacter lineage. The Genome segments present in both Salinicola genomes, but absent from genomes of all Chromohalobacter spp., (9) Genomic islands predicted in the SMB35 T genome. The genome comparisons were visualized using the BRIG software [26]. The inner scales designate the coordinates in kilobase pairs contribution of the otsAB genes to the stress response was demonstrated in C. salexigens DSM 3043 T where trehalose produced via the TPS/TPP pathway was characterized as a secondary solute involved in osmo-and thermoprotection [35].
We also conducted proteome comparisons to identify genes that are specifically associated with Salinicola or Chromohalobacter. Results of these analyses revealed 399 genes that were present in S. socius SMB35 T and Salinicola sp. MH3R3-1, but absent from the genomes of Chromohalobacter spp. (Fig. 3). Almost half of these genes (188 genes, or 47%) encoded conserved hypothetical proteins. Among Salinicola-specific genes with predicted functions were genes for the alternative trehalose synthesis pathway that involves the conversion of the terminal unit of a glucose polymer from a α- (1)(2)(3)(4) to an α,α-(1-1) configuration, and then the release of the resulting trehalosyl moiety as free trehalose [36]. The key enzymes of this pathway, maltooligosyl trehalose synthase and maltooligosyltrehalose trehalohydrolase, are encoded by treY and treZ genes, which were identified in Salinicola strains SMB35 T and MH3R3-1 but not in genomes of SMB17 and other Chromohalobacter strains. In contrast to the TPS/TPP pathway, the importance of the treY-treZ pathway for the salinity and temperature tolerance of the Halomonadaceae remains to be determined. The rest of Salinicola-specific genes encoded putative regulatory proteins (transcriptional regulators, a diguanylate cyclase/phosphodiesterase with PAC/PAS sensor domain, and a RpoN-like sigma factor) and different enzymes, some of which constitute pathways for metabolism of phosphonate and the polyamine  (8) Genome segments present in all Chromohalobacter genomes, but absent from genomes of Salinicola spp., (9) Genomic islands predicted in the Chromohalobacter sp. SMB17 genome. The genome comparisons were visualized using the BRIG software [26]. The inner scales designate the coordinates in kilobase pairs spermidine. The Salinicola-specific transporter proteins were predicted to function in the transport of sodium, potassium, di-and tricarboxylic acids, and provide resistance to heavy metals such as copper, lead, cadmium, zinc and mercury. Interestingly, SMB35 T also carried the gene for aminocyclopropane-1-carboxylic acid deaminase, which encodes for an enzyme that converts aminocyclopropane carboxylate into ammonia and αketobutyrate. In plants, ACC is the immediate precursor of the hormone ethylene that mediates many developmental processes and response to stress. Stressed plants produce excessive amounts of ethylene, which results in smaller roots and delayed aging, abscission, and senescence [37]. Soil bacteria with ACC deaminase lower plant ethylene levels by catabolizing aminocyclopropane carboxylate, which stimulates root growth and improves plant tolerance to biotic and abiotic stress.
We identified a total of 187 genes that were uniquely present in Chromohalobacter spp. and had no counterparts in the sequenced Salinicola genomes (Fig. 4). A significant proportion (37%) of these genes belonged to the conserved hypothetical category. The genes with defined functions encoded regulators (transcriptional factors and components of two-component signal transduction circuits), components of type IV fimbriae, a VapC-like toxin/ antitoxin system, and various enzymes, including subunits of a formate dehydrogenase complex and components of a cyanophycin biosynthesis pathway. Other Chromohalobacter-specific proteins were predicted to function in stress response (detoxification of reactive oxygen and resistance to arsenic) or transport phosphate, di-tripeptides, nitrate, branched chain amino acids, and other solutes.
Finally, the three sequenced genomes were screened for the presence of genomic islands using the Island-Viewer 3 pipeline [25]. Results of the analysis revealed that Chromohalobacter sp. SMB17 had the highest proportion of DNA assigned to genomic islands (252 kbp, or 6.7%) (Fig. 4), which encoded, among other functions, a betaine-binding subunit of ABC transporter, a tricarboxylate transport system, and several O-antigen biosynthesis enzymes. Other features associated with GIs included multiple transposons, a retron element, and seven integrases, three of which were associated with prophages. Salinicola socius SMB35 T and Salinicola sp. MH3R3-1 had, respectively, 233.8 kbp and 129.0 kbp of DNA assigned to genomic islands, which represents 5.6% and 3.1% of the genome of each strain (Fig. 3). These GIs encoded multiple conserved hypothetical proteins, various enzymes, transport and regulatory proteins. In addition, each strain harbored a unique assortment of intact and decaying mobile genetic elements. For example, S. socius SMB35 T harbored three prophages that collectively encompassed 120.6 kbp of the genome and contained genes for structural bacteriophage proteins, DNA metabolism enzymes, transcriptional regulators, and lytic enzymes. All three SMB35 T prophages contained integrase genes and two of these elements were integrated into tRNA-Gly (CCC) and tRNA-Thr (CGT). The genome of Salinicola sp. MH3R3-1 contained two islands (35.7 and 24.9 kbp) that contained genes for plasmid replication, partitioning, and conjugation and exhibited similarity to integrative and conjugative elements, a class of hybrid genomic islands with elements of both temperate phages and conjugative plasmids [38].

Conclusions
This study provides new insights into the genomic diversity of the family Halomonadaceae by contributing three genome sequences of moderately halophilic heterotrophic bacteria of the Salinicola -Chromohalobacter group. The comparative analysis of genomes of S. socius SMB35 T , Salinicola sp. MH3R3-1, and Chromohalobacter sp. SMB17 underscores the importance of two classes of osmoprotectants, ectoines, and quaternary ammonium compounds, to the capacity of these bacteria to tolerate wide ranges of solute concentrations and adapt to hypersaline environments. The sequenced genomes share highly conserved genes for the synthesis of ectoine (ectABC) and hydroxyectoine (ectD), and conversion of choline to GB (betIBA). The three strains also are capable of achieving osmoprotection by accumulating ectoines and quaternary amines via uptake and share an ectoine-specific ABC-type transporter (EhuABCD), two QA-specific ABC-type transporters (ProVWX and OsmF-YehYXW), and several transporters of the Betaine/Carnitine/Choline-Transport family. The three sequenced strains are further capable of utilizing ectoines and quaternary ammonium compounds as sources of C and N and share a cluster of ectoine catabolism genes (doeABXCD), as well as three operons (gbcAB, dgcAB, and soxABDG) that control the degradation of GB to aspartic acid. SMB35 T , MH3R3-1, and SMB17 also carry otsAB genes that encode enzymes for the synthesis of trehalose, which in this group of bacteria functions as a secondary compatible solute involved in osmotic and thermal protection. Finally, results of this study revealed that Chromohalobacter and Salinicola share a core genome of approximately 2800 genes, which represents 75-90% of the predicted proteome of each strain. However, each genus also harbored a set of unique protein-coding genes, which was particularly significant in Salinicola and amounted to approximately 0.5 Mbp. The genusspecific genome segments encoded enzymes, transcriptional regulators, transporters, conserved hypothetical proteins, and mobile genetic elements. We hypothesize that this flexible gene pool may contribute to the