Draft Genomes of Halophilic Chromohalobacter and Halomonas Strains Isolated from Brines of The Carpathian Foreland, Poland

Chromohalobacter and Halomonas are genera of bacterial microorganisms belonging to the group of halophiles. They are characterized by high diversity and the ability to produce bioproducts of biotechnological importance, such as ectoine, biosurfactants and carotenoids. Here, we report three draft genomes of Chromohalobacter and two draft genomes of Halomonas isolated from brines. The length of the genomes ranged from 3.6 Mbp to 3.8 Mbp, and GC content was in the 60.11%-66.46% range. None of the analysed genomes has been assigned to any previously known species of the genus Chromohalobacter or Halomonas. Phylogenetic analysis revealed that Chromohalobacter 296-RDG and Chromohalobacter 48-RD10 belonged to the same species, and Chromohalobacter 11-W is more distantly related to the other two analysed strains than to Chromohalobacter canadensis. Halomonas strains 11-S5 and 25-S5 were clustered together and located close to Halomonas ventosae. Functional analysis revealed BGCs related to ectoine production in all genomes analysed. This study increases our overall understanding of halophilic bacteria and is also consistent with the notion that members of this group have significant potential as useful natural product producers.


Introduction
Hypersaline ecosystems have been widely explored mainly because of their unique biodiversity and biotechnological potential. It is related to the adaptation of halophiles to the conditions of high osmotic pressure, limited availability of energy resources and other unfavourable environmental circumstances (1). Novel halophilic microorganisms have been isolated in recent years from habitats such as saline lakes, salt mines, saline soils or fermented foods (2)(3)(4)(5)(6). A great variety of saline environments related to factors other than salinity itself, such as pH, temperature or the availability of nutrients, results in a considerable diversity of inhabiting microorganisms, both in terms of genetics and metabolism.
Halomonadaceae is a family of halophilic Gammaproteobacteria, including Chromohalobacter, Halomonas and 12 other genera (7,8). Currently, Chromohalobacter genus includes eight validly published species isolated from salterns, seas and food products. The process of validating the publication of new species of prokaryotes is related to the fulfilment of all of the requirements set out in the "International Code of Nomenclature of Prokaryotes" (9). Moreover, the new species Chromohalobacter moromii sp. nov. isolated from lupine-based moromi fermentation has been described and is pending validation (6). Microorganisms belonging to the genus Chromohalobacter, for example, Chromohalobacter salexigens, have been identified as producers of biotechnologically valuable compounds and, due to their genomic characteristics, could become a useful metabolic engineering tool for the overproduction of ectoines (10).
On the other hand, the Halomonas genus includes 117 validly published species and new species like Halomonas alkalisoli sp. nov are waiting for validation Ivyspring International Publisher (2). Most of these species are widely distributed in saline habitats, such as salt lakes, marine environments, and saline soils (11,12). Halomonas strains exhibit high metabolic and physiological versatility, thus a wide range of bioproducts, such as ectoine, glycine betaine and polyhydroxyalkanoates (PHA) can be produced (12,13).
This study presents the characteristics of five genomes of microorganisms isolated from brines, sources of which are located in the southern part of the Carpathian Foreland in Poland near Kraków city. Three genome sequences of Chromohalobacter sp. and two draft genome sequences of Halomonas sp are reported.
All sequencing data are publicly available from the National Institutes of Health under BioProject accessions PRJNA899688, PRJNA899690, PRJNA89 9692, PRJNA899693, PRJNA899694.

Results and discussion
Each draft genome was composed of between 38 and 321 contigs, with genome sizes ranging 3.6-3.9 Mbps. The overall genome completeness was estimated at between 98.71-99.86%, with contamination in the range of 0.54-8.42 and GC content in the 60.11-66.46% range. The summary is presented in Table 1.
Preliminary taxonomic annotation of genomes using gtdb-tk, assigned isolates 11-W, 296-RDG, and 48-RD10 to Chromohalobacter genus, and isolates 11-S5, 25-S5 to Halomonas genus. The assignment to the species level was impossible because of too high differences in genome sequences between the analysed strains and the previously described genomes available in the databases. A phylogenetic analysis was performed in order to deepen knowledge about the relationship between the analysed isolates and other species. A phylogenetic tree, based on the 16S rRNA gene sequences was built. The resulting tree confirmed that the isolates 11-W, 296-RDG, and 48-RD10 fell within a cluster comprising members of the genus Chromohalobacter and the strains 11-S5, 25-S5 fell within a cluster including members of the genus Halomonas (Figure 1). However, in both cases, the analysed strains were separated from the other species included in the analysis. The closest species for Chromohalobacter strains 296-RDG and 48-RD10 was Chromohalobacter canadensis, and for strain 11-W, it was Chromohalobacter sarecensis. In the case of Halomonas strains, the closest taxon was Halomonas sediminicola. This initial phylogenetic analysis, using a comparison of 16S rRNA gene sequences, was then deepened through the construction of a further, whole-genome sequence-based phylogenetic tree build using Genome BLAST Distance Phylogeny approach (GBDP) created on the TYGS platform ( Figure 2). The obtained phylogenetic tree confirmed the observations made at an earlier stage. On the genomewide scale, it was noticed that Chromohalobacter 11-W is more distant from the other two analysed strains than from Chromohalobacter canadensis. That may suggest genomes assignment to two different species of Chromohalobacter. This observation was confirmed by digital DNA-DNA hybridization (dDDH) evaluation, where the similarity between Chromohalobacter 296-RDG and 48-RD10 was 87.5% (d4 method), between 11-W and 296-RDG it was 42.7 % and between 11-W and 48-RD10 it reached 42.9%. Halomonas strains 11-S5 and 25-S5 were clustered together on the whole-genome tree. Allocation to the same species was confirmed by dDDH which was 89.7%. The closest related species to the analysed strains was Halomonas ventosae.   Functional annotation of genomes revealed that they all contained numerous genes involved in the biosynthesis of secondary metabolites (Table 2). However, both Halomonas strains, 11-S5 and 25-S5, had a higher number of genes belonging to this category (99 and 101 genes, respectively) than isolates belonging to the genus Chromohalobacter, which consisted of 69-89 such genes, depending on the strain. This observation is consistent with previous reports that Halomonas and Chromohalobacter have a high diversity of biosynthetic processes (10,12,13) Based on these results, the annotation of BGCs with antiSMASH was performed. Interestingly, as a result, more BGCs were identified in Chromohalobacter strains than Halomonas, despite a smaller number of genes associated with processes identified during the analysis. Moreover, BGCs related to ectoine production have been identified in all genomes. Most likely, it is related to the adaptation of the studied microorganisms to conditions of high salinity. Ectoine produced by the analysed strains is one of the most important compatible solutes that protects the cell against high osmotic pressure (26). In the 11-W, 296-RDG, 48-RD10, and 11-S5 strains, a complete operon ectABC was identified. In the 25-S5 strain, only the ectC gene, essential for ectoine production, was identified. In Halomonas isolates, BGCs associated with ectoine production were the only BGCs identified in the genomes. In Chromohalobacter strains, BGCs related to the production of siderophores, redox-cofactors, and arylopolyenes, were also identified. Table 3 summarizes the information on the identified BGCs in each of the strains.
To summarize, the draft genomes of three Chromohalobacter strains and two Halomonas strains expand the genomic representation in the tree of life. The strains analysed were isolated from the hitherto unexplored saline environment, which allows a deeper understanding of their biodiversity.