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. 2026 Jan 16;27:181. doi: 10.1186/s12864-025-12431-x

Genomic analysis of a novel isolate Vreelandella Titanicae sp. Zn11_249 from the chaotropic environment Salar de Uyuni (Bolivia)

Esteban Sabroso 1,2, Concepción Abrusci 1, Nuria Rodríguez 3, Ricardo Amils 1,2,3, José M Martínez 1,2,✉
PMCID: PMC12892813  PMID: 41540327

Abstract

Background

This article describes the isolation of Vreelandella titanicae Zn11_249 from the Salar de Uyuni (Bolivia). The genome of the bacterium is described and new information about the species Vreelandella titanicae is added to the database. However, the aim is not only to present this information, but also to provide the ecological approach of the isolate to the extreme ecosystem in which it develops and genes sequences that could improve the biotechnological capacities around this strain.

Results

Vreelandella titanicae Zn11_249 was isolated from samples of a whirlpool from the Salar de Uyuni. The nucleotide sequence of the genome has been determined and analysed. The genome contains 5,575,580 bp and has a G + C content of 54.61%. This analysis identified 4,995 coding sequences, 62 tRNA genes, 1 tmRNA gene, 12 rRNA, 1 CRSPR region and 1 repeat region. Annotation of the Vreelandella titanicae Zn11_249 genome found the metabolic genes necessary for the correct functioning of the bacterium in the niche it inhabits. On the one hand, the metabolic pathways found are related to the assimilation of carbon, nitrogen, phosphorus and sulphur. The assimilation of these compounds for an adequate development of the bacteria was related to the ecological role of Vreelandella titanicae Zn11_249 to understand its possible function and importance in the Salar de Uyuni. On the other hand, sequences encoding resistance genes were identified, possibly due to the stresses the bacteria are subjected to in this ecosystem. The sequences found included heavy metal resistance genes, genes related to oxidative stress response or to osmotic stress among others.

Conclusions

The genes involved in the metabolism of Vreelandella titanicae Zn11_249 enable it to live on in the extreme conditions of Salar de Uyuni. This bacterium plays a crucial role in the biogeochemical cycles of carbon, nitrogen, and sulphur. Its metabolic adaptability allows them to thrive under high salinity and harsh conditions, making it a key player in the transformation and mobility of essential elements. Although based on a single genome, this primally study provides a solid foundational framework for future transcriptomic, proteomic, and experimental investigations into the ecological role and biotechnological potential of Vreelandella titanicae Zn11_249.

Data summary

The authors confirm all supporting data, code, and protocols have been provided within the article or through Supporting Information data files.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12864-025-12431-x.

Keywords: Salar de uyuni, Biogeochemistry, Chaotropicity, Vreelandella Titanicae Zn11_249., Genotypic analysis, Adaptation mechanisms

Introduction

Hypersaline environments, characterised by high salt concentrations (2.5–5.2 M) [1], host halophilic microorganisms, such as Halolactobacillus halophiles, which require salt for growth. Additionally, halotolerant microorganisms, like Oceanobacillus iheyensis, can survive in hypersaline conditions without requiring salt for growth [2]. The family Halomonadaceae, of the order Oceanospirillales, is the most important family of halophiles [3]. Microorganisms belonging to this family have been isolated from different saline habitats such as seawater, hypersaline and/or saline lakes, saline sediments, hydrothermal environments, food, and plants among others [1, 2]. The genus Halomonas manage to develop under osmotic stress due to their ability to accumulate inorganic ions, compatible organic solutes (osmolytes) or the formation of resistance structures [4]. Recently this family has been redistributed to clarify the genetic relationship, phylogeny, and taxonomy of species and genera within this family [5].

Salar de Uyuni (SdU) is located at an altitude of 3,653 m above sea level in the southwestern region of Potosí, in the Bolivian Altiplano depression. This salt flat is an endorheic basin and is considered the largest salt flat on the Earth, occupying an area of 10,582 Km2 [6]. The salt crust appears during the dry-cold season and is primarily composed of sodium chloride salt in the northern zone, while lithium, magnesium are more prevalent in the southern zone [2, 7]. The mineralogy of the saline strata is composed mainly of halite with small amounts of gypsum, volcanic detrital materials, and clay minerals [8].

The present study aims to comprehend the adaptations and ecological role of the bacterium Vreelandella titanicae Zn11_249, belonging to the family Halomonadaceae, isolated from a sample of the non-industrial area of SdU, collected during the dry-cold season.

Methods

The strain Zn11_249

The strain Zn11_249 was isolated on marine agar with a salinity of 3.5% from a salt sample collected from a whirlpool located in the natural non-anthropogenic area of SdU (latitude 20°10’23.3”S and longitude 67°43’20.7”O) during the dry-cold season (austral winter; July 2013). The isolate Zn11_249 grew in LB medium at a salinity range from 0 to 4.5 M, with the best growth rates observed above 1.5 M. The characterisation of the sample was conducted as described in Martinez et al. 2021 [8]. Halite as the main mineral and presence of gypsum accumulations. Elemental analysis showed that the chemical composition of this sampling site contains 0.001 g/L K+, 294.48 g/L Na+, 1.28 g/L Cl−, 0.009 g/L S, 0.345 g/L Mg2+, 0.01 g/L Li+, 0.01 g/L Ca2+ and 0.0002 g/L Br− as main ions.

DNA extraction and 16 S rRNA gene amplification

Bacteria was grown in Tryptic Soy Agar (TSA; Sigma Aldrich) up to exponential state. DNA extraction was using the CTAB-phenol-chloroform protocol [9]. The DNA concentration was analysed by fluorimetry (Qubit v.2.0, Invitrogen, USA) and its integrity was verified through a 1% (w/v) agarose gel (Conda, Spain) diluted in 0.5x TBE (70 V for 20 min). Genomic DNA was preserved at −20 °C.

Identification of the isolate was performed by amplification of the 16 S rRNA gene as was described in García et al. (2018), using the MEGA 11 version [10].

Sequencing, annotation and comparative genome analysis

DNA library preparation and genome sequencing was provided by MicrobesNG (Birmingham, UK). Genomic DNA libraries were prepared using the Kit Nextera CT Prep (Illumina, USA) as recommended by the manufacturer. Sequencing was performed using the Illumina NovaSeq 6000 platform (Illumina, USA) using the 250 bp paired end-of-fact protocol. Reads quality were analysed using FastQC v0.11.9, and low-quality reads and adapters were filtered with Trimmomatic v0.30 [11] using the default parameters (SLIDINGWINDOW Q15). De novo assembly was performed using SPAdes v3.15.5 with default parameters and the “--careful” option [12]. Assembly quality was tested by Quast v5.2.0 [13].

Gene prediction and annotation were achieved with PROKKA v1.14.6 software with default parameters [14] and the RAST platform [15] taking the genome of Vreelandella titanicae BH1 as a reference [16]. Genome graphing was performed using the Proksee software [17]. In addition, complementary methods were used to contrast information with different software’s: CRISPR Recognition Tool (CRT) was used to search for CRISPR genes [18], PlasmidSpades was used to screen for possible plasmids [19], and Abricate was used to analyse possible virulence and antimicrobial genes using NCBI dataset [20, 21].

Comparative genomics was performance by OAT (Orthologous Average Nucleotide Identity Tool) v0.93.10 using Original ANI and OrthoANI [22]. DDH (DNA-DNA hybridization) was performance by TYGS (Type Genome Server) v400 [23]. The prediction of orthologous groups was performed using Orthofinder on the Galaxy Europe server v2.5.5, using the predicted proteome [24]. These analyses were conducted using 30 species from the family Halomonadaceae, including the genera Vreelandella, Salinicola, Billgrantia, Onhisia, Kushneria, Modicisalibacterium, Halovibrio, Frazmannia, and Salinibacter, with Haloarcula salina YGHS18 included as an outgroup. All data were retrieved from the NCBI database (Supporting Information Table 3) [16].

Ammonium assimilation and metal resistance assays

For ammonium assimilation assay, U1 medium was prepared as described in Sabroso et al.. (2025) [25]. Heavy metal resistance assays were performed as described by Mateos et al.. (2022) using NiCl2, CrCl3, HgCl2, PbCl2 [26].

Results

Draft genome sequence, annotation results and comparative genome analysis

Sequencing of the genomic DNA library resulted in a total number of 2,669,328 paired-end reads with an average insertion size of 624 bp and an average coverage of 231.832X. The draft genome assembly of Vreelandella titanicae Zn11_249 comprised 5,575,580 bp (Fig. 1) divided into a total of 496 contigs. The GC content resulted in 54.61% (Fig. 1). N50 and L50 values of 302,507 and 7 bp, respectively, were achieved.

Fig. 1.

Fig. 1

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Graphical representation of Vreelandella titanicae Zn11_249 genome. Genes on the forward strand are represented on the outer circle and reverse strand on the inner circle, identify key genes distribution for the essential nitrogen (green), sulphur (orange) and phosphorus (grey) metabolism of the isolate (produced using Proksee)

Genome annotation identified a total of 4,995 coding sequences, 62 tRNA genes, 1 tmRNA gene, 12 rRNA genes (6 copies and 1 partial sequence of the 5 S rRNA gene, 1 sequence and 1 partial sequence of the 16 S rRNA gene and 3 partial copies of the 23 S rRNA gene), 1 CRISPR region and 1 repeat region (Fig. 1). Among these 4,995 coding sequences, genes essential for the normal metabolic functioning of the bacterium in the environment were found. Some of these identified genes are involved in the denitrification pathway; inorganic assimilation of sulphur, phosphorus (Fig. 1) and potassium; and metabolism of aromatic compounds; cell regulation and signalling; DNA repair and resistance to heavy metals, among others. Likewise, genes related to the response to oxidative and osmotic stress were identified that may be related to the extreme conditions of the ecosystem in which the bacteria live.

No plasmids were found within the Vreelandella titanicae Zn11_249 genome. Also, no virulence and antimicrobial gene encoding sequences were identified.

With the aim of identifying the strain Zn11_249 within the genus Vreelandella, its genome was compared with other genomes from the family Halomonadaceae using different methods. First, a similarity with the species Vreelandella titanicae BH1 of 97.29% (Fig. 2a) was observed using OriginalANI and 97.51% by OrtoANI (Fig. 2b).

Fig. 2.

Fig. 2

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Results obtained using OAT (Orthologous Average Nucleotide Identity) tools analysing ten species of the genus Vreelandella and Halarcula salina YGHS18 as an outgroup: (a) Original ANI values (%); (b) OrthoANI values (%). ANI values of ≥ 96% indicate a high degree of similarity between species

Furthermore, DNA-DNA hybridisation (DDH) predicted closer similarity between strain Zn11_249 and strains Vreelandella titanicae SOB56 (73.10%) and Vreelandella titanicae BH1 (72.90%) (Table 1). Finally, the study of orthologous genes showed that 95.3% of the genes of strains Zn11_249, SOB56, and BH1 constitute a core genome (Table 2). Strain Zn11_249 has a total of 233 different genes (Table 2) related to the genomes of strains BH1 and SOB56.

Table 1.

Results of DNA-DNA hybridization (DDH) obtained using TYGS. DDH values of ≥ 70% indicate the same species

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Table 2.

Results obtained using OrthoFinder to identify orthologous gene groups. A value of ≥ 95% of genes in orthogroups indicates a high level of genomic similarity between species

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Metabolic potential

In this study, an in silico analysis of the metabolic potential of strain Zn11_249 in relation to the characteristics of the SdU was conducted. Thus, genes related to adaptation mechanisms to the extreme conditions of the SdU were identified. Within the carbon metabolism, glycolysis pathway genes were identified with key enzymes such as glucokinase (EHLJMEHL_02835), fructokinase (EHLJMEHL_02833), or three copies of pyruvate kinase (EHLJMEHL_02797; EHLJMEHL_03523; EHLJMEHL_04873). The complete glucolysis pathway was not identified, but even so, the genes encoding the enzymes responsible for the peripheric reactions of the pathway, such as phosphoenolpyruvate carboxylase (EHLJMEHL_02067) or glucose-6-phosphate isomerase (EHLJMEHL_02791), were found. With these results, the search for genes responsible for the synthesis and regulation of phosphoenolpyruvate, ppsA (EHLJMEHL_04110) and ppsR (EHLJMEHL_04111), was completed. Regarding the tricarboxylic acid cycle (TCA), most of the enzymes that perform it were identified, such as citrate synthetase (EHLJMEHL_03671), succinyl-CoA synthetase (EHLJMEHL_03662; EHLJMEHL_03663); fumarate hydratase class two (EHLJMEHL_04137) or malate dehydrogenase (EHLJMEHL_04167) (Supporting Information Table 1). In this case, the coding sequence of korABCD (2-oxoglutarate) was not found. Genes of the Wood-Ljungdahl pathway were also included in the identification, and identified as were fdhA (EHLJMEHL_02668), fdhB1 (EHLJMEHL_03863), folD (EHLJMEHL_01245), metF (EHLJMEHL_04847), and several copies of acsA (EHLJMEHL_03251; EHLJMEHL_03952; EHLJMEHL_04358).

Coding sequences of genes involved in different fermentations such as lactate, acetate, or ethanol fermentation, among others, were identified (Supporting Information Table 1). In addition, coding sequences of enzymes responsible for peripheric reactions of the beta-ketoadipate pathway were found through the catechol branch and protocatechuate-four together with the enzyme p-hydroxybenzoate hydrolase (Supporting Information Table 1). On the other hand, coding sequences for complexes two, three, four, and five of oxidative phosphorylation were found as an energy producing mechanism for the bacterium (Supporting Information Table 1).

Regarding the nitrogen cycle, essential genes for oxidative denitrification were highlighted by finding the sequences responsible for the membrane nitrate oxidoreductase NAR, narG (EHLJMEHL_01213), the periplasmic nitrate oxide reductase NAP (EHLJMEHL_03864), the cytochrome c nitric oxide reductase NOR (EHLJMEHL_01194; EHLJMEHL_01193) and the nitrous oxide reductase NOS (EHLJMEHL_01173). Genomic analysis also revealed the presence of assimilatory nitrate reduction genes (nas), showing nitrate reductase (EHLJMEHL_00865), transcriptional regulator (EHLJMEHL_00890), and glutamine/glutamate synthetases (EHLJMEHL_02347; EHLJMEHL_03189). Consistent with the function of glnA and gltD, experimental assays demonstrated that the strain actively assimilated ammonium from the culture medium under aerobic conditions. In addition, genes encoding two-component systems for the regulation of nitrate assimilation were identified as nitrate and/or nitrite sensors narX (EHLJMEHL_01165) and narL (EHLJMEHL_01164) (Fig. 1).

Sulphur metabolism was also found with representative genes encoding for thiosulphate dehydrogenase, tsdA (EHLJMEHL_04577), the flavoproteins NADPH-sulphite reductase, cysJ (EHLJMEHL_01188) and cysI (EHLJMEHL_01187); sarcosine oxidase, sox (EHLJMEHL_02658; EHLJMEHL_02660; EHLJMEHL_02659; EHLJMEHL_02657); transcriptional activator soxR (EHLJMEHL_02660) and the sulphite reductase sir (EHLJMEHL_02886) (Fig. 2). For phosphorus metabolism, phosphate transport genes such as ugpA (EHLJMEHL_01037, EHLJMEHL_02327), or the phosphate regulon sensor phoR (EHLJMEHL_00134) were identified (Fig. 1).

Putative adaptative genetic mechanisms

To complement the metabolic analysis of Vreelandella titanicae Zn11_249, potential genes associated with adaptation to hypersaline conditions were also examined. The bacterium possesses 168 subsystems involved in genomic DNA repair, CRISPR systems, and responses to various environmental stresses.

In the general stress response of Vreelandella titanicae Zn11_249, sequences encoding Hfl (hflX or hflC), bacterial hemoglobins (dosC and hisM), and carbon starvation (csrA, ssp.A, and rspB) were identified (Supporting Information Table 2). Regarding DNA repair systems, the genome of Vreelandella titanicae Zn11_249 contains the basic components, including exonuclease uvrA, uvrB, and uvrC for nucleotide excision repair (NER); the genes lexA, umuD, and umuC, responsible for the SOS response; glycosylase mutY for base excision repair (BER); genes involved in the mismatch repair mechanism such as mutL and mutS; and genes involved in recombination such as recBCD (Supporting Information Table 2). In addition to these mechanisms, sequences of proteins cys1 (EHLJMEHL_02358), cys2 (EHLJMEHL_02357), and cys3 (EHLJMEHL_02356), endonucleases cas1 (EHLJMEHL_02362) and cas6f (EHLJMEHL_02355), and nuclease/helicase cas3 (EHLJMEHL_02361) were found to be involved in the CRISPR system.

Genes involved in the synthesis and transport of osmolytes, potentially contributing to osmotic stress tolerance, were identified. First, sequences corresponding to the osmolyte glycine betaine (N, N,N-trimethylglycine) were analysed, which included the consumption of choline sulphatase as a precursor of glycine betaine synthesis, and choline transport systems. Additionally, orthologues for the biosynthesis of choline sulphatase were found, such as phosphatidylcholine synthase (EHLJMEHL_03896), phosphatidylserine decarboxylase (EHLJMEHL_01888), and phosphatidylglycerol phosphate synthase (EHLJMEHL_03999) (Supporting Information Table 2). Vreelandella titanicae Zn11_249 also contains genes for the essential system for choline uptake like betT (EHLJMEHL_03488) and ABC transporters with high affinity for choline. To complete the glycine betaine synthesis pathway, genes responsible for its synthesis betAB (EHLJMEHL_01041; EHLJMEHL_01042) were identified.

Furthermore, the studied microorganism possesses the complete operon responsible for the synthesis of ectoine ectABC (EHLJMEHL_01291; EHLJMEHL_01292; EHLJMEHL_01293). The strain Zn11_249 also has genes for the synthesis of osmolytes carnitine and proline, including the operon for the carnitine protein, L-carnitine dehydrogenase, pyrroline-5-carboxylate reductase, and glutamate 5-kinase, which are responsible for the synthesis of these two molecules (Supporting Information Table 2).

The genomic analysis of Vreelandella titanicae Zn11_249 allowed to detect 27 genes involved in the oxidative stress response. These include genes for the expression of superoxide dismutase, catalase, bacterioferritin, glutaredoxin, thioredoxin, peroxiredoxin, and organic hydroperoxide resistance protein (Supporting Information Table 2).

Finally, genes involved in resistance to toxic compounds such as heavy metals and metalloids were identified, including those encoding proteins for resistance to arsenate, copper, cobalt, zinc, and cadmium (pcoC, copB, copA, pcoC, arsC, arsH, czcD, czcA), and various efflux pumps and transporters for manganese, cobalt, zinc, and cadmium (mntP, corA, corC, chrA). In agreement with these genomic predictions, experimental assays confirmed that the strain is also tolerant to NiCl₂, CrCl₃, HgCl₂, and PbCl₂. Efflux pumps contributing to multidrug resistance were also found, including mdtL, nedtC, mdtH, mexB, mexA, acrB, and ifrA, among others (Supporting Information Table 2).

Discussion

The strain Zn11_249, isolated from the northern region of SdU, was first identified with a 99.95% similarity to the 16 S rRNA gene of Halomonas titanicae BH1 (taxonomic identification NCBI: txid1204738). The family Halomonadaceae, included in the class Gammaproteobacteria, is the largest family of halophiles, comprising over 160 species with published names. Recently, this family has been reclassified, including Halomonas titanicae, to clarify the genetic relationships, phylogeny, and taxonomy of the species and genera [5]. Through comparative genomic analysis, Vreelandella titanicae BH1 was considered the closest species to Zn11_249. The study strain showed an 95.3% of orthologous genes. The differing origins of the two isolates represent a key distinguishing factor, underscoring the relevance of the genomic study of isolate Zn11_249. Vreelandella titanicae BH1 has been described as a moderately halophilic, Gram-negative, heterotrophic, aerobic, non-spore-forming, peritrichously flagellated bacterium. The reference strain BH1 was first discovered in residual samples taken from the RMS Titanic wreck. The BH1 strain exhibits optimal 30–37 °C, and pH 7–7.5 [27]. The strain BH1 grew in a range from 0 to 4 M NaCl [27], similar to the strain Zn11_249. However, the strain Zn11_249 grew best between 1.5 and 4.5 M, and the strain BH1 between 0.3 and 1 M [27].

In recent years, the genus Halomonas has been studied for its important implication in saline environments with respect to the maintenance and function of biogeochemical cycles. Taxonomic identification of Halomonas titanicae BH1 determined that this strain could be a heterotrophic bacterium [27]. The genome of Vreelandella titanicae Zn11_249 encodes key genes for glycolysis and the tricarboxylic acid cycle, consistent with its experimentally confirmed ability to grow on glucose as the sole carbon source [25]. Genes involved in autotrophic pathways such as Wood-Ljungdahl pathway for the potential utilisation of inorganic compounds for energy generation were identified. Furthermore, the partially complete peripheral pathway of beta-ketoadipate degradation with coding sequences for catechol 1,2-dioxygenase, muconate lactonizing enzyme and muconolactone isomerase to finally transform catechol into beta-ketoadipate was found (Supporting Information Table 1). Finally, the corresponding sequence of beta-ketoadipylthiolase was found, which possibly converts this product into acetyl-CoA [28]. This degradation could be associated with the protocatechuate-four degradation pathway found in the genome with the enzyme 3-oxoadipate enol-lactonase 2 (EHLJMEHL_02636) associated with the two pathways [28, 29]. The degradation of these compounds in bacteria of the genus Halomonas, such as Halomonas organivorans or Halomonas sp. KHS3 has already been demonstrated and could be an evolutive strategy in the carbon scarcity in hypersaline environments such as in SdU [28, 30].

In addition, the existence of coding sequences of genes responsible for various fermentation and oxidative phosphorylation complexes were identified. This could be attributed to the decrease in oxygen concentrations occurring during the wet-warm season (austral summer) of SdU, thus revealing genes involved in both aerobic and anaerobic energy-yielding pathways (Supporting Information Table 1) [31, 32]. Vreelandella titanicae Zn11_249 encode complexes II-V of the respiratory chain including succinate dehydrogenase (sdhABCD, complex II), cytochrome bc complex (petABC, complex III), cytochrome c oxidase (ctaCD, complex IV). The presence of atpABCDEFH genes indicates that the respiratory chain is associated with an ATPase F01F1 that generates ATP (Supporting Information Table 1) [33]. These characteristics were also analysed in bacteria of the same family, such as Halomonas piezotolerants NBT06E8T, which could use this metabolic potential to overcome periods of low oxygen concentration [32]. These results confirm the possible heterotrophic metabolism of Vreelandella titanicae Zn11_249, already described in other studies on the same specie [27]. However, the presence of sequences encoding genes for autotrophy suggest that it could use both in aerobic and anaerobic conditions [5].

Within the efforts to investigate the putative ecological role of the genus Halomonas in extreme environments, the role of nitrogen metabolism has been identified as an important metabolism to consider [34]. According to the genome of strain Zn11_249, this bacterium may be involved in the nitrogen cycle owing to the presence of genes related to oxidative denitrification and assimilatory nitrate reduction.

Furthermore, the presence of complete gene sets for denitrification in Vreelandella titanicae Zn11_249, including nitrate, nitrite, nitric oxide, and nitrous oxide reductases, suggests its active role in the nitrogen cycle under the extreme conditions of SdU (Supporting Information Table 1). To complete denitrification, the nos group of enzymes would be responsible for reducing nitrous oxide to atmospheric nitrogen [35, 36]. Additionally, the detection of genes involved in nitrate assimilation reinforces the potential adaptive versatility of this organism in oligotrophic, nitrogen-variable environments (Fig. 3) [26, 37]. In other studies, results showed that these enzymes can participate in both described metabolisms by the existence of nitrate transcriptional regulator nasR and two-component systems narX-narL acting as a sensor regulated by nitrite and nitrate changes, found within the genome [34]. High salinity stress inhibits soil microbial communities that mediate nitrogen cycling. Studies show that in these ecosystems, the presence of high ionic Na+ concentrations decrease the capacity for denitrification and nitrogen assimilation due to the rapid mobilization of NH4+ in the substrate [38, 39]. The low availability of ammonium could indicate that the presence of complete denitrification and assimilation pathways constitutes a life-saving strategy in the SdU. This characteristic suggests the potential coexistence with atmospheric nitrogen-fixing bacteria already found in other samples collected in this ecosystem as cyanobacteria and nitrifying bacteria, forming possible microbial consortia [8, 36, 40].

Fig. 3.

Fig. 3

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Schematic representation of nitrogen (blue) and sulfur (orange) metabolic pathways in Vreelandella titanicae Zn11_249. Key enzymes involved in denitrification (nar, nap, nir, nor, nos) and assimilatory nitrate reduction to ammonium (nas, nir, glnA, gltD) are shown, along with regulatory systems (nasR, narX/narL). Sulfur metabolism includes thiosulfate oxidation (tsdA, sox), sulfate uptake (cysZ), and assimilatory reduction to hydrogen sulfide for cysteine synthesis (cysDNHCJI, sir, cysM)

Sulphur metabolism is present in the genome of Vreelandella titanicae Zn11_249. The presence of genes for thiosulfate and sulphate transformation pathways, including thiosulfate oxidation and assimilatory sulphate reduction, suggests that Vreelandella titanicae Zn11_249 participates in sulphur cycling through energy generation and biosynthesis, which is ubiquitous in the genus Halomonas. The presence of tsdA is also consistent with the possible ability of bacteria of the genus Halomonas to obtain energy from oxidizing thiosulphate as complementary source of energy, showing their heterotrophic sulphur-oxidizing activity [34]. Another feature of sulphur metabolism is the presence of the sulphate scavenger permease cysZ. In Vreelandella titanicae Zn11_249, genes involved in the assimilation of sulphur from sulphate already found in Halomonas titanicae SOB56 were identified. Among the genes coding for the thiosulphate oxidation to sulphate soxABDG and from thiosulphate to sulphite by thiosulphate sulphurtransferase rdhA were identified. Sequences coding for the reaction from sulphate to APS (adenosine 5’-phosphosulphate), by cysDN and APS to PAPS (3’-phosphoadenylyl-sulphate), by cysC were also identified. Subsequently, the metabolism of sulphur involved the reduction of PAPS to sulphite, coding by the assimilatory sulphite reductase. Later, sulphite is reduced to hydrogen sulphide by cysIJ and sir (ferredoxin) that finally assimilate sulphur into cysteine by cysM (Fig. 3) [33, 41]. In addition, sequences responsible for genes encoding transcriptional regulators and permeases required for this metabolism were identified (Supporting Information Table 1). Due to the presence of gypsum mineral in the sampling area, it would be expected that genes related to sulphur metabolism are present in the genome of the bacterium. This metabolism, which is present in Vreelandella titanicae Zn11_249, indicate its participation in the biogeochemical cycle of sulphur by its assimilation.

Phosphate is an essential micronutrient for microorganisms, as it is involved in energy-requiring reactions through adenosine phosphates or NADPH, and as an important element of the DNA and RNA molecules, proteins, polyphosphates, and phospholipids [42]. The geological characteristics of SdU indicate an important deficiency of phosphate, making this micronutrient the primary limiting factor for bacterial growth. Previous studies in oligotrophic saline environments have shown that, depending on phosphate concentration, moderate halophilic bacteria can alter the composition of their cellular compartments [43]. An example of this is the cell membrane, where phospholipids can be replaced by sulphur (sulpholipids), betaines (genes involved in the synthesis of betaine lipids) or amino acids (aminolipids) [43, 44]. The presence of genes encoding phosphate transporters and ugpA, the inorganic phosphate regulator phoR, and sequences coding for the synthesis of osmolytes such as ectABC or betAB (discussed later) along with non-phosphorus lipid precursors like metK, suggest an adaptive mechanism of Vreelandella titanicae Zn11_249 in response to phosphate deficiency in the SdU [43].

Necessary genetic characteristics of Vreelandella titanicae Zn11_249 for its potential adaptation to the extremophilic environment of SdU

The extreme conditions existing in SdU require the presence of essential genes in microorganisms for development and survival in such environments. Identifying these genes provide insights into the strategies that Vreelandella titanicae Zn11_249 could be used to adapt to the SdU polyextremophilic conditions [1]. In terms of general stress, the genome of this bacterium includes essential components involved in membrane integrity and stress tolerance, bacterial hemoglobins, important in managing oxidative and nitrosative stress, and carbon starvation (Supporting Information Table 2) [45, 46]. Also, elements for DNA repair mechanisms, for example, nucleotide excision repair (NER), the genes lexA, the SOS response [47], the base excision repair (BER), genes involved in mismatch repair such as mutL and mutS, and recombination genes like recBCD, were identified [48]. Given that these mechanisms are involved in responding to oxidative damage to DNA, the genomic analysis revealed specific gene sequences that assist in repairing mechanisms under oxidative stress. The study identified 27 genes responsible for oxidative stress response, including superoxide dismutase, catalase, bacterioferritin, glutaredoxin, thioredoxin, peroxiredoxin, and the organic hydroperoxide resistance protein, along with its transcriptional regulator (Supporting Information Table 2) [32]. The presence of reactive oxides in SdU creates a highly oxidizing environment, as reflected in the redox potential of the salt flat [7, 49]. An example is the existence of sulphates, precursors to gypsum formation, which generate free radicals through UV radiation. These radicals could react with hydroxyl groups from the environment to form highly oxidizing hydroxyl radicals (1) [49, 50]. Additionally, due to the presence of halogen oxides such as BrO, already identified in the SdU by Hönninger [49], there could exist reactions with sulphate radicals, acting as free radicals (2) or reacting with the formed hydroxyl radicals (3) [51, 52].

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In the SdU, diverse viral communities might exist, with virus concentrations ranging from 1,55 × 107 – 2,23 × 108 CPL ml− 1 typical of hypersaline environments [53]. The presence of these viruses could be related with the presence of the CRISPR immune system identified in the genome of Vreelandella titanicae Zn11_249 [54].

The hypersalinity present in the SdU requires Vreelandella titanicae Zn11_249 to develop adaptive strategies to cope with these conditions. The fact that strain Zn11_249 is halotolerant suggests that it may contain genes related to osmolyte synthesis. The analysis revealed various gene sequences responsible for the synthesis and transport of the osmolyte glycine betaine (N, N,N-trimethylglycine). These sequences could cover the use of choline sulfatase as a precursor of glycine betaine, as well as the synthesis and transport systems of choline into the cell. The identified sequences related to choline synthesis were identical to those reported for Sinorhizobium meliloti [55]. Additionally, orthologues of choline sulfatase biosynthesis, were also identified. Vreelandella titanicae Zn11_249 also contains genes that comprise the essential system for choline uptake and high-affinity ABC transporters for its transport (Supporting Information Table 2). To complete the possible glycine betaine synthesis pathway, genes responsible for its synthesis (betAB) were identified [56], although genes linked to de novo synthesis of choline sulfatase were not identified. This may be due to the existence of hypothetical proteins that have not yet been identified as the enzymes responsible for this process, although the high energy cost of de novo choline biosynthesis should be also considered. Based on this second hypothesis, there were the possibility that choline transport mechanisms are the main support for osmolyte synthesis in the isolate, as has been observed in previous studies [57]. Continuing with osmoregulatory solutes, Vreelandella titanicae Zn11_249 possesses the complete operon responsible for ectoine synthesis, a well-studied compound due to its applications in skincare and medicine; the osmolyte carnitine; and proline synthesis with proA [4, 58]. Given that SdU is a polyextremophilic environment with factors such as low water activity, high salinity, and high ultraviolet radiation, the ability of these osmolytes to attract water, stabilize macromolecules and whole cells, protect DNA from ionizing radiation, and prevent UV damage is essential for development and survival [8, 59].

Finally, the presence of high concentrations of very toxic compounds such as arsenic, bromine, or lithium makes it of interest to identify sequences encoding proteins responsible for metal tolerance. Among these tolerance mechanisms, efflux pumps, resistance proteins, or P-type ATPases were found (Supporting Information Table 2) [60, 61]. The existence of efflux pumps for multiple compounds is particularly interesting in these environments. The presented data suggest that selective pressure caused by resistance to these heavy metals may lead to co-selection for antibiotic resistance [62]. Therefore, it can be hypothesized that the stress imposed on Vreelandella by the high concentration of heavy metals, like Li+, could be causing cross-resistance, conferring resistance to various types of drugs as well.

Conclusions

The analysis of Vreelandella titanicae Zn11_249 genome suggests that this bacterium is potentially able to utilize diverse carbon, nitrogen, sulphur and phosphate sources, which play a key role in sustaining life in the oligotrophic conditions of Salar de Uyuni. In addition, the isolate has genes potentially involved in fermentation and respiratory systems when oxygen decreased in warm-humid season. The study identified genes for osmolyte synthesis, DNA repair strategies or efflux pumps associated with osmotic stress, oxidative stress, high ultraviolet radiation or high concentrations of heavy metals, extreme environmental conditions that the isolate has to overcome. This work not only provides genomic characteristics that could be responsible for the survival of the isolate in this extreme environment, but also the presence of coding sequences of genes with biotechnological interest for the possible industrial potential of the isolate. This study provides a foundational genomic characterization of Vreelandella titanicae Zn11_249, highlighting genes potentially involved in metabolic versatility and adaptation to extreme environmental stresses characteristic of SdU. While functional and ecological interpretations are limited by the focus on a single genome, our results offer valuable hypotheses and a platform for future transcriptomic, proteomic, and experimental studies to elucidate the ecological role and biotechnological potential of the isolate. Therefore, the information of this research opens up possibilities for new studies to understand the ecological role and biotechnological potential of Vreelandella titanicae Zn11_249.

Supplementary Information

Supplementary Material 1 (22KB, xlsx)
Supplementary Material 2 (14.9KB, xlsx)
Supplementary Material 3 (17.9KB, xlsx)

Abbreviations

Salar de Uyuni

SdU

Tryptic Soy Agar

TSA

CTAB

cetyltrimethylammonium bromide

APS

adenosine 5’-phosphosulphate

PAPS

3’-phosphoadenylyl-sulphate

Authors’ contributions

Conceptualizations: E. S., J-M. M., R. A., Data curation: E. S., J-M. M., Formal analysis: E.S., J-M. M., Methodology: E. S., J-M. M., N. R., R. A., Investigation: E. S., J-M. M., Visualization: E. S., J-M. M., N. R., Writing – original draft: E. S., J-M. M, Writing – review & editing: J-M. M, C. A., R. A., Supervision: J-M. M., C. A., R. A., Funding acquisition: R. A., Project administration: R. A., Resources: R. A., N. R.

Funding information

This research was supported by the Spanish Ministry of Science, Innovation and University projects CGL2015-66242-R and PID2022-136607NB-I00. The authors would like to thank the Gerencia Nacional de Recursos Evaporíticos, COMIBOL, Estado Plurinacional de Bolivia, for facilitating the acquisition of the samples.

Data availability

Reads were deposited at ENA/GenBank/DDBJ under the accession number [ERR13925816](https:/www.ebi.ac.uk/ena/browser/view/ERR13925816), and the complete genome sequences were deposited under the accession numbers [CAYELD000000000](https:/www.ebi.ac.uk/ena/browser/view/CAYELD000000000). All of them are included under study number [PRJEB81508](https:/www.ebi.ac.uk/ena/browser/view/PRJEB81508).

Declarations

Ethics approval and consent to participate

This work is not subject to ethical conflicts or data protection of third parties.

Consent for publication

Not Applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1 (22KB, xlsx)
Supplementary Material 2 (14.9KB, xlsx)
Supplementary Material 3 (17.9KB, xlsx)

Data Availability Statement

Reads were deposited at ENA/GenBank/DDBJ under the accession number [ERR13925816](https:/www.ebi.ac.uk/ena/browser/view/ERR13925816), and the complete genome sequences were deposited under the accession numbers [CAYELD000000000](https:/www.ebi.ac.uk/ena/browser/view/CAYELD000000000). All of them are included under study number [PRJEB81508](https:/www.ebi.ac.uk/ena/browser/view/PRJEB81508).

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