https://orcid.org/0000-0002-4249-0488
Figures
Abstract
Halomonas elongata strain ml10562, was isolated from hypersaline that was collected from Acos Peru. Average Nucleotide Identity (ANI) and dDDH (digital DNA-DNA Hybridization) values between strain ml10562 and type strains of Halomonas elongata species were 71.0–78.4% and 18.8–21.5%, respectively. The draft genome, spanning 4,075,440 base pairs, has a GC content of 64.2% and contains 3,912 genes. Functional characterization revealed the strain’s ability to tolerate and resist increasing concentrations of sodium arsenate, with a minimum inhibitory concentration of 25 mM. Bioinformatic analysis revealed the presence of two operons, arsR-arsH-arsB and arsJ-gapdh-arsC, in the genome of strain ml10562, which could play a crucial role in arsenic resistance through transporter-mediated mechanisms. Overall, these results emphasize the potential adaptability of H. elongata ml10562 to arsenic-containing environments and extend our understanding of bacterial arsenic resistance mechanisms, allowing promising applications in bioremediation.
Citation: Pila-Lacuta S, Pauccar D, Rojas-Vargas J, Rodríguez-Cruz UE, Sierra JL, Castelán-Sánchez HG, et al. (2025) Isolation of a potentially arsenic-resistant Halomonas elongata strain (ml10562) from hypersaline systems in the Peruvian Andes, Cusco. PLoS ONE 20(4): e0320639. https://doi.org/10.1371/journal.pone.0320639
Editor: Debasis Mitra, Graphic Era Institute of Technology: Graphic Era Deemed to be University, India
Received: September 23, 2024; Accepted: February 22, 2025; Published: April 16, 2025
Copyright: © 2025 Pila-Lacuta et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: The sequence data are available at NCBI under the accession number JANIAS000000000, the version described in this paper is JANIAS010000000. BioProject accession number PRJNA862055, and Sequence Read Archive (SRA) accession numbers SAMN29936603.
Funding: This research was supported by Canon Funds “Yachayninchis Wiñarinanpaq” Research Strengthening Program, Resolution No. R-1149-2019-UNSAAC (Contract No. 022-2019-UNSAAC).
Competing interests: The authors have declared that no competing interests exist.
Introduction
The genus Halomonas is a group of Gram-negative bacteria with 180 recognized species [1]. Most species are widely distributed in saline habitats such as salt lakes, marine environments, and saline sands as well as soils [2]. It is one of the most widespread groups of halophilic microorganisms found in different geographical locations, perhaps because of its great metabolic and physiological versatility [3–5].
Within this genus, Halomonas elongata is an aerobic and moderately halophilic bacillus that belongs to the Halomonadaceae familiy. H. elongata thrives in environments with high salt concentrations [2,6]. The main mechanism by which H. elongata can grow in high-salt environments is the accumulation of the compatible solvent ectoin. Ectoine can be produced by bacteria either through de novo synthesis from aspartate or by uptake from the environment via the ectoine-specific, osmoregulated TeaABC (transmembrane ectoine ABC) transporter. This transporter system comprises three genes that encode distinct proteins: a large transmembrane protein (TeaC), a small transmembrane protein (TeaB), and a periplasmic substrate-binding protein (TeaA). The TeaA component is part of the tripartite ATP-independent periplasmic (TRAP) transporter family, specialized for ectoine transport in response to osmotic conditions [7–9].
Halomonas species, including H. elongata, have been found to have the ability to tolerate and even accumulate heavy metals such as arsenic (As) and lead (Pb) in their cells [5,10]. Some arsenic-resistance genes have been described, and their behavior in arsenic-contaminated media has also been reported [4,11,12]. Thus, this species could be a candidate for decontamination of arsenic pollution [12], which has adverse effects on human health [13].
The ability to tolerate and accumulate heavy metals is of great interest for environmental bioremediation [14–16]. H. elongata could potentially be used to remove heavy metals from contaminated soils and waters, thereby reducing the negative impact of such pollutants on the environment. In Peru, this is important because various mineral-rich areas suffer from significant arsenic contamination in their natural groundwater and surface drinking water, which can stem from both volcanic activity (natural pollution) and human-induced mining operations (anthropogenic pollution) [17].
The genus Halomonas possesses the arsenic resistance operon (ars operon), consisting of the genes arsR, arsB, and arsC (collectively known as arsRBC), which work together to reduce arsenate and expel it from the cell. More complex operons, such as arsRDABC which contains five genes arsR, arsD, arsA, arsB, arsC., have also been identified in other bacterial strains [18]. Recent studies have further illuminated a novel pathway for arsenate detoxification involving glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ArsJ, an organoarsenical efflux permease. GAPDH, traditionally known for its role in glycolysis, has been shown to interact synergistically with ArsJ to facilitate the efflux of organoarsenicals from the cell. This discovery suggests a broader role for GAPDH in arsenic resistance beyond its metabolic functions [14,19–22].
The arsR gene produces a regulatory protein that binds to arsenate and activates the expression of the operon when the arsenate concentration is high. The arsD gene encodes a trans-acting repressor of the arsenical operon. The arsA gene codes for an intracellular ATPase protein associated with the arsenic efflux pump encoded by the arsB gene. Additionally, the arsC gene encodes a protein with reductase activity, which converts arsenate into arsenite, released into the external environment through the arsenic efflux pump. Some bacteria contain the arsH gene instead of the arsC gene, which also encodes for an arsenate reductase.
However, the arsRDABC operon is not the sole mechanism conferring resistance to arsenic. In the case of Halomonas sp. strain GFAJ-1, its genome harbors two additional genes: gapdh and arsJ. These genes encode the enzymes GAPDH (glyceraldehyde 3-phosphate dehydrogenase) and a novel MFS transporter, ArsJ, respectively. Both genes suggested a new pathway for arsenic resistance (Wu et al., 2018).
In the present work, we report the isolation and characterization of H. elongata strain ml10562 from hypersaline water at high altitude in Cusco, Peru. Phylogenetic analyses and taxonomic criteria confirmed that strain ml10562 belongs to the species H. elongata. Given the extreme environmental conditions in its habitat, we hypothesized that strain ml10562 possesses genetic traits that contribute to arsenic resistance. To confirm this, we performed phenotypic tests to assess its tolerance to arsenite (As III) and arsenate (As V), and genomic analyses to identify potential determinants of arsenic resistance. While arsenite (As III) is generally considered more toxic due to its higher reactivity with cellular components, arsenate (As V) is more stable under oxygen-rich conditions in the environment and remains a significant contaminant in drinking water sources, posing significant risks to ecosystems and human health [23]. Our results show that strain ml10562 is tolerant to elevated concentrations of both arsenite and arsenate, highlighting its adaptability to arsenic-contaminated environments. Genomic analysis revealed two arsenic resistance operons, arsR-arsH-arsB and arsJ-gapdh-arsC, which may be crucial for arsenic detoxification through transporter-mediated mechanisms. These results suggest that strain ml10562 could be a valuable candidate for bioremediation in arsenic-contaminated waters and soils.
Materials and methods
Bacterial strains and culture conditions
Strain ml10562 was isolated from a hypersaline spring, located at 13°57′5″S, 71°44′18″W, 3,088 m elevation, in the Acos district, Acomayo province, Cusco region of Perú. We collected freshwater samples in Acos, an area that is not designated as a protected natural zone by the “Servicio Nacional de Áreas Naturales Protegidas” (Sernanp). The site is publicly accessible, and there are no specific laws or regulations in Peru that restrict sampling in this location. Twenty liters of water were taken from the natural environment, filtered through 0.22 um, and cultured in modified Sea water (SW) agar [Sodium chloride (NaCl) 25%, Magnesium chloride (MgCl2) 0.5%, Magnesium sulfate (MgSO4) 0.583%, Potassium chloride (KCl) 0.117%, yeast extract 0.05%, Sodium bicarbonate (NaHCO3) 0.003%, Calcium chloride (CaCl2) 0.0083%, pH:8.0] at 37 °C for seven days. Morphologically-distinct colonies were observed, all of which were newly streaked in SW plates for purification [24].
Genome sequencing and control quality
Total genomic DNA was extracted from the strain ml10562, which was cultured in SW medium, using the GenElute kit from Sigma-Aldrich (St. Louis, MO, United States). Sequencing reactions were performed using the Illumina MiSeq platform and the Nextera XT DNA library kit, following the protocol (San Diego, CA, United States). Raw sequences were made available in the SRA database under accession number SRR20740642.
The quality of reads were evaluated using FASTQC [25], and the raw sequences were trimmed with a quality ≥Q29 using TrimGalore [26]. Sequence duplicates were removed using CD-HIT-dup [27], and the reads were de novo assembly with SPAdes v3.13.0 [28]. Finally, the assembly quality was evaluated with CheckM v1.2.2 [29] with the “genus Halomonas” option. The bacterial genome was annotated using RAST tool kit [30]. The genome has been deposited into NCBI under accession number ASM2450512v2.
Genome assembly, identification and annotation
To conduct the phylogenetic analysis and taxonomic identification, we retrieved twenty-six genomes of the Halomonas genus from the NCBI portal (accessed on March 2, 2024). Eight genomes of H. elongata were available in the NCBI database, and based on the results of GTDB-Tk v2.0.0, we also included the 18 genomes most closely related to our strain and identified in the NCBI database [31]. In addition, the genomes of Chromohalobacter canadensis strain DSM 6769 and Chromohalobacter sarecensis strain DSM 15547 were included as outgroups in the phylogenetic tree.
For the phylogenetic reconstruction, we used Proteinortho v6.0.3 to identify the core genome of the twenty-nine set of genomes, using the -identity=50 option [32]. To the core-genome was defined as the single copy orthologous gene families shared by all the genomes. Amino acid sequences were aligned with MAFFT v7 [33], and all positions in the alignment with gaps in 5% or more of the sequences were removed using trimAl v1.4.rev22 [34]. A maximum-likelihood (ML) tree was estimated with IQ-TREE v2.1.2 [35] based on a total of 1,000 bootstrap replicates, using the LG+F+R5 model suggested by the IQ-TREE program.
For taxonomic identification, a whole genome comparison was performed, between our genome and the genome of the type strain H. elongata DSM strain 2581, using PyANI [36] and TYGS [37]. PyANI analysis calculates the average nucleotide identity based on MUMmer (ANIm > 95% for species delineation), and TYGS determines the digital DNA-DNA hybridization (dDDH > 70% for species delineation). The genome annotation of the genome of H. elongata strain ml10562 was made with prokka v1.14.6 [38], following the default parameters.
Identification of genes related to resistance to Arsenic
We also performed genome mining on the selected genomes to identify operons associated with arsenic resistance. Specifically, we targeted the arsR-arsH-arsB and arsJ-gapdh-arsC operons. To achieve this, we conducted an iterative search using PSI–BLAST on the annotated amino acid sequences, applying a cut-off threshold of E < 0.01 to include relevant sequences. The resulting sequences were mapped onto a pre-constructed phylogenetic tree and visualized using ITOL [39].
Growth in arsenic H. elongata ml10562
H. elongata strain ml10562 was cultured in a modified SW medium containing 5% NaCl and pH 7.8 ± 0.2, to which sodium arsenate (Na3AsO4) was added at the following concentrations: 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, and 45 mM. Incubation was performed at 30 °C for 72 hours at 80 rpm, with absorbance measurements taken every 8 hours at a wavelength of 600 nm. The minimal inhibitory concentration (MIC) of sodium arsenate of the strain was the lowest concentration that inhibited their growth. A culture without sodium arsenate was used as a negative control (S1 Table).
Results
Genome characterization and arsenic-related genes in Halomonas sp. strain ml10562
Strain ml10562 was assembled into 683 contigs with a genome size of 4,075,440 bp and a GC content of 63.25% from 329,142 reads, with a total of 77,677,512 bases sequenced, resulting in a coverage of 10x with an N50 value of 9,225 bp. The genome size and the GC content are similar to the values of the genome of the type strain H. elongata strain DSM 2581 (Table 1). The assembly has 95.58% completeness, and 6.77% contamination, according to the CheckM tool analysis. A total of 4,273 coding genes were predicted, representing an increase of 11% compared to the number of coding genes found in the type strain of Halomonas and the genome has been deposited into NCBI under accession number ASM2450512v2.
Genome annotation reveals the presence of genes critical for arsenic resistance in two operons (Fig 1A). The first contains the arsR-arsH-arsB genes, encoding for the regulator ArsR, the arsenical resistance reductase protein ArsH, and the arsenic transporter ArsB. The second harboring the arsJ-gapdh-arsC genes, encoding the MFS transporter ArsJ, the ArsJ-associated glyceraldehyde-3-phosphate dehydrogenase, and the arsenate reductase ArsC.
A. Circular plot of the genome. Reading from the center outwards, genome size, GC skew, GC content, CDS reverse, and CDS forward. The two arsenic-resistance operons are in red arrows, indicating their synteny. B. ML tree of the core genome identified by Proteinortho. The tree was obtained by IQ-TREE with 1,000 bootstrapping. The bacterial genomes were recovered from the NCBI portal (March 2, 2024).
Phylogenetic classification analysis
The phylogenetic tree of H. elongata strain ml10562 (Fig 1B) was derived from different species of the genus Halomonas using the 842 single copy gene families identified, representing the core genome of the used genomes set. The tree was reconstructed with the LG+F+R5 model suggested by the IQ-TREE program. It shows that the closest relatives to ml10562 belong to H. elongata species, isolates obtained from soil/water saltern samples. These species form a monophyletic clade, that is well-supported and independent of other Halomonas species. H. elongata strain 153B falls outside the H. elongata species clade, consistent with its NCBI “taxonomy inconclusive” status, suggesting it may belong to a novel Halomonas species.
To confirm whether the isolated bacteria was H. elongata, ANI and dDDH analyses were performed against the H. elongata type strain DSM 2581 genome. ANIm value of 98.47% and dDDH value of 84.80%, above the thresholds for species delineation (ANIm>95%, dDDH>70%), confirm that our strain belongs to H. elongata species. In conclusion, the phylogenetic analysis and ANI/dDDH values strongly support the identification of the isolated strain ml10562 as H. elongata.
Identification of ars operons in Halomonas genomes
We used genome mining to identify genes responsible for arsenic resistance encoded by operons in 29 bacterial genomes. Our analysis focused on seven proteins that are encoded by the genes: arsR, arsH, arsB, arsK, arsJ, gapdh, and arsC. The first operon includes arsR, arsH, and arsB, while the second operon includes arsK, arsJ, gapdh, and arsC [20].
Our results show that the proteins encoded by the arsR and arsH genes are ubiquitous and present in all genomes analyzed. However, the ArsB protein, a transmembrane As(III) efflux permease responsible for the export of arsenite from the cytosol, was identified in only 7 of the 29 genomes. Remarkably, the H. elongata ml10562 strain contains all three proteins in the first operon (Fig 2). The ArsR protein functions as an arsenite-responsive repressor that regulates transcription of the ars operon. The ArsH protein functions as a reductase with various substrates, including arsenate, which contribute to arsenic resistance by converting arsenate to arsenite and aiding in its subsequent detoxification [20]. The absence of the ArsB protein in many genomes suggests alternative mechanisms for arsenic efflux in these bacteria.
Halomonas elongata ml10562 strain have present two operons, arsR-arsH-arsB, and arsJ-gapdh-arsC.
The ArsK protein, which is involved as an arsenic transport protein in the detoxification of arsenic [40], was identified in the genomes of Halomonas binhaiensis Y2R2 and Halomonas organivorans CECT 5995 [41]. This suggests that these bacteria may have additional arsenic resistance mechanisms beyond the arsJ-gapdh-arsC operon. The arsJ-gapdh-arsC operon associated with arsenic resistance was almost completely present in all genomes analyzed, except for Halomonas organivorans CECT 5995 and Halomonas halophila NBRC 102604.
These results suggest a diversity of genetic strategies for arsenic resistance within the genus Halomonas, with some strains having a full complement of arsenic resistance proteins, while others rely on alternative mechanisms.
Arsenite operon genes in arsenic-resistant ml10562 strain
Primers targeting to arsenite transporter gene (arsB) successfully amplified a single amplicon of the expected size (approximately 800 pb) and the sequences showed 73% - 80.5% nucleotide sequence identity to putative arsenite efflux pump of Pseudomonas sp. and Halomonas sp. The length of arsB reported was approximate 750 pb, in case of H. elongata ml10562 the sequence was 806 pb, and also was similar to the others arsB well characterized bacteria sequences with PCR product about 850 pb [42].
The fragment of arsenate reductase (arsC), a member of the second operon (arsC-gapdh-arsJ), was amplified by new design primers, and the amplicon was 86 pb. Sequence analysis revealed 87.5% similarity to the arsenate reductase (glutaredoxin) of Onishia taeanensis strain USBA-857 (WP_112055332.1), previously classified within the genus Halomonas [43]. This similarity suggests that strain ml10562 contains this reductase gene. The nucleotide sequences obtained in this study were deposited in the GenBank database with number of access PQ331186.
H. elongata ml10562 has the ability to grow and tolerate different concentrations of sodium arsenate
H. elongata strain ml10562 grown in broth at different concentrations of sodium arsenate during different time periods, and the curves with higher absorbance values are seen at decreasing concentrations of 5 > 10 > 15 > 20 mM Na3AsO4, clearly showing that tolerance to this compound decreases as the concentration to which this strain is exposed increases (Fig 3, S1 Fig).
Strain ml10562 had growth from 8 h with 5 mM, while 10 mM and 15 mM had exponential growth after 32 hrs.
The 25 mM and 30 mM curves show a die-off phase, as evidenced by decreased absorbance values. This suggests that the minimum inhibitory concentration of sodium arsenate is 25 mM, as this is the lowest concentration that prevents bacterial growth.
The results show that the strain ml10562 tolerates and is resistant to increasing concentrations of sodium arsenate (Na3AsO4). As the sodium arsenate concentration increased, a decline in growth was observed, with higher absorbance values corresponding to lower concentrations. Upon gradually increasing the concentration of sodium arsenate, it was observed that at 25 mM, strain ml10562 tolerated the arsenate without any increase in OD for the first 24 hours. After 48 hours, the OD values declined close to zero (OD < 0.0056).
Discussion
Arsenic contamination represents a severe ecological and health hazard, exacerbated by industrial activities such as mining that release arsenic into water and soil. Bacteria play a crucial role in the biogeochemical cycling of arsenic, acting as both mobilizers and sinks in these contaminated ecosystems. Understanding the genetic basis of arsenic resistance in bacteria provides insights into their potential applications in bioremediation and makes arsenic-tolerant microorganisms valuable resources for the mitigation of arsenic pollution [44,45].
The Peruvian mining sector, particularly in regions such as Cerro de Pasco, has contributed to significant arsenic contamination in interconnected aquifers, some of which include hypersaline and freshwater systems in the Andes [46,47]. Hypersaline environments, often characterized by extreme conditions, are known to harbor a variety of arsenic-resistant bacteria, as has been reported from saline lakes such as Lake Van, Mono Lake, Searles Lake, and Dali Lake [44]. In this study, we isolated H. elongata strain ml10562 from a hypersaline region in the Peruvian Andes, an environment in which resistance to heavy metals, including arsenic, could provide a survival advantage. The adaptation of this strain to both hypersaline and arsenic-contaminated conditions suggests its potential as a model organism for understanding arsenic resistance in extreme environments and highlights its potential utility for bioremediation specifically in hypersaline, arsenic-contaminated ecosystems.
Genome sequencing and assembly confirmed that strain ml10562 belongs to H. elongata, a species that is well adapted to high salinity and has been shown to exhibit arsenic tolerance. A comparative genomic analysis revealed two arsenic resistance operons: arsR-arsH-arsB and arsJ-gapdh-arsC. These operons encode critical functions for arsenic detoxification. The arsR-arsH-arsB operon comprises arsR, a regulatory gene encoding a repressor that controls expression of the operon in response to arsenic stress [10,11,48,49]; arsB (also known as acr3), which functions as an efflux pump and facilitates the export of arsenite [As(III)][50]; and arsH, which may reduce arsenate to arsenite, providing additional detoxification support [20]. The arsJ-gapdh-arsC operon also includes arsenate reductase (arsC), which is essential for the conversion of arsenate [As(V)] to arsenite, which can then be actively exported from the cell [19]. The arsJ gene, which is also present in other arsenic-resistant Halomonas strains, is thought to encode a transporter involved in arsenite export, while gapdh may contribute to both arsenic resistance and general cellular metabolism, indicating a dual function [20]. Notably, eight of the twenty-nine Halomonas genomes analyzed contain the arsB gene for arsenic export, indicating that strains without this gene may rely on alternative efflux mechanisms.
Interestingly, we identified the arsK gene in two Halomonas genomes. The arsK gene encodes an arsenic efflux transporter previously characterized in Agrobacterium tumefaciens strain GW4, where it helps to expel arsenic compounds from cells to reduce toxicity [40]. The presence of arsK in Halomonas suggests possible arsenic resistance mechanisms that should be further investigated to confirm its function and role in detoxification. The absence of arsK in strain ml10562 suggests that it relies on alternative arsenic efflux systems or mainly on the arsJ-gapdh-arsC operon for arsenic export and detoxification.
Previous studies on arsenic resistance in Halomonas species have shown varying degrees of tolerance, suggesting species- or strain-specific adaptations to arsenic. For example, Halomonas sp. strain A3H3 showed resistance to 29 mM As(III) and more than 106 mM As(IV) [11], while Halomonas sp. strain MG showed a tolerance level of 10.68 mM arsenic [12]. In contrast, strain ml10562 showed moderate tolerance to arsenic, with an MIC at 25 mM sodium arsenate. Although this resistance is lower than that of strain A3H3, it is higher than that of strain MG. These results suggest that strain ml10562 may have adaptations unique to its hypersaline origin, where selection pressure favors intermediate levels of arsenic resistance. In addition, differences in the genetic make-up of arsenic resistance mechanisms, such as variations in operons or the presence or absence of specific transporters, could be responsible for the diversity in arsenic tolerance between Halomonas strains.
Conclusions
In conclusion, the genomic analysis of H. elongata strain ml10562 revealed key insights into its arsenic resistance mechanisms and phylogenetic relationships within the Halomonas genus. The strain exhibited a high degree of genomic completeness and similarity to other strains of H. elongata and phylogenetic analysis confirmed the classification of strain ml10562 as a member of the H. elongata species. Furthermore, the strain demonstrated tolerance and resistance to increasing concentrations of sodium arsenate, with the minimum inhibitory concentration determined to be 25 mM. This resistance was supported by the presence of arsenic detoxification genes such as arsH and arsJ, which play crucial roles in mitigating the cellular accumulation of arsenate.
Overall, these findings highlight the adaptability of H. elongata strain ml10562 to arsenic-rich environments and contribute to our understanding of the diverse mechanisms bacteria employ to survive in such challenging conditions.
Further directions
Our study identified two operons, arsR-arsH-arsB and arsJ-gapdh-arsC, associated with arsenic resistance in H. elongata ml10562. While their presence suggests a potential mechanism for arsenic detoxification, further functional characterization is warranted. Techniques like gene knockout and overexpression could elucidate their specific roles in arsenic resistance. Additionally, qPCR or RNA-seq could be used to quantify the expression of these genes (arsR, arsH, arsB, arsJ, gapdh, arsC) under arsenic exposure. This would confirm if they are upregulated in response to arsenic stress. However, the primary objective of this study was to provide a comprehensive genomic analysis of H. elongata ml10562, highlighting its potential for future applications, such as bioremediation of arsenic-contaminated environments.
Supporting information
S1 Fig. Growth of H. elongata strain ml10562 at different sodium arsenate concentrations on a logarithmic scale.
Strain ml10562 showed growth from 8 hours at 5 mM, while at 10 mM and 15 mM exponential growth was observed after 32 hours. The data are presented on a logarithmic scale to emphasize the differences in growth patterns.
https://doi.org/10.1371/journal.pone.0320639.s001
(TIF)
S1 Table. Curve of growth of H. elongata strain ml10562.
https://doi.org/10.1371/journal.pone.0320639.s002
(XLS)
Acknowledgments
We thank Juan Manuel Hurtado Ramírez for his support in the installation of bioinformatics programs.
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