Physiological and genomic insights into abiotic stress of halophilic archaeon Natrinema altunense 4.1R isolated from a saline ecosystem of Tunisian desert

Abstract

Halophilic archaea are polyextremophiles with the ability to withstand fluctuations in salinity, high levels of ultraviolet radiation, and oxidative stress, allowing them to survive in a wide range of environments and making them an excellent model for astrobiological research. Natrinema altunense 4.1R is a halophilic archaeon isolated from the endorheic saline lake systems, Sebkhas, located in arid and semi-arid regions of Tunisia. It is an ecosystem characterized by periodic flooding from subsurface groundwater and fluctuating salinities. Here, we assess the physiological responses and genomic characterization of N. altunense 4.1R to UV-C radiation, as well as osmotic and oxidative stresses. Results showed that the 4.1R strain is able to survive up to 36% of salinity, up to 180 J/m² to UV-C radiation, and at 50 mM of H₂O₂, a resistance profile similar to Halobacterium salinarum, a strain often used as UV-C resistant model. In order to understand the genetic determinants of N. altunense 4.1R survival strategy, we sequenced and analyzed its genome. Results showed multiple gene copies of osmotic stress, oxidative stress, and DNA repair response mechanisms supporting its survivability at extreme salinities and radiations. Indeed, the 3D molecular structures of seven proteins related to responses to UV-C radiation (excinucleases UvrA, UvrB, and UvrC, and photolyase), saline stress (trehalose-6-phosphate synthase OtsA and trehalose-phosphatase OtsB), and oxidative stress (superoxide dismutase SOD) were constructed by homology modeling. This study extends the abiotic stress range for the species N. altunense and adds to the repertoire of UV and oxidative stress resistance genes generally known from haloarchaeon.

Keywords: Genomic analysis; Haloarchaea; Molecular modeling; Natrinema altunense; Osmotic stress; Oxidative stress; UV-C radiation.

Fig. 1

Phylogenetic tree of N. altunense 4.1R strain with its closest relative species based on 16S rRNA gene sequences. The evolutionary history was inferred using the UPGMA method. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) are shown next to the branches. The tree was rooted with Halobacterium salinarum (LC556329.1). Evolutionary analyses were conducted in MEGAX v10.2.6. GenBank Accession number of sequences are shown in parenthesis

Fig. 2

Physiological tolerance of N. altunense 4.1R to abiotic stress. A Heat map plots of physiological tolerance to pH (at 37 °C), salinity (at 37 °C and pH 7.4), and temperature (at pH 7.4). B Survival cells after exposition to increasing UV-C radiation after incubation in light and dark conditions. C Percentage of cells viability at different concentrations of H₂O₂. The bars represent the standard error, which is not shown at 40 and 50 mM H₂O₂ due to their small value

Fig. 3

Comparative genome analysis. A Venn diagram showing the distribution of shared gene families (orthologous clusters) among N. altunense 4.1R, N. altunense AJ2, N. altunense JCM 12,890 and N. altunense 1A4-DGR. B Totals of orthologs in each genome that were used to generate the Venn diagram. C Sum of the number of genes shared between 4 genomes (total of 3146 genes), between 3 genomes (total of 337 genes), between 2 genomes (total of 130 genes), and a total of 18 singletons specific genes identified for N. altunense 4.1R (14 genes) and N. altunense 1A4-DGR (4 genes)

Fig. 4

Schematic representation of salt and oxidative stress tolerance mechanisms in N. altunense 4.1R inferred from whole genome analysis. A Genes involved in trehalose, Glutamate, and proline biosynthesis. Various ion transporters such as the potassium transporter (kdpB), Na + /H + antiporter, potassium uptake protein (TrkAH), Na⁺/proline symporters (Opua), potassium channel protein (PCP), sodium/calcium antiporter and Kef-type K⁺ transporter systems were detected to maintain the internal ion homeostasis. B Genes involved the oxidative stress tolerance include enzymatic and nonenzymatic antioxidant proteins. Primary enzymatic antioxidants include superoxide dismutase (SOD), catalase (Cat), catalase/peroxidase HPI (Cat/Prx HPI), glutaredoxins (GRx), peroxiredoxins (PRxs), glutathione S-transferases (GST), and alkyl hydroperoxide reductase (AhpC). Primary nonenzymatic antioxidants contain glutathione, thioredoxin, and glutaredoxin

Fig. 5

Predicted network interactions of proteins implicated in responses to Uvr-C radiation, osmotic and oxidative stresses of N. altunense 4.1R using tools and databases that can predict protein function (PPI enrichment p-value: PPI enrichment p-value: < 1.0 $\times$ ⁻¹⁶). Colored edges represent the evidence of protein–protein associations where turquoise and pink edges represent known interactions; green, red, and blue edges represent predicted interactions, and the rest represent other interactions like homology prediction. Colored nodes indicate the different metabolic pathways

Fig. 6

Molecular models of the proteins from N. altunense 4.1R involved in responses to UV-C radiation. A UvrA. Dimeric structure, where subunits are represented in red and blue. Zn binding sites are indicated in circles and shown in detail right panel. B UvrB. Overall structure and details of the ADP and polythyimine trinucleotide binding sites. Ligands are represented with carbon atoms in green, oxygen in red, and nitrogen in blue. C UvrC. Overall structure where the alpha helixes are represented in orange, while beta strands in purple. D Photolyase. Overall structure and details of the FAD binding site, where the ligand is represented with carbon atoms in green, oxygen in red, and nitrogen in blue

Fig. 7

Molecular models of the proteins from N. altunense 4.1R involved in responses to saline (OtsA and OtsB) and oxidative stress (SOD). A OtsA. Tetrameric structure, subunit, and details of the T6P and UDP binding sites. In the tetramer, each subunit is represented by a different color. In the right-top a subunit is shown where a flexible loop (Ser44-Gly99) is remarked. In the right-down, the T6P and UDP are represented with carbon atoms in green, oxygen in red, and nitrogen in blue. B OtsB. Overall structure and details of the Mg binding site. Notice the conserved aspartates coordinating the Mg binding. C SOD. Tetramer, details of the subunit and Mn binding site. Each subunit is represented by a different color in the tetramer. Notice that the Mn binding site is coordinated by three conserved histidines and one aspartate