Historical Methods

DNA methylation modification regulates gene expression during temperature stress. The adaptation mechanisms of cold-adapted microorganisms to low temperatures have been explained at the gene and metabolic levels. However, considering the important epigenetic modification in cells, the role of genomic modification in cold-adapted microorganisms remains underexplored. This study aims to discuss the regulatory role of DNA methylation in the cold response of psychrotroph Exiguobacterium undae TRM 85608. Methylome analysis shows that the methylation level of most genes in the bacterium decreases under cold stress. Combined with transcriptome results, the expression of important cold-response genes such as ABC transporter permease and ATP-binding proteins increases, but their methylation levels decrease, which is associated with a reduction of DNA adenine methyltransferase. We believe that the reduction in genomic methylation modification caused by low temperature is a major factor in stabilizing the normal growth of the cell. The bacterium counteracts cold stress by reducing the expression of methylation modification enzymes and weakening the inhibition of cold-response gene modification. These findings provide new insights into how psychrophilic organisms adapt to low temperatures.

Studies of microorganisms in extreme Mars-analog environments have generally overlooked fungi. Here, we document fungi in lake waters, slime, and halite of the acid-saline Lakes Magic and Gneiss in Western Australia with pH 1.4–3.5 and 7–32% total dissolved solids (TDS). Both extremotolerant fungi, including ascomycete Parengyodontium torokii, and relatively common fungi (mesophilic), including Penicillium breviocompactum and Trametes pubescens, were present. Our discovery of P. torokii in halite is among the first known fungal examples of such preservation, and we propose that it has the biological traits of a generalist species. Nine strains of the dominant P. torokii fungi were tested for growth on diverse salts. The presence of mesophilic fungal saprotrophs in these lakes, along with extremophilic fungi, algae, bacteria, and archaea, suggests transport of the former into indigenous lake populations. This reveals a distinction between habitability and preservation potential; not all biosignatures in lake waters or their halite represent organisms that were active in situ. Our results suggest that searches for biosignatures in extreme waters and salt minerals on Earth and Mars should include the possibility of fungi. Additionally, interpretations of microbial communities in both modern brines and the rock record should consider the likelihood of mixed indigenous and transported taxa.

To fish-out novel salt-tolerance genes, metagenomic DNA of moderately saline sediments of India’s largest hypersaline Sambhar Lake was cloned in fosmid. Two functionally-picked clones helped the Escherichia coli host to tolerate 0.6 M NaCl. Deep sequencing of their fosmid DNA insert revealed 32–37% of genes to encode transporters, mostly belonging to ABC (ATP-Binding Cassette)-type, but none specific to channel Na+. The complete metagenome sequence of Sambhar Lake brines, and reanalysed data of twelve other hypersaline metagenome sequences, however, have only around 5% transporter genes, suggesting metagenomic DNA fragments being biasedly-cloned during functional screening. Almost half of the ~ 40 Kb inserts in the two clones was shared, and encode several transporters, and some transposase. This advocates that these transporter-loaded DNA lengths are shuttled among microorganisms of hypersaline environments. Interestingly, one clone showed retarded growth with prominent cell disruptions in scanning electron microscopic images, when fosmid copy number was increased or transporters were NaCl-induced. Its cloned insert exclusively has three genes, encoding a structurally functional ATP-binding protein and its efflux component, whose possible overexpression led to membrane crowding and cell rupture. Thus, microorganisms thriving in hypersaline lakes have plentiful ABC transporters that are mutually shared among themselves. These novel salt tolerance genes have future agricultural biotechnological potential.

Four halophilic archaeal strains were isolated from sea salt and a saline lake in China. Based on phylogenetic and phylogenomic analyses, the four strains are related to the genera of Halobellus, Halobaculum, and Halorarum within the family Haloferacaceae. The four strains possess genes responsible for carotenoid synthesis, maintenance of a high internal salt concentration, as well as diverse enzymes with biotechnological potential. The average nucleotide identity (ANI), average amino acid identity (AAI), and digital DNA-DNA hybridization (dDDH) values among these four strains and their related species were lower than the established thresholds proposed for species demarcation. Strains DFY28T, ZY16T, QDC11T, and XH14T were distinguished from related species based on a variety of phenotypic characteristics. The major polar lipids of these four strains were similar to those of respective relatives within the genera Halobellus, Halobaculum, and Halorarum. The phenotypic, phylogenetic, and genome-based analyses suggest that strains DFY28T (= CGMCC 1.17470T = JCM 34310T), ZY16T (= CGMCC 1.17476T = JCM 34311T), QDC11T (= MCCC 4K00127T = KCTC 4308T), and XH14T (= CGMCC 1.17028T = JCM 34145T) represent four novel species of the genera Halobellus, Halobaculum and Halorarum, for which the names Halobellus marinus sp. nov., Halobellus ordinarius sp. nov., Halobaculum rarum sp. nov., and Halorarum halobium sp. nov. are proposed.

Abstract Rhodothalassium (Rts.) salexigens is a halophilic purple nonsulfur bacterium and the sole species in the genus Rhodothalassium, which is itself the sole genus in the family Rhodothalassiaceae and sole family in the order Rhodothalassiales (class Alphaproteobacteria). The genome of this phylogenetically unique phototroph comprises 3.35 Mb and is highly chimeric, with nearly half of its genes originating from families other than the Rhodothalassiaceae, many of which lack phototrophic species. Photosynthesis genes in Rts. salexigens are not arranged in a typical photosynthesis gene cluster but are scattered across the genome, suggesting an origin from horizontal transfers. Despite an encoded RuBisCO, autotrophy has not been observed in Rts. salexigens, and enzymes that oxidize common inorganic electron donors are not encoded. Phospholipid biosynthesis in Rts. salexigens is restricted, and phosphoglycerolipids are the only phospholipids present in its intracytoplasmic membranes. Rts. salexigens fixes nitrogen using a Mo-containing nitrogenase and uses ammonia despite previous results that indicated it was a glutamate auxotroph. Glycine betaine is the sole osmolyte in Rts. salexigens, and enzymes are encoded that facilitate both its uptake and its biosynthesis from glycine. The genomic data also support chemotactic swimming motility, growth over a range of salinities, and the production of membrane-strengthening hopanoids.

Azo dye wastewater has garnered significant attention from researchers because of its association with high-temperature, high-salt, and high-alkali conditions. In this study, consortium ZZ efficiently decolorized brown D3G under halophilic and thermophilic conditions. he results indicated that consortium ZZ, which was mainly dominated by Marinobacter, Bacillus, and Halomonas, was achieved decolorization rates ranging from 1 to 10% at temperatures between 40 °C and 50 °C, while maintaining a pH range of 7 to 10 for direct brown D3G degradation. Through the comprehensive utilization of UV–vis spectral analysis, Fourier transform infrared (FTIR), gas chromatography mass spectrometric (GC–MS) techniques, as well as metagenomic analysis, the decolorization and degradation pathway of direct brown by consortium ZZ was proposed. The azo dye reductase, lignin peroxidase, and laccase were also highly expressed in the decolorization process. Additionally, phytotoxicity tests using seeds of Cucumis sativus and Oryza sativa revealed that the intermediates generated showed no significant toxicity compared with distilled water. This investigation elucidated the pivotal contribution of consortium ZZ to azo dye degradation and provided novel theoretical insights along with practical guidance for azo dye treatment at halo-thermophilic conditions.

Since prophages can play a multifaceted role in bacterial evolution, this study aims to characterize the virome of Rummeliibacillus stabekisii, a bacterium isolated from different environments, including Antarctic soil and NASA spacecraft floors. From the analyses, it was found that the Antarctic strain, PP9, had the largest number of prophages, including intact ones, indicating potential benefits for survival in adverse conditions. In contrast, other strains harbored predominantly degenerate prophages, suggesting a dynamic process of gene gain and loss during evolution. Furthermore, strain PP9 exhibited polylysogeny, a strategy capable of increasing its competitive advantage by providing a broader spectrum of defensive mechanisms. In addition, evidence demonstrates that prophage regions in PP9 act as hotspots for recombination events, favoring the insertion of different phages and possible antimicrobial resistance genes. Finally, lytic cycle induction experiments revealed at least two intact prophages active in PP9. In this way, understanding the interaction between viruses and bacteria can provide valuable information about microbial evolution and adaptation in extreme environments, such as Antarctica.

Polyhydroxyalkanoates (PHAs) are intracellular polymers that enhance bacterial fitness against various environmental stressors. Pseudomonas extremaustralis 14-3b is an Antarctic bacterium capable of accumulating, short-chain-length PHAs (sclPHAs), composed of C3–C5 monomers, as well as medium-chain-length PHAs (mclPHAs) containing ≥ C6 monomers. Since pH changes are pivotal in bacterial physiology, influencing microbial growth and metabolic processes, we propose that accumulated PHA increases P. extremaustralis fitness to cope with pH changes. To test this, we analyzed the production of sclPHA and mclPHA at different pH levels and its effect on bacterial survival against pH stress. P. extremaustralis was able to grow and accumulate PHA when the culture media pH ranged from 6.0 to 9.5, showing a marked loss of viability outside this range. Additionally, based on the analysis of different PHA-deficient mutants, we found that when exposed to both acidic and alkaline conditions, sclPHA and mclPHA conferred different protection against pH stress, with sclPHA making the main contribution. These results highlight the importance of PHA in supporting survival in pH-stressful environments.

Among the many ice-binding proteins (IBPs) found in microorganisms (bacteria, archaea, fungi and algae), the canonical DUF3494 beta-barrel type is the most common. Until now, little variation has been found in this structure: an initial coil leads into an alpha helix that directs the following coils into a reverse stack, with the final coil ending up next to the initial coil. Here, I show that there exist many bacterial proteins whose AlphaFold-predicted structures deviate from the DUF3494 structure so that they are not recognized as belonging to an existing DUF or Pfam family. In these non-canonical DUF3494 (ncDUF3494) proteins, the number of coils in the alpha helix is highly variable, often being as high as 14. The putative ice-binding sides of each of 13 proteins modeled have a well-aligned row of hydrophilic residues, with spacings that are close to the repeat distance on the ice a-axis. A recombinant protein made for one of the proteins showed that it had ice-binding activity, even in the µg/ml range. The ncDUF3494 proteins appear to be found only in bacteria, the great majority of which live in icy habitats. C-terminal PEP-Cterm motifs, which are rare in DUF3494s, are present in most of the ncDUF3494s, possibly indicating a secretory function. The relatively narrow distribution of ncDUF3494 proteins suggests that they are a later development in DUF3494 evolution.

AbstractThe novel esterase gene lipP1, which encodes HjEstP1, was discovered in the genome of the extremely halophilic archaeon Haloarcula japonica. A homology search and sequence alignment revealed that HjEstP1 is a member of family IV esterases with conserved GXSXG and HGGG motifs. lipP1 was expressed in its parental strain, and recombinant HjEstP1 was purified and characterized. Optimal pH and temperature of HjEstP1 were 6.0 and > 60 °C, respectively. HjEstP1 showed higher activity with increasing NaCl concentration, and optimal NaCl concentration was > 4.5 M. Furthermore, HjEstP1 preferentially hydrolyzed pNP and glycerol esters with short chain fatty acids. To our knowledge, this is the first report of an esterase from an extremely halophilic archaeon obtained via homologous expression.

As the textile wastewater is highly saline and has high pH it is important to employ extremophilic microbes to survive in harsh conditions and provide effective bioremediation of textile dyes. This study aims to find a sustainable solution for dye removal by investigating the potential of an indigenously isolated bacterium, Nesterenkonia lacusekhoensis EMLA3 (halo-alkaliphilic) for treatment of an azo dye, methyl orange (MO) and textile effluent. MO dye decolorization studies were conducted using mineral salt media (MSM) by varying incubation time (0–120 h), initial dye concentration (50–350 mg/L), pH (7.0–12.0), inoculum dose (3–10%), agitation (stationary, 100 rpm and 200 rpm), and temperature (20–55 °C). Dye removal by the bacterium for 50 mg/L of dye was > 97.0% within 72 h of incubation at pH 11.0 in stationary condition. Bacterium had excellent reusability i.e. > 97% dye removal for up to 5 cycles. Moreover, bacterium has the potential for co-removal of chromium (VI) (3.5–28 mg/L), and also almost complete dye removal in presence of high amount of NaCl. Liquid chromatography-mass spectrometry showed degradation as the mechanism of dye removal. Application of the bacterium to MO dye spiked real textile wastewater showed excellent dye removal. Phyto-toxicity assessment conducted on Vigna radiata and Triticum aestivum seeds, showed 100% germination of biotreated textile wastewater indicating its reuse potential.

Among extremophiles, thermophile microorganisms from geothermal sites have been widely studied. Nevertheless, our knowledge is still relatively poor on microbial communities colonizing fumaroles, which are super-ephemeral habitats, characterized by an only intermittent presence of water. Here we characterized by metabarcoding both bacterial and archaeal communities from hot spring waters and biofilms, together with dry and wet fumaroles, of a geothermal basin in central Italy. Taxa composition of the analyzed samples mirrored that of previous studies, with Thermoproteota dominating among Archaea, while high percentages of thermophiles and spore-forming organisms were retrieved for Bacteria. Cyanobacteriota were the dominant group in biofilms. Community structure was different in the two domains, with highly selected communities of Archaea, less diversified than bacterial ones. Linear regression analyses highlighted significant correlations between diversity and environmental parameters in dry, but not in wet fumaroles. Although ASV numbers displayed different trends for the two different prokaryotic domains (positive correlation with pH for Bacteria, negative correlation for both pH and T for Archaea), such results indicate that even an extremely ephemeral presence of water can influence the importance of temperature and pH as drivers for microbial community structure.

Numerous psychrophiles inhabit the cold environments that are prevalent across the global biosphere. The adaptation of psychrophiles to cold conditions has been widely studied in strains from the archaeal phylum Euryarchaeota and the bacterial class Gamma-proteobacteria. However, given the vast diversity of microorganisms in cold environments, many microbial lineages with potentially unique cold-adaptation strategies remain largely unexplored. This study investigates the cold responses of the Antarctic strain Poseidonibacter antarcticus SM1702T, a cold-adapted bacterium belonging to the class Epsilon-proteobacteria within the phylum Campylobacterota. Proteomic analysis revealed that this strain responds to low temperatures by overexpressing proteins involved in energy production and amino acid transport. Experimental results confirmed that intracellular ATP concentrations increased at low temperatures compared to higher temperatures. Low temperatures significantly reduced the strain’s amino acid absorption rates, a condition that was mitigated by increased expression of membrane transporters. We propose that the impairment of membrane protein function due to low temperatures is the primary factor affecting cell growth. As a result, the strain enhances ATP synthesis and upregulates membrane transporter expression to counteract cold stress. These findings contribute to a deeper understanding of cold adaptation strategies in psychrophiles.