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Understanding protein stability is critical for the application of enzymes in biotechnological processes. The structural basis for the stability of thermally adapted chitinases has not yet been examined. In this study, the amino acid sequences and X-ray structures of psychrophilic, mesophilic, and hyperthermophilic chitinases were analyzed using computational and molecular dynamics (MD) simulation methods. From the findings, the key features associated with higher stability in mesophilic and thermophilic chitinases were fewer and/or shorter loops, oligomerization, and less flexible surface regions. No consistent trends were observed between stability and amino acid composition, structural features, or electrostatic interactions. Instead, unique elements affecting stability were identified in different chitinases. Notably, hyperthermostable chitinase had a much shorter surface loop compared to psychrophilic and mesophilic homologs, implying that the extended floppy surface region in cold-adapted and mesophilic chitinases may have acted as a “weak link” from where unfolding was initiated. MD simulations confirmed that the prevalence and flexibility of the loops adjacent to the active site were greater in low-temperature-adapted chitinases and may have led to the occlusion of the active site at higher temperatures compared to their thermostable homologs. Following this, loop “hot spots” for stabilizing and destabilizing mutations were also identified. This information is not only useful for the elucidation of the structure–stability relationship, but will be crucial for designing and engineering chitinases to have enhanced thermoactivity and to withstand harsh industrial processing conditions

Tardigrada, a phylum of meiofaunal organisms, have been at the center of discussions of the evolution of Metazoa, the biology of survival in extreme environments, and the role of horizontal gene transfer in animal evolution. Tardigrada are placed as sisters to Arthropoda and Onychophora (velvet worms) in the superphylum Panarthropoda by morphological analyses, but many molecular phylogenies fail to recover this relationship. This tension between molecular and morphological understanding may be very revealing of the mode and patterns of evolution of major groups. Limnoterrestrial tardigrades display extreme cryptobiotic abilities, including anhydrobiosis and cryobiosis, as do bdelloid rotifers, nematodes, and other animals of the water film. These extremophile behaviors challenge understanding of normal, aqueous physiology: how does a multicellular organism avoid lethal cellular collapse in the absence of liquid water? Meiofaunal species have been reported to have elevated levels of horizontal gene transfer (HGT) events, but how important this is in evolution, and particularly in the evolution of extremophile physiology, is unclear. To address these questions, we resequenced and reassembled the genome of H. dujardini, a limnoterrestrial tardigrade that can undergo anhydrobiosis only after extensive pre-exposure to drying conditions, and compared it to the genome of R. varieornatus, a related species with tolerance to rapid desiccation. The 2 species had contrasting gene expression responses to anhydrobiosis, with major transcriptional change in H. dujardini but limited regulation in R. varieornatus. We identified few horizontally transferred genes, but some of these were shown to be involved in entry into anhydrobiosis. Whole-genome molecular phylogenies supported a Tardigrada+Nematoda relationship over Tardigrada+Arthropoda, but rare genomic changes tended to support Tardigrada+Arthropoda.

Nitrifying microorganisms occur across a wide temperature range from 4 to 84 °C and previous studies in geothermal systems revealed their activity under extreme conditions. Archaea were detected to be responsible for the first step of nitrification, but it is still a challenging issue to clarify the identity of heat-tolerant nitrite oxidizers. In a long-term cultivation approach, we inoculated mineral media containing ammonium and nitrite as substrates with biofilms and sediments of two hot springs in Yellowstone National Park (USA). The nitrifying consortia obtained at 70 °C consisted mostly of novel Chloroflexi as revealed by metagenomic sequencing. Among these, two deep-branching novel Chloroflexi were identified as putative nitrite-oxidizing bacteria (NOB) by the presence of nitrite oxidoreductase encoding genes in their genomes. Stoichiometric oxidation of nitrite to nitrate occurred under lithoautotrophic conditions, but was stimulated by organic matter. Both NOB candidates survived long periods of starvation and the more abundant one formed miniaturized cells and was heat resistant. This detection of novel thermophilic NOB exemplifies our still incomplete knowledge of nitrification, and indicates that nitrite oxidation might be an ancient and wide-spread form of energy conservation.

The molecular basis of convergent phenotypes is often unknown. However, convergence at a genomic level is predicted when there are large population sizes, gene flow among diverging lineages or strong genetic constraints. We used whole-genome resequencing to investigate genomic convergence in fishes ( Poecilia spp.) that have repeatedly colonized hydrogen sulfide (H 2 S)-rich environments in Mexico. We identified genomic similarities in both single nucleotide polymorphisms (SNPs) and structural variants (SVs) among independently derived sulfide spring populations, with approximately 1.2% of the genome being shared among sulfidic ecotypes. We compared these convergent genomic regions to candidate genes for H 2 S adaptation identified from transcriptomic analyses and found that a significant proportion of these candidate genes (8%) were also in regions where sulfidic individuals had similar SNPs, while only 1.7% were in regions where sulfidic individuals had similar SVs. Those candidate genes included genes involved in sulfide detoxification, the electron transport chain (the main toxicity target of H 2 S) and other processes putatively important for adaptation to sulfidic environments. Regional genomic similarity across independent populations exposed to the same source of selection is consistent with selection on standing variation or introgression of adaptive alleles across divergent lineages. However, combined with previous analyses, our data also support that adaptive changes in mitochondrially encoded subunits arose independently via selection on de novo mutations. Pressing questions remain on what conditions ultimately facilitate the independent rise of adaptive alleles at the same loci in separate populations, and thus, the degree to which evolution is repeatable or predictable. This article is part of the theme issue ‘Convergent evolution in the genomics era: new insights and directions'.

Three bacterial strains producing blue-violet pigmented colonies on R2A agar were isolated from a wet rock wall and lakes in the deglaciated northern part of James Ross Island, Antarctica. The isolated strains inhibited phytopathogenic Gram-positive bacteria Clavibacter spp., Curtobacterium flacumfaciens , and Paenarthrobacter ilicis . Phylogenetic analysis based on the 16S rRNA gene indicated that the isolates belonged to the genus Massilia and the closest relatives were Massilia violaceinigra B2 T , Massilia rubra CCM 8692 T , Massilia frigida CCM 8695 T , Massilia antarctica CCM 8941 T , and Massilia aquatica CCM 8693 T . A polyphasic taxonomic study based on lep A genes sequencing, automated ribotyping, MALDI-TOF MS, chemotaxonomy analyses, extensive biotyping, average nucleotide identity, and digital DNA-DNA hybridization calculations based on whole-genome sequences proved that the isolates represent a novel Massilia species for which the names Massilia pseudoviolaceinigra sp. nov. and Massilia scottii sp. nov. are suggested, with the type strains P3689 T (= CCM 9206 T  = LMG 33568 T ) and P5043 T (= CCM 9029 T  = LMG 32502 T ), respectively. These two bioactive metabolite-producing species may play an important role in shaping the composition of fresh-water Antarctic microbiomes due to the inhibition of various Gram-positive bacteria.

The Dallol geothermal area in the northern part of the Danakil Depression (up to 124–155 meter below sea level) is deemed one of the most extreme environments on Earth. The area is notable for being part of the Afar Depression, an incipient seafloor-spreading center located at the triple junction, between Nubian, Somali and Arabian plates, and for hosting environments at the very edge of natural physical-chemical extremities. The northern part of the Danakil Depression is dominated by the Assale salt plain (an accumulation of marine evaporite deposits) and hosts the Dallol volcano. Here, the interaction between the evaporitic deposit and the volcanisms have created the unique Dallol hot springs, which are highly acidic (pH ~ 0) and saline (saturation) with maximum temperatures ranging between 90 and 109 °C. Here we report for the first time evidence of life existing with these hot springs using a combination of morphological and molecular analyses. Ultra-small structures are shown to be entombed within mineral deposits, which are identified as members of the Order Nanohaloarchaea. The results from this study suggest the microorganisms can survive, and potential live, within this extreme environment, which has implications for understanding the limits of habitability on Earth and on (early) Mars.

Freshwater sulfide springs have extreme environmental conditions that only few vertebrate species can tolerate. These species often develop a series of morphological and molecular adaptations to cope with the challenges of life under the toxic and hypoxic conditions of sulfide springs. In this paper, we described a new fish species of the genus Jenynsia, Anablepidae, from a sulfide spring in Northwestern Argentina, the first in the family known from such extreme environment. Jenynsia sulfurica n. sp. is diagnosable by the lack of scales on the pre-pelvic area or the presence of a single row of scales, continuous or not, from the isthmus to the bases of the pelvic fins. Additionally, it presents a series of morphological and molecular characteristics that appear convergent with those seen in other fish species (e.g., Poeciliids) inhabiting sulfide springs. Most notably, J. sulfurica has an enlarged head and postorbital area compared to other fish of the genus and a prognathous lower jaw with a hypertrophied lip, thought to facilitate respiration at the air-water interface. Analyses of cox1 sequence showed that J. sulfurica has two unique mutations resulting in amino acid substitutions convergent to those seen in Poeciliids from sulfide springs and known to provide a physiological mechanism related to living in sulfide environments. A phylogenetic analysis, including molecular and morphological characters, placed J. sulfurica as sister taxa to J. alternimaculata, a species found in nearby, non-sulfide habitats directly connected to the sulfide springs. Thus, it can be inferred that the selection imposed by the presence of H2S has resulted in the divergence between these two species and has potentially served as a barrier to gene flow.

The red microalga Cyanidioschyzon merolae inhabits extreme environments with high temperatures (40–56 °C), high acidity (pH 0.05–4), and high concentrations of heavy metals that are lethal to most forms of life. However, information is scarce on the precise adaptation mechanisms of this extremophile to such hostile conditions. Gaining such knowledge is important for understanding the evolution of microorganisms in the early stages of life on Earth characterized by such extreme environments. Through an analysis of the re-programming of the global transcriptome upon the long-term (up to 15 days) exposure of C. merolae to extremely high concentrations of nickel (1 and 3 mM), the key adaptive metabolic pathways and associated molecular components were identified. Our work shows that the long-term Ni exposure of C. merolae leads to the lagged metabolic switch demonstrated via the transcriptional upregulation of the metabolic pathways critical for cell survival. DNA replication, cell cycle, and protein quality control processes were upregulated, while downregulation occurred with energetically costly processes, including the assembly of the photosynthetic apparatus and lipid biosynthesis. This study paves the way for future multi-omic studies of the molecular mechanisms of abiotic stress adaptation in phototrophs, as well as the future development of rational approaches to the bioremediation of contaminated aquatic environments.

ABSTRACT Photovoltaic panels can be colonized by a highly diverse microbial diversity, despite life-threatening conditions. Although they are distributed worldwide, the microorganisms living on their surfaces have never been profiled in tropical regions using 16S rRNA high-throughput sequencing and PICRUst metagenome prediction of functional content. In this work, we investigated photovoltaic panels from two cities in southeast Brazil, Sorocaba and Itatiba, using these bioinformatics approach. Results showed that, despite significant differences in microbial diversity (p < 0.001), the taxonomic profile was very similar for both photovoltaic panels, dominated mainly by Proteobacteria, Bacteroidota and lower amounts of Cyanobacteria phyla. A predominance of Hymenobacter and Methylobacterium-Methylorubrum was observed at the genus level. We identified a microbial common core composed of Hymenobacter, Deinococcus, Sphingomonas, Methylobacterium-Methylorubrum, Craurococcus-Caldovatus, Massilia, Noviherbaspirillum and 1174-901-12 sharing genera. Predicted metabolisms focused on specific genes associated to radiation and desiccation resistance and pigments, were detected in members of the common core and among the most abundant genera. Our results suggested that taxonomic and functional profiles investigated were consistent with the harsh environment that photovoltaic panels represent. Moreover, the presence of stress genes in the predicted functional content was a preliminary evidence that microbes living there are a possibly source of metabolites with biotechnological interest. Microbial diversity on photovoltaic panel surfaces in a tropical environment.

Background Extremophiles evolved capacities to survive extended exposure to harsh environmental conditions such as complete desiccation (anhydrobiosis) and freezing (cryobiosis). Accumulation of the three-carbon polyhydric alcohol glycerol is commonly observed in anhydrobiotic organisms, although it is considered to preferentially enhance cryobiosis rather than anhydrobiosis. Results Here, using dormant stages of the halophilic extremophile crustacean Artemia franciscana , we show that this role is reversed. We find that A. franciscana and related branchiopods evolved co-opted entomoglyceroporin (Eglp)-like aquaporin-type channels previously only characterized in hexapods. Phylogenomic and site-directed mutagenesis analyses indicate that EglpL orthologs likely evolved during the early Cambrian in the common ancestor of the Pancrustacea. RNAi-mediated knockdown experiments show that the A. franciscana EglpL glycerol transporter is subfunctionally co-regulated with canonical aquaglyceroporins (Glps) to mediate glycerol accumulation in the diapause cysts. Termination of diapause using either desiccation or hydrogen peroxide and further exposure of the cysts to freezing suggest that the acquired glycerol plays a more critical role in anhydrobiosis rather than cryobiosis. Conclusions These findings uncover the essential role of evolutionary divergent aquaporin-type glycerol channels in the accrual of glycerol in an anhydrobiotic organism and reveal a previously overlooked function of this polyol for desiccation tolerance.