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Rapid fluctuation of environmental conditions can impose severe stress upon living organisms. Surviving such episodes of stress requires a rapid acclimation response, e.g., by transcriptional and post-transcriptional mechanisms. Persistent change of the environmental context, however, requires longer-term adaptation at the genetic level. Fast-growing unicellular aquatic eukaryotes enable analysis of adaptive responses at the genetic level in a laboratory setting. In this study, we applied continuous cold stress (28°C) to the thermoacidophile red alga G. sulphuraria, which is 14°C below its optimal growth temperature of 42°C. Cold stress was applied for more than 100 generations to identify components that are critical for conferring thermal adaptation. After cold exposure for more than 100 generations, the cold-adapted samples grew ∼30% faster than the starting population. Whole-genome sequencing revealed 757 variants located on 429 genes (6.1% of the transcriptome) encoding molecular functions involved in cell cycle regulation, gene regulation, signaling, morphogenesis, microtubule nucleation, and transmembrane transport. CpG islands located in the intergenic region accumulated a significant number of variants, which is likely a sign of epigenetic remodeling. We present 20 candidate genes and three putative cis-regulatory elements with various functions most affected by temperature. Our work shows that natural selection toward temperature tolerance is a complex systems biology problem that involves gradual reprogramming of an intricate gene network and deeply nested regulators.

Verrucomicrobial methanotrophs are a group of aerobic bacteria isolated from volcanic environments. They are acidophiles, characterized by the presence of a particulate methane monooxygenase (pMMO) and a XoxF-type methanol dehydrogenase (MDH). Metagenomic analysis of DNA extracted from the soil of Favara Grande, a geothermal area on Pantelleria Island, Italy, revealed the presence of two verrucomicrobial Metagenome Assembled Genomes (MAGs). One of these MAGs did not phylogenetically classify within any existing genus. After extensive analysis of the MAG, we propose the name of " Candidatus Methylacidithermus pantelleriae" PQ17 gen. nov. sp. nov. The MAG consisted of 2,466,655 bp, 71 contigs and 3,127 predicted coding sequences. Completeness was found at 98.6% and contamination at 1.3%. Genes encoding the pMMO and XoxF-MDH were identified. Inorganic carbon fixation might use the Calvin-Benson-Bassham cycle since all genes were identified. The serine and ribulose monophosphate pathways were incomplete. The detoxification of formaldehyde could follow the tetrahydrofolate pathway. Furthermore, " Ca. Methylacidithermus pantelleriae" might be capable of nitric oxide reduction but genes for dissimilatory nitrate reduction and nitrogen fixation were not identified. Unlike other verrucomicrobial methanotrophs, genes encoding for enzymes involved in hydrogen oxidation could not be found. In conclusion, the discovery of this new MAG expands the diversity and metabolism of verrucomicrobial methanotrophs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Picone, Blom, Hogendoorn, Frank, van Alen, Pol, Gagliano, Jetten, D’Alessandro, Quatrini and Op den Camp.)

The Favara Grande is a geothermal area located on Pantelleria Island, Italy. The area is characterized high temperatures in the top layer of the soil (60°C), low pH (3-5) and hydrothermal gas emissions mainly composed of carbon dioxide (CO 2 ), methane (CH 4 ), and hydrogen (H 2 ). These geothermal features may provide a suitable niche for the growth of chemolithotrophic thermoacidophiles, including the lanthanide-dependent methanotrophs of the phylum Verrucomicrobia. In this study, we started enrichment cultures inoculated with soil of the Favara Grande at 50 and 60°C with CH 4 as energy source and medium containing sufficient lanthanides at pH 3 and 5. From these cultures, a verrucomicrobial methanotroph could be isolated via serial dilution and floating filters techniques. The genome of strain AP8 was sequenced and based on phylogenetic analysis we propose to name this new species Methylacidimicrobium thermophilum AP8. The transcriptome data at μ max (0.051 ± 0.001 h -1 , doubling time ~14 h) of the new strain showed a high expression of the pmoCAB 2 operon encoding the membrane-bound methane monooxygenase and of the gene xoxF 1, encoding the lanthanide-dependent methanol dehydrogenase. A second pmoCAB operon and xoxF 2 gene were not expressed. The physiology of strain AP8 was further investigated and revealed an optimal growth in a pH range of 3-5 at 50°C, representing the first thermophilic strain of the genus Methylacidimicrobium . Moreover, strain AP8 had a K S(app) for methane of 8 ± 1 μM. Beside methane, a type 1b [NiFe] hydrogenase enabled hydrogen oxidation at oxygen concentrations up to 1%. Taken together, our results expand the knowledge on the characteristics and adaptations of verrucomicrobial methanotrophs in hydrothermal environments and add a new thermophilic strain to the genus Methylacidimicrobium ., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Picone, Blom, Wallenius, Hogendoorn, Mesman, Cremers, Gagliano, D’Alessandro, Quatrini, Jetten, Pol and Op den Camp.)

Volcanic and geothermal areas are hot and often acidic environments that emit geothermal gasses, including H 2 , CO and CO 2 . Geothermal gasses mix with air, creating conditions where thermoacidophilic aerobic H 2 - and CO-oxidizing microorganisms could thrive. Here, we describe the isolation of two Kyrpidia spormannii strains, which can grow autotrophically by oxidizing H 2 and CO with oxygen. These strains, FAVT5 and COOX1, were isolated from the geothermal soils of the Favara Grande on Pantelleria Island, Italy. Extended physiology studies were performed with K. spormannii FAVT5, and showed that this strain grows optimally at 55°C and pH 5.0. The highest growth rate is obtained using H 2 as energy source (μ max 0.19 ± 0.02 h -1 , doubling time 3.6 h). K. spormannii FAVT5 can additionally grow on a variety of organic substrates, including some alcohols, volatile fatty acids and amino acids. The genome of each strain encodes for two O 2 -tolerant hydrogenases belonging to [NiFe] group 2a hydrogenases and transcriptome studies using K. spormannii FAVT5 showed that both hydrogenases are expressed under H 2 limiting conditions. So far no Firmicutes except K. spormannii FAVT5 have been reported to exhibit a high affinity for H 2 , with a K s of 327 ± 24 nM. The genomes of each strain encode for one putative CO dehydrogenase, belonging to Form II aerobic CO dehydrogenases. The genomic potential and physiological properties of these Kyrpidia strains seem to be quite well adapted to thrive in the harsh environmental volcanic conditions., (Copyright © 2020 Hogendoorn, Pol, Picone, Cremers, van Alen, Gagliano, Jetten, D’Alessandro, Quatrini and Op den Camp.)