Identification of an Amylomaltase from the Halophilic Archaeon Haloquadratum walsbyi by Functional Metagenomics: Structural and Functional Insights

Amylomaltases are prokaryotic 4-α-glucanotransferases of the GH77 family. Thanks to the ability to modify starch, they constitute a group of enzymes of great interest for biotechnological applications. In this work we report the identification, by means of a functional metagenomics screening of the crystallization waters of the saltern of Margherita di Savoia (Italy), of an amylomaltase gene from the halophilic archaeon Haloquadratum walsbyi, and its expression in Escherichia coli cells. Sequence analysis indicated that the gene has specific insertions yet unknown in homologous genes in prokaryotes, and present only in amylomaltase genes identified in the genomes of other H. walsbyi strains. The gene is not part of any operon involved in the metabolism of maltooligosaccharides or glycogen, as it has been found in bacteria, making it impossible currently to assign a precise role to the encoded enzyme. Sequence analysis of the H. walsbyi amylomaltase and 3D modelling showed a common structure with homologous enzymes characterized in mesophilic and thermophilic bacteria. The recombinant H. walsbyi enzyme showed starch transglycosylation activity over a wide range of NaCl concentrations, with maltotriose as the best acceptor substrate compared to other maltooligosaccharides. This is the first study of an amylomaltase from a halophilic microorganism.

Evaluating the potential for Haloarchaea to serve as ice nucleating particles

Aerosols play a crucial role in cloud formation. Biologically derived materials from bacteria, fungi, pollen, lichen, viruses, algae, and diatoms can serve as ice nucleating particles (INPs), some of which initiate glaciation in clouds at relatively warm freezing temperatures. However, determining the magnitude of the interactions between clouds and biologically derived INPs remains a significant challenge due to the diversity and complexity of bioaerosols and limited observations of such aerosols facilitating cloud ice formation. Additionally, microorganisms from the domain Archaea have, to date, not been evaluated as INPs. Here, we present the first results reporting the ice nucleation activity of four species in the class Haloarchaea. Intact cells of Halococcus morrhuae and Haloferax sulfurifontis demonstrated the ability to induce immersion freezing at temperatures up to −18 ∘C, while lysed cells of Haloquadratum walsbyi and Natronomonas pharaonis were unable to serve as immersion INPs. Exposure to heat and peroxide digestion indicated that the INPs of intact cells were driven by organic (H. morrhuae and H. sulfurifontis) and possibly also heat labile materials (H. sulfurifontis only). While halophiles are prominent in hypersaline environments such as the Great Salt Lake and the Dead Sea, other members of the Archaea, such as methanogens and thermophiles, are prevalent in anoxic systems in seawater, sea ice, marine sediments, glacial ice, permafrost, and other cold niches. Archaeal extremophiles are both diverse and highly abundant. Thus, it is important to assess their ability to serve as INPs as it may lead to an improved understanding of biological impacts on clouds.

Evaluating the potential for Archaea to serve as ice nucleating particles

Aerosols play a crucial role in cloud formation. Biologically-derived materials from microorganisms such as plant bacteria, fungi, pollen, and various vegetative detritus can serve as ice nucleating particles (INPs), some of which initiate glaciation in clouds at relatively warm freezing temperatures. However, determining the magnitude of the interactions between clouds and biologically-derived INPs remains a significant challenge due to the diversity and complexity of bioaerosols, and limited observations of such aerosols to facilitate cloud ice formation. Additionally, microorganisms from the domain Archaea have to date not been evaluated as INPs. Here, we present the first results reporting the ice nucleation activity of a subset of archaeal cells from Haloarchaea. Intact cells of Halococcus morrhuae and Haloferax sulfurifontis demonstrated the ability to induce freezing as warm as −18 ˚C, while lysed cells of Haloquadratum walsbyi and Natronomonas pharaonis were unable to serve as warm temperature INPs. Exposure to heat and peroxide digestion indicated that the INPs of intact cells were driven by organic (H. morrhuae and H. sulfurifontis) and possibly also heat-labile materials (H. sulfurifontis only). While halophiles are prominent in hypersaline environments such as the Great Salt Lake and the Dead Sea, other members of the Archaea, such as methanogens and thermophiles, are prevalent in anoxic systems in seawater, sea ice, marine sediments, glacial ice, permafrost, and other cold niches. Archaeal extremophiles are both diverse and highly abundant. Thus, assessing their ability to become airborne, and their abilities to impact cloud formation, is necessary to improve understanding of biological impacts on clouds.