Interplay of intracellular and trans-cellular DNA methylation in natural archaeal consortia.

DNA methylation serves a variety of functions across all life domains. In this study, we investigated archaeal methylomics within a tripartite xylanolytic halophilic consortium. This consortium includes Haloferax lucertense SVX82, Halorhabdus sp. SVX81, and an ectosymbiotic Candidatus Nanohalococcus occultus SVXNc, a nano-sized archaeon from the DPANN superphylum. We utilized PacBio SMRT and Illumina cDNA sequencing to analyse samples from consortia of different compositions for methylomics and transcriptomics. Endogenous cTAG methylation, typical of Haloferax, was accompanied in this strain by methylation at four other motifs, including GDGcHC methylation, which is specific to the ectosymbiont. Our analysis of the distribution of methylated and unmethylated motifs suggests that autochthonous cTAG methylation may influence gene regulation. The frequency of GRAGAaG methylation increased in highly expressed genes, while CcTTG and GTCGaGG methylation could be linked to restriction-modification (RM) activity. Generally, the RM activity might have been reduced during the evolution of this archaeon to balance the protection of cells from intruders, the reduction of DNA damage due to self-restriction in stressful environments, and the benefits of DNA exchange under extreme conditions. Our methylomics, transcriptomics and complementary electron cryotomography (cryo-ET) data suggest that the nanohaloarchaeon exports its methyltransferase to methylate the Haloferax genome, unveiling a new aspect of the interaction between the symbiont and its host.

Functional diversity of nanohaloarchaea within xylan-degrading consortia.

Extremely halophilic representatives of the phylum Candidatus Nanohaloarchaeota (members of the DPANN superphyla) are obligately associated with extremely halophilic archaea of the phylum Halobacteriota (according to the GTDB taxonomy). Using culture-independent molecular techniques, their presence in various hypersaline ecosystems around the world has been confirmed over the past decade. However, the vast majority of nanohaloarchaea remain uncultivated, and thus their metabolic capabilities and ecophysiology are currently poorly understood. Using the (meta)genomic, transcriptomic, and DNA methylome platforms, the metabolism and functional prediction of the ecophysiology of two novel extremely halophilic symbiotic nanohaloarchaea (Ca. Nanohalococcus occultus and Ca. Nanohalovita haloferacivicina) stably cultivated in the laboratory as members of a xylose-degrading binary culture with a haloarchaeal host, Haloferax lucentense, was determined. Like all known DPANN superphylum nanoorganisms, these new sugar-fermenting nanohaloarchaea lack many fundamental biosynthetic repertoires, making them exclusively dependent on their respective host for survival. In addition, given the cultivability of the new nanohaloarchaea, we managed to discover many unique features in these new organisms that have never been observed in nano-sized archaea both within the phylum Ca. Nanohaloarchaeota and the entire superphylum DPANN. This includes the analysis of the expression of organism-specific non-coding regulatory (nc)RNAs (with an elucidation of their 2D-secondary structures) as well as profiling of DNA methylation. While some ncRNA molecules have been predicted with high confidence as RNAs of an archaeal signal recognition particle involved in delaying protein translation, others resemble the structure of ribosome-associated ncRNAs, although none belong to any known family. Moreover, the new nanohaloarchaea have very complex cellular defense mechanisms. In addition to the defense mechanism provided by the type II restriction-modification system, consisting of Dcm-like DNA methyltransferase and Mrr restriction endonuclease, Ca. Nanohalococcus encodes an active type I-D CRISPR/Cas system, containing 77 spacers divided into two loci. Despite their diminutive genomes and as part of their host interaction mechanism, the genomes of new nanohaloarchaea do encode giant surface proteins, and one of them (9,409 amino acids long) is the largest protein of any sequenced nanohaloarchaea and the largest protein ever discovered in cultivated archaea.

Nanohaloarchaea as beneficiaries of xylan degradation by haloarchaea.

Climate change, desertification, salinisation of soils and the changing hydrology of the Earth are creating or modifying microbial habitats at all scales including the oceans, saline groundwaters and brine lakes. In environments that are saline or hypersaline, the biodegradation of recalcitrant plant and animal polysaccharides can be inhibited by salt-induced microbial stress and/or by limitation of the metabolic capabilities of halophilic microbes. We recently demonstrated that the chitinolytic haloarchaeon Halomicrobium can serve as the host for an ectosymbiont, nanohaloarchaeon 'Candidatus Nanohalobium constans'. Here, we consider whether nanohaloarchaea can benefit from the haloarchaea-mediated degradation of xylan, a major hemicellulose component of wood. Using samples of natural evaporitic brines and anthropogenic solar salterns, we describe genome-inferred trophic relations in two extremely halophilic xylan-degrading three-member consortia. We succeeded in genome assembly and closure for all members of both xylan-degrading cultures and elucidated the respective food chains within these consortia. We provide evidence that ectosymbiontic nanohaloarchaea is an active ecophysiological component of extremely halophilic xylan-degrading communities (although by proxy) in hypersaline environments. In each consortium, nanohaloarchaea occur as ectosymbionts of Haloferax, which in turn act as scavenger of oligosaccharides produced by xylan-hydrolysing Halorhabdus. We further obtained and characterised the nanohaloarchaea-host associations using microscopy, multi-omics and cultivation approaches. The current study also doubled culturable nanohaloarchaeal symbionts and demonstrated that these enigmatic nano-sized archaea can be readily isolated in binary co-cultures using an appropriate enrichment strategy. We discuss the implications of xylan degradation by halophiles in biotechnology and for the United Nation's Sustainable Development Goals.

Scientific novelty beyond the experiment.

Practical experiments drive important scientific discoveries in biology, but theory-based research studies also contribute novel-sometimes paradigm-changing-findings. Here, we appraise the roles of theory-based approaches focusing on the experiment-dominated wet-biology research areas of microbial growth and survival, cell physiology, host-pathogen interactions, and competitive or symbiotic interactions. Additional examples relate to analyses of genome-sequence data, climate change and planetary health, habitability, and astrobiology. We assess the importance of thought at each step of the research process; the roles of natural philosophy, and inconsistencies in logic and language, as drivers of scientific progress; the value of thought experiments; the use and limitations of artificial intelligence technologies, including their potential for interdisciplinary and transdisciplinary research; and other instances when theory is the most-direct and most-scientifically robust route to scientific novelty including the development of techniques for practical experimentation or fieldwork. We highlight the intrinsic need for human engagement in scientific innovation, an issue pertinent to the ongoing controversy over papers authored using/authored by artificial intelligence (such as the large language model/chatbot ChatGPT). Other issues discussed are the way in which aspects of language can bias thinking towards the spatial rather than the temporal (and how this biased thinking can lead to skewed scientific terminology); receptivity to research that is non-mainstream; and the importance of theory-based science in education and epistemology. Whereas we briefly highlight classic works (those by Oakes Ames, Francis H.C. Crick and James D. Watson, Charles R. Darwin, Albert Einstein, James E. Lovelock, Lynn Margulis, Gilbert Ryle, Erwin R.J.A. Schrödinger, Alan M. Turing, and others), the focus is on microbiology studies that are more-recent, discussing these in the context of the scientific process and the types of scientific novelty that they represent. These include several studies carried out during the 2020 to 2022 lockdowns of the COVID-19 pandemic when access to research laboratories was disallowed (or limited). We interviewed the authors of some of the featured microbiology-related papers and-although we ourselves are involved in laboratory experiments and practical fieldwork-also drew from our own research experiences showing that such studies can not only produce new scientific findings but can also transcend barriers between disciplines, act counter to scientific reductionism, integrate biological data across different timescales and levels of complexity, and circumvent constraints imposed by practical techniques. In relation to urgent research needs, we believe that climate change and other global challenges may require approaches beyond the experiment.

Thermophilic Carboxylesterases from Hydrothermal Vents of the Volcanic Island of Ischia Active on Synthetic and Biobased Polymers and Mycotoxins.

Hydrothermal vents are geographically widespread and host microorganisms with robust enzymes useful in various industrial applications. We examined microbial communities and carboxylesterases of two terrestrial hydrothermal vents of the volcanic island of Ischia (Italy) predominantly composed of Firmicutes, Proteobacteria, and Bacteroidota. High-temperature enrichment cultures with the polyester plastics polyhydroxybutyrate and polylactic acid (PLA) resulted in an increase of Thermus and Geobacillus species and to some extent Fontimonas and Schleiferia species. The screening at 37 to 70°C of metagenomic fosmid libraries from above enrichment cultures identified three hydrolases (IS10, IS11, and IS12), all derived from yet-uncultured Chloroflexota and showing low sequence identity (33 to 56%) to characterized enzymes. Enzymes expressed in Escherichia coli exhibited maximal esterase activity at 70 to 90°C, with IS11 showing the highest thermostability (90% activity after 20-min incubation at 80°C). IS10 and IS12 were highly substrate promiscuous and hydrolyzed all 51 monoester substrates tested. Enzymes were active with PLA, polyethylene terephthalate model substrate, and mycotoxin T-2 (IS12). IS10 and IS12 had a classical α/ß-hydrolase core domain with a serine hydrolase catalytic triad (Ser155, His280, and Asp250) in their hydrophobic active sites. The crystal structure of IS11 resolved at 2.92 Å revealed the presence of a N-terminal ß-lactamase-like domain and C-terminal lipocalin domain. The catalytic cleft of IS11 included catalytic Ser68, Lys71, Tyr160, and Asn162, whereas the lipocalin domain enclosed the catalytic cleft like a lid and contributed to substrate binding. Our study identified novel thermotolerant carboxylesterases with a broad substrate range, including polyesters and mycotoxins, for potential applications in biotechnology. IMPORTANCE High-temperature-active microbial enzymes are important biocatalysts for many industrial applications, including recycling of synthetic and biobased polyesters increasingly used in textiles, fibers, coatings and adhesives. Here, we identified three novel thermotolerant carboxylesterases (IS10, IS11, and IS12) from high-temperature enrichment cultures from Ischia hydrothermal vents and incubated with biobased polymers. The identified metagenomic enzymes originated from uncultured Chloroflexota and showed low sequence similarity to known carboxylesterases. Active sites of IS10 and IS12 had the largest effective volumes among the characterized prokaryotic carboxylesterases and exhibited high substrate promiscuity, including hydrolysis of polyesters and mycotoxin T-2 (IS12). Though less promiscuous than IS10 and IS12, IS11 had a higher thermostability with a high temperature optimum (80 to 90°C) for activity and hydrolyzed polyesters, and its crystal structure revealed an unusual lipocalin domain likely involved in substrate binding. The polyesterase activity of these enzymes makes them attractive candidates for further optimization and potential application in plastics recycling.

Wintertime Simulations Induce Changes in the Structure, Diversity and Function of Antarctic Sea Ice-Associated Microbial Communities.

Antarctic sea-ice is exposed to a wide range of environmental conditions during its annual existence; however, there is very little information describing the change in sea-ice-associated microbial communities (SIMCOs) during the changing seasons. It is well known that during the solar seasons, SIMCOs play an important role in the polar carbon-cycle, by increasing the total photosynthetic primary production of the South Ocean and participating in the remineralization of phosphates and nitrogen. What remains poorly understood is the dynamic of SIMCO populations and their ecological contribution to carbon and nutrient cycling throughout the entire annual life of Antarctic sea-ice, especially in winter. Sea ice at this time of the year is an extreme environment, characterized by complete darkness (which stops photosynthesis), extremely low temperatures in its upper horizons (down to -45 °C) and high salinity (up to 150-250 psu) in its brine inclusions, where SIMCOs thrive. Without a permanent station, wintering expeditions in Antarctica are technically difficult; therefore, in this study, the process of autumn freezing was modelled under laboratory conditions, and the resulting 'young ice' was further incubated in cold and darkness for one month. The ice formation experiment was primarily designed to reproduce two critical conditions: (i) total darkness, causing the photosynthesis to cease, and (ii) the presence of a large amount of algae-derived organic matter. As expected, in the absence of photosynthesis, the activity of aerobic heterotrophs quickly created micro-oxic conditions, which caused the emergence of new players, namely facultative anaerobic and anaerobic microorganisms. Following this finding, we can state that Antarctic pack-ice and its surrounding ambient (under-ice seawater and platelet ice) are likely to be very dynamic and can quickly respond to environmental changes caused by the seasonal fluctuations. Given the size of Antarctic pack-ice, even in complete darkness and cessation of photosynthesis, its ecosystem appears to remain active, continuing to participate in global carbon-and-sulfur cycling under harsh conditions.

Anaerobic carboxydotrophy in sulfur-respiring haloarchaea from hypersaline lakes.

Anaerobic carboxydotrophy is a widespread catabolic trait in bacteria, with two dominant pathways: hydrogenogenic and acetogenic. The marginal mode by direct oxidation to CO2 using an external e-acceptor has only a few examples. Use of sulfidic sediments from two types of hypersaline lakes in anaerobic enrichments with CO as an e-donor and elemental sulfur as an e-acceptor led to isolation of two pure cultures of anaerobic carboxydotrophs belonging to two genera of sulfur-reducing haloarchaea: Halanaeroarchaeum sp. HSR-CO from salt lakes and Halalkaliarchaeum sp. AArc-CO from soda lakes. Anaerobic growth of extremely halophilic archaea with CO was obligatory depended on the presence of elemental sulfur as the electron acceptor and yeast extract as the carbon source. CO served as a direct electron donor and H2 was not generated from CO when cells were incubated with or without sulfur. The genomes of the isolates encode a catalytic Ni,Fe-CODH subunit CooS (distantly related to bacterial homologs) and its Ni-incorporating chaperone CooC (related to methanogenic homologs) within a single genomic locus. Similar loci were also present in a genome of the type species of Halalkaliarchaeum closely related to AArc-CO, and the ability for anaerobic sulfur-dependent carboxydotrophy was confirmed for three different strains of this genus. Moreover, similar proteins are encoded in three of the four genomes of recently described carbohydrate-utilizing sulfur-reducing haloarchaea belonging to the genus Halapricum and in two yet undescribed haloarchaeal species. Overall, this work demonstrated for the first time the potential for anaerobic sulfur-dependent carboxydotrophy in extremely halophilic archaea.

Calm and Frenzy: marine obligate hydrocarbonoclastic bacteria sustain ocean wellness.

According to current estimates, the annual volume of crude oil entering the ocean due to both anthropogenic activities and naturally occurring seepages reaches approximately 8.3 million metric tons. Huge discharges from accidents have caused large-scale environmental disasters with extensive damage to the marine ecosystem. The natural clean-up of petroleum spills in marine environments is carried out primarily by naturally occurring obligate hydrocarbonoclastic bacteria (OHCB). The natural hosts of OHCB include a range of marine primary producers, unicellular photosynthetic eukaryotes and cyanobacteria, which have been documented as both, suppliers of hydrocarbon-like compounds that fuel the 'cryptic' hydrocarbon cycle and as a source of isolation of new OHCB. A very new body of evidence suggests that OHCB are not only the active early stage colonizers of plastics and hence the important component of the ocean's 'plastisphere' but also encode an array of enzymes experimentally proven to act on petrochemical and bio-based polymers.

Cultivation of a vampire: 'Candidatus Absconditicoccus praedator'.

Halorhodospira halophila, one of the most-xerophilic halophiles, inhabits biophysically stressful and energetically expensive, salt-saturated alkaline brines. Here, we report an additional stress factor that is biotic: a diminutive Candidate-Phyla-Radiation bacterium, that we named 'Ca. Absconditicoccus praedator' M39-6, which predates H. halophila M39-5, an obligately photosynthetic, anaerobic purple-sulfur bacterium. We cultivated this association (isolated from the hypersaline alkaline Lake Hotontyn Nur, Mongolia) and characterized their biology. 'Ca. Absconditicoccus praedator' is the first stably cultivated species from the candidate class-level lineage Gracilibacteria (order-level lineage Absconditabacterales). Its closed-and-curated genome lacks genes for the glycolytic, pentose phosphate- and Entner-Doudoroff pathways which would generate energy/reducing equivalents and produce central carbon currencies. Therefore, 'Ca. Absconditicoccus praedator' is dependent on host-derived building blocks for nucleic acid-, protein-, and peptidoglycan synthesis. It shares traits with (the uncultured) 'Ca. Vampirococcus lugosii', which is also of the Gracilibacteria lineage. These are obligate parasitic lifestyle, feeding on photosynthetic anoxygenic Gammaproteobacteria, and absorption of host cytoplasm. Commonalities in their genomic composition and structure suggest that the entire Absconditabacterales lineage consists of predatory species which act to cull the populations of their respective host bacteria. Cultivation of vampire : host associations can shed light on unresolved aspects of their metabolism and ecosystem dynamics at life-limiting extremes.

Halapricum desulfuricans sp. nov., carbohydrate-utilizing, sulfur-respiring haloarchaea from hypersaline lakes.

Nine pure cultures of neutrophilic haloaloarchaea capable of anaerobic growth by carbohydrate-dependent sulfur respiration were isolated from hypersaline lakes in southwestern Siberia and southern Russia. According to phylogenomic analysis the isolates were closely related to each other and formed a new species within the genus Halapricum (family Haloarculaceae). They have three types of catabolism: fermentative, resulting in H2 formation; anaerobic respiration using sulfur compounds as e-acceptors and aerobic respiration. Apart from elemental sulfur, all isolates can also use three different sulfoxides as acceptors and the type strain also grows with thiosulfate, reducing it partially to sulfide and sulfite. All strains utilized sugars and glycerol as the e-donors and C source for anaerobic growth and some can also grow with alpha-glucans, such as starch and dextrins. The major respiratory menaquinones are MK-8:8 and MK-8:7, but 5-19% consists of "thermoplasmata" quinones (MMK-8:8 and MMK-8:7), whose occurrence in haloarchaea is unprecedented. On the basis of their unique physiological properties and results of phylogenomic analysis, the isolates are suggested to be classified into a novel species Halapricum desulfuricans sp. nov. (type strain HSR12-2T = JCM 34032T = UNIQEM U1001T).

High concentrations of dissolved biogenic methane associated with cyanobacterial blooms in East African lake surface water.

The contribution of oxic methane production to greenhouse gas emissions from lakes is globally relevant, yet uncertainties remain about the levels up to which methanogenesis can counterbalance methanotrophy by leading to CH4 oversaturation in productive surface waters. Here, we explored the biogeochemical and microbial community variation patterns in a meromictic soda lake, in the East African Rift Valley (Kenya), showing an extraordinarily high concentration of methane in oxic waters (up to 156 µmol L-1). Vertical profiles of dissolved gases and their isotopic signature indicated a biogenic origin of CH4. A bloom of Oxyphotobacteria co-occurred with abundant hydrogenotrophic and acetoclastic methanogens, mostly found within suspended aggregates promoting the interactions between Bacteria, Cyanobacteria, and Archaea. Moreover, aggregate sedimentation appeared critical in connecting the lake compartments through biomass and organic matter transfer. Our findings provide insights into understanding how hydrogeochemical features of a meromictic soda lake, the origin of carbon sources, and the microbial community profiles, could promote methane oversaturation and production up to exceptionally high rates.

Astrobiology of life on Earth.

Astrobiology is mistakenly regarded by some as a field confined to studies of life beyond Earth. Here, we consider life on Earth through an astrobiological lens. Whereas classical studies of microbiology historically focused on various anthropocentric sub-fields (such as fermented foods or commensals and pathogens of crop plants, livestock and humans), addressing key biological questions via astrobiological approaches can further our understanding of all life on Earth. We highlight potential implications of this approach through the articles in this Environmental Microbiology special issue 'Ecophysiology of Extremophiles'. They report on the microbiology of places/processes including low-temperature environments and chemically diverse saline- and hypersaline habitats; aspects of sulphur metabolism in hypersaline lakes, dysoxic marine waters, and thermal acidic springs; biology of extremophile viruses; the survival of terrestrial extremophiles on the surface of Mars; biological soils crusts and rock-associated microbes of deserts; subsurface and deep biosphere, including a salticle formed within Triassic halite; and interactions of microbes with igneous and sedimentary rocks. These studies, some of which we highlight here, contribute to our understanding of the spatiotemporal reach of Earth'sfunctional biosphere, and the tenacity of terrestrial life. Their findings will help set the stage for future work focused on the constraints for life, and how organisms adapt and evolve to circumvent these constraints.

Corrigendum: High Representation of Archaea Across All Depths in Oxic and Low-pH Sediment Layers Underlying an Acidic Stream.

[This corrects the article DOI: 10.3389/fmicb.2020.576520.].

Carbohydrate-dependent sulfur respiration in halo(alkali)philic archaea.

Archaea are environmentally ubiquitous on Earth, and their extremophilic and metabolically versatile phenotypes make them useful as model systems for astrobiology. Here, we reveal a new functional group of halo(natrono)archaea able to utilize alpha-d-glucans (amylopectin, amylose and glycogen), sugars, and glycerol as electron donors and carbon sources for sulfur respiration. They are facultative anaerobes enriched from hypersaline sediments with either amylopectin, glucose or glycerol as electron/carbon sources and elemental sulfur as the terminal electron acceptor. They include 10 strains of neutrophilic haloarchaea from circum pH-neutral lakes and one natronoarchaeon from soda-lake sediments. The neutrophilic isolates can grow by fermentation, although addition of S0 or dimethyl sulfoxide increased growth rate and biomass yield (with a concomitant decrease in H2 ). Natronoarchaeal isolate AArc-S grew only by respiration, either anaerobically with S0 or thiosulfate as the terminal electron acceptor, or aerobically. Through genome analysis of five representative strains, we detected the full set of enzymes required for the observed catabolic and respiratory phenotypes. These findings provide evidence that sulfur-respiring haloarchaea partake in biogeochemical sulfur cycling, linked to terminal anaerobic carbon mineralization in hypersaline anoxic habitats. We discuss the implications for life detection in analogue environments such as the polar subglacial brine-lakes of Mars.

High Representation of Archaea Across All Depths in Oxic and Low-pH Sediment Layers Underlying an Acidic Stream.

Parys Mountain or Mynydd Parys (Isle of Anglesey, United Kingdom) is a mine-impacted environment, which accommodates a variety of acidophilic organisms. Our previous research of water and sediments from one of the surface acidic streams showed a high proportion of archaea in the total microbial community. To understand the spatial distribution of archaea, we sampled cores (0-20 cm) of sediment and conducted chemical analyses and taxonomic profiling of microbiomes using 16S rRNA gene amplicon sequencing in different core layers. The taxonomic affiliation of sequencing reads indicated that archaea represented between 6.2 and 54% of the microbial community at all sediment depths. Majority of archaea were associated with the order Thermoplasmatales, with the most abundant group of sequences being clustered closely with the phylotype B_DKE, followed by "E-plasma," "A-plasma," other yet uncultured Thermoplasmatales with Ferroplasma and Cuniculiplasma spp. represented in minor proportions. Thermoplasmatales were found at all depths and in the whole range of chemical conditions with their abundance correlating with sediment Fe, As, Cr, and Mn contents. The bacterial microbiome component was largely composed in all layers of sediment by members of the phyla Proteobacteria, Actinobacteria, Nitrospirae, Firmicutes, uncultured Chloroflexi (AD3 group), and Acidobacteria. This study has revealed a high abundance of Thermoplasmatales in acid mine drainage-affected sediment layers and pointed at these organisms being the main contributors to carbon, and probably to iron and sulfur cycles in this ecosystem.

The Structure of the Lipid A of Gram-Negative Cold-Adapted Bacteria Isolated from Antarctic Environments.

Gram-negative Antarctic bacteria adopt survival strategies to live and proliferate in an extremely cold environment. Unusual chemical modifications of the lipopolysaccharide (LPS) and the main component of their outer membrane are among the tricks adopted to allow the maintenance of an optimum membrane fluidity even at particularly low temperatures. In particular, the LPS' glycolipid moiety, the lipid A, typically undergoes several structural modifications comprising desaturation of the acyl chains, reduction in their length and increase in their branching. The investigation of the structure of the lipid A from cold-adapted bacteria is, therefore, crucial to understand the mechanisms underlying the cold adaptation phenomenon. Here we describe the structural elucidation of the highly heterogenous lipid A from three psychrophiles isolated from Terra Nova Bay, Antarctica. All the lipid A structures have been determined by merging data that was attained from the compositional analysis with information from a matrix-assisted laser desorption ionization (MALDI) time of flight (TOF) mass spectrometry (MS) and MS2 investigation. As lipid A is also involved in a structure-dependent elicitation of innate immune response in mammals, the structural characterization of lipid A from such extremophile bacteria is also of great interest from the perspective of drug synthesis and development inspired by natural sources.

Hydrocarbon-Degrading Bacteria Alcanivorax and Marinobacter Associated With Microalgae Pavlova lutheri and Nannochloropsis oculata.

Marine hydrocarbon-degrading bacteria play an important role in natural petroleum biodegradation processes and were initially associated with man-made oil spills or natural seeps. There is no full clarity though on what, in the absence of petroleum, their natural niches are. Few studies pointed at some marine microalgae that produce oleophilic compounds (alkanes, long-chain fatty acids, and alcohols) as potential natural hosts of these bacteria. We established Dansk crude oil-based enrichment cultures with photobioreactor-grown marine microalgae cultures Pavlova lutheri and Nannochloropsis oculata and analyzed the microbial succession using cultivation and SSU (16S) rRNA amplicon sequencing. We found that petroleum enforced a strong selection for members of Alpha- and Gamma-proteobacteria in both enrichment cultures with the prevalence of Alcanivorax and Marinobacter spp., well-known hydrocarbonoclastic bacteria. In total, 48 non-redundant bacterial strains were isolated and identified to represent genera Alcanivorax, Marinobacter, Thalassospira, Hyphomonas, Halomonas, Marinovum, Roseovarius, and Oleibacter, which were abundant in sequencing reads in both crude oil enrichments. Our assessment of public databases demonstrated some overlaps of geographical sites of isolation of Nannochloropsis and Pavlova with places of molecular detection and isolation of Alcanivorax and Marinobacter spp. Our study suggests that these globally important hydrocarbon-degrading bacteria are associated with P. lutheri and N. oculata.

Symbiosis between nanohaloarchaeon and haloarchaeon is based on utilization of different polysaccharides.

Nano-sized archaeota, with their small genomes and limited metabolic capabilities, are known to associate with other microbes, thereby compensating for their own auxotrophies. These diminutive and yet ubiquitous organisms thrive in hypersaline habitats that they share with haloarchaea. Here, we reveal the genetic and physiological nature of a nanohaloarchaeon-haloarchaeon association, with both microbes obtained from a solar saltern and reproducibly cultivated together in vitro. The nanohaloarchaeon Candidatus Nanohalobium constans LC1Nh is an aerotolerant, sugar-fermenting anaerobe, lacking key anabolic machinery and respiratory complexes. The nanohaloarchaeon cells are found physically connected to the chitinolytic haloarchaeon Halomicrobium sp. LC1Hm. Our experiments revealed that this haloarchaeon can hydrolyze chitin outside the cell (to produce the monosaccharide N-acetylglucosamine), using this beta-glucan to obtain carbon and energy for growth. However, LC1Hm could not metabolize either glycogen or starch (both alpha-glucans) or other polysaccharides tested. Remarkably, the nanohaloarchaeon's ability to hydrolyze glycogen and starch to glucose enabled growth of Halomicrobium sp. LC1Hm in the absence of a chitin. These findings indicated that the nanohaloarchaeon-haloarchaeon association is both mutualistic and symbiotic; in this case, each microbe relies on its partner's ability to degrade different polysaccharides. This suggests, in turn, that other nano-sized archaeota may also be beneficial for their hosts. Given that availability of carbon substrates can vary both spatially and temporarily, the susceptibility of Halomicrobium to colonization by Ca Nanohalobium can be interpreted as a strategy to maximize the long-term fitness of the host.

Bioprospecting Reveals Class III ω-Transaminases Converting Bulky Ketones and Environmentally Relevant Polyamines.

Amination of bulky ketones, particularly in (R) configuration, is an attractive chemical conversion; however, known ω-transaminases (ω-TAs) show insufficient levels of performance. By applying two screening methods, we discovered 10 amine transaminases from the class III ω-TA family that were 38% to 76% identical to homologues. We present examples of such enzymes preferring bulky ketones over keto acids and aldehydes with stringent (S) selectivity. We also report representatives from the class III ω-TAs capable of converting (R) and (S) amines and bulky ketones and one that can convert amines with longer alkyl substituents. The preference for bulky ketones was associated with the presence of a hairpin region proximal to the conserved Arg414 and residues conforming and close to it. The outward orientation of Arg414 additionally favored the conversion of (R) amines. This configuration was also found to favor the utilization of putrescine as an amine donor, so that class III ω-TAs with Arg414 in outward orientation may participate in vivo in the catabolism of putrescine. The positioning of the conserved Ser231 also contributes to the preference for amines with longer alkyl substituents. Optimal temperatures for activity ranged from 45 to 65°C, and a few enzymes retained ≥50% of their activity in water-soluble solvents (up to 50% [vol/vol]). Hence, our results will pave the way to design, in the future, new class III ω-TAs converting bulky ketones and (R) amines for the production of high-value products and to screen for those converting putrescine.IMPORTANCE Amine transaminases of the class III ω-TAs are key enzymes for modification of chemical building blocks, but finding those capable of converting bulky ketones and (R) amines is still challenging. Here, by an extensive analysis of the substrate spectra of 10 class III ω-TAs, we identified a number of residues playing a role in determining the access and positioning of bulky ketones, bulky amines, and (R)- and (S) amines, as well as of environmentally relevant polyamines, particularly putrescine. The results presented can significantly expand future opportunities for designing (R)-specific class III ω-TAs to convert valuable bulky ketones and amines, as well as for deepening the knowledge into the polyamine catabolic pathways.

Decoding the ocean's microbiological secrets for marine enzyme biodiscovery.

A global census of marine microbial life has been underway over the past several decades. During this period, there have been scientific breakthroughs in estimating microbial diversity and understanding microbial functioning and ecology. It is estimated that the ocean, covering 71% of the earth's surface with its estimated volume of about 2 × 1018 m3 and an average depth of 3800 m, hosts the largest population of microbes on Earth. More than 2 million eukaryotic and prokaryotic species are thought to thrive both in the ocean and on its surface. Prokaryotic cell abundances can reach densities of up to 1012 cells per millilitre, exceeding eukaryotic densities of around 106 cells per millilitre of seawater. Besides their large numbers and abundance, marine microbial assemblages and their organic catalysts (enzymes) have a largely underestimated value for their use in the development of industrial products and processes. In this perspective article, we identified critical gaps in knowledge and technology to fast-track this development. We provided a general overview of the presumptive microbial assemblages in oceans, and an estimation of what is known and the enzymes that have been currently retrieved. We also discussed recent advances made in this area by the collaborative European Horizon 2020 project 'INMARE'.