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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.

Hydrothermal vents have a widespread geographical distribution and are of high interest for investigating microbial communities and robust enzymes for 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 (Geobacillus and Brevibacillus spp.), Proteobacteria and Bacteroidota. High-temperature enrichment cultures with the polyester plastics polyhydroxybutyrate (PHB) and polylactic acid (PLA) resulted in an increase of Thermus and Geobacillus spp., and to some extent, Fontimonas and Schleiferia spp. The screening at 37-70ºC of metagenomic fosmid library from above enrichment cultures resulted in identification and successful production in Escherichia coli of three hydrolases (IS10, IS11 and IS12), all derived from yet uncultured Chloroflexota and showing low sequence identity (33-56%) to characterized enzymes. Enzymes exhibited maximal esterase activity at temperatures 70-90ºC, with IS11 showing the highest thermostability (90% activity after 20 min incubation at 80ºC). IS10 and IS12 were highly substrate-promiscuous and hydrolysed all 51 monoester substrates tested. Enzymes were active with polyesters (PLA and polyethylene terephthalate model substrate, 3PET) and mycotoxin T-2 (IS12). IS10 and IS12 had a classical α/β hydrolase core domain with a serine hydrolase catalytic triad (Ser155, His280, and Asp250) in the hydrophobic active sites. The crystal structure of IS11 resolved at 2.92 Å revealed the presence of the N-terminal β-lactamase-like domain and C-terminal lipocalin domain. The catalytic cleft of IS11 includes catalytic residues Ser68, Lys71, Tyr160, and Asn162, whereas the lipocalin domain encloses the catalytic cleft like a lid contributing to substrate binding. Thus, this study has identified novel thermotolerant carboxylesterases with a broad substrate range including polyesters and mycotoxins for potential applications in biotechnology.IMPORTANCEHigh-temperature-active microbial enzymes are important biocatalysts for many industrial applications including recycling of synthetic and biobased polyesters increasingly used in textiles, fibres, coatings and adhesives. Here, we have discovered three novel thermotolerant carboxylesterases (IS10, IS11 and IS12) from high-temperature enrichment cultures from the Ischia hydrothermal vents 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 compared to IS10 and IS12, IS11 had a higher thermostability with high temperature optimum (80-90 ºC) for activity, hydrolysed polyesters, and its crystal structure revealed an unusual lipocalin domain likely involved in substrate binding. The polyesterase activity in these enzymes makes them attractive candidates for further optimisation and potential application in plastics recycling.

© 2020 the Author(s)., 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., This study was partially supported by grants from the Italian Ministry of University and Research under the RITMARE Flagship Project (2012–2016) and the INMARE Project (Contract H2020-BG-2014-2634486), funded by the European Union’s Horizon 2020 Research Program. P.N.G. and O.V.G. acknowledge support from the Centre for Environmental Biotechnology Project, partly funded by the European Regional Development Fund via the Welsh Assembly Government. D.Y.S. was supported by SYAM-Gravitation Program of the Dutch Ministry of Education and Science Grant 24002002 and Russian Foundation for Basic Research Grant 19-04-00401). S.V.T. acknowledges Kurchatov Centre for Genome Research, and his work was supported by Ministry of Science and Higher Education of Russian Federation Grant 075-15-2019-1659.

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.

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. eng

International audience; Evolution of virulence traits from adaptation to environmental niches other than the host is probably a common feature of marine microbial pathogens, whose knowledge might be crucial to understand their emergence and pathogenetic potential. Here we report genome sequence analysis of a novel marine bacterial species, Vibrio bathopelagicus sp. nov., isolated from warm bathypelagic waters (3309m depth) of the Mediterranean Sea. Interestingly, V. bathopelagicus sp. nov. is closely related to coastal Vibrio strains pathogenic to marine bivalves. V. bathopelagicus sp. nov. genome encodes genes involved in environmental adaptations to the deep-sea but also in virulence, such as the R5.7 element, MARTX toxin cluster, Type VI secretion system and zinc-metalloprotease, previously associated with Vibrio infection in farmed oysters. The results of functional in vitro assays on immunocytes (hemocytes) of the Mediterranean mussel Mytilus galloprovincialis and the Pacific oyster Crassostrea gigas, and of the early larval development assay in Mytilus support strong toxicity of V. bathopelagicus sp. nov. towards bivalves. V. bathopelagicus sp. nov., isolated from a remote Mediterranean bathypelagic site, is an example of a planktonic marine bacterium with genotypic and phenotypic traits associated with animal pathogenicity, that might have played an evolutionary role in the origin of coastal marine pathogens.

Marine sediments represent an important sink of harmful petroleum hydrocarbons after an accidental oil spill. Electrobioremediation techniques, which combine electrokinetic transport and biodegradation processes, represent an emerging technological platform for a sustainable remediation of contaminated sediments. Here, we describe the results of a long-term mesocosm-scale electrobioremediation experi- ment for the treatment of marine sediments contaminated by crude oil. A dimensionally stable anode and a stainless-steel mesh cathode were employed to drive seawater electrolysis at a fixed current density of 11 A/m2. This approach allowed establishing conditions conducive to contaminants biodeg- radation, as confirmed by the enrichment of Alcanivorax borkumensis cells harboring the alkB-gene and other aerobic hydrocarbonoclastic bacteria. Oil chemistry analyses indicated that aromatic hydrocarbons were primarily removed from the sediment via electroosmosis and low molecular weight alkanes (nC6 to nC10) via biodegradation., This work was financially supported by the European Commission within the Seventh Framework Programme under Grant Agreement No. 312139 (“Kill-Spill: Integrated biotechnological solutions for combating marine oil spills”).

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. Funding was provided by the Biotechnology and Biological Sciences Research Council (BBSRC, United Kingdom) project BBF003471. Peerreview

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.

International audience; A novel anaerobic methylotrophic halophilic methanogen strain SLHTYRO T was isolated from a deep hypersaline anoxic basin called "Tyro" located in the Eastern Mediterranean Sea. Cells of SLHTYRO T were motile cocci. The strain SLHTYRO T grew between 12 and 37 °C (optimum 30 °C), at pH between 6.5 and 8.2 (optimum pH 7.5) and salinity from 45 to 240 g L −1 NaCl (optimum 135 g L −1). Strain SLHTYRO T was methylotrophic methanogen able to use methylated compounds (trimethylamine, dimethylamine, monomethylamine and methanol). Strain SLHTYRO T was able to grow at in situ hydrostatic pressure and temperature conditions (35 MPa, 14 °C). Phylogenetic analysis based on 16S rRNA gene and mcrA gene sequences indicated that strain SLHTYRO T was affiliated to genus Methanohalophilus within the order Methanosarcinales. It shared >99.16% of the 16S rRNA gene sequence similarity with strains of other Methanohalophilus species. Based on ANIb, AAI and dDDH measurements, and the physiological properties of the novel isolate, we propose that strain SLHTYRO T should be classified as a representative of a novel species, for which the name Methanohalophilus profundi sp. nov. is proposed; the type strain is SLHTYRO T (=DSM 108854 = JCM 32768 = UBOCC-M-3308).

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.

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&rsquo, 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.

Marine bacterium Oleiphilus messinensis ME102 (DSM 13489T) isolated from the sediments of the harbor of Messina (Italy) is a member of the order Oceanospirillales, class Gammaproteobacteria, representing the physiological group of marine obligate hydrocarbonoclastic bacteria (OHCB) alongside the members of the genera Alcanivorax, Oleispira, Thalassolituus, Cycloclasticus and Neptunomonas. These organisms play a crucial role in the natural environmental cleanup in marine systems. Despite having the largest genome (6.379.281 bp) among OHCB, O. messinensis exhibits a very narrow substrate profile. The alkane metabolism is pre-determined by three loci encoding for two P450 family monooxygenases, one of which formed a cassette with ferredoxin and alcohol dehydrogenase encoding genes and alkane monoxygenase (AlkB) gene clustered with two genes for rubredoxins and NAD+-dependent rubredoxin reductase. Its genome contains the largest numbers of genomic islands (15) and mobile genetic elements (140), as compared with more streamlined genomes of its OHCB counterparts. Among hydrocarbon-degrading Oceanospirillales, O. messinensis encodes the largest array of proteins involved in the signal transduction for sensing and responding to the environmental stimuli (345 vs 170 in Oleispira antarctica, the bacterium with the second highest number). This must be an important trait to adapt to the conditions in marine sediments with a high physico-chemical patchiness and heterogeneity as compared to those in the water column.

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 andformed 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 U1001 T).

An aerobic hyperthermophilic CO-oxidizing archaeon, Sulfolobus sp. strain ETSY, was isolated and characterized. Presently, it is the only known representative of both hyperthermophiles and Archaea that is capable of aerobic oxidation of CO, a gas of global importance for atmospheric chemistry and of local importance as one of the substrates for the microbial communities of hydrothermal vents. In the genome of Sulfolobus sp. ETSY we found genetic determinants of aerobic CO oxidation: a coxFMSLDE gene cluster and two separately located coxG genes. We also found such gene clusters in the genomes of certain strains of Sulfolobus islandicus and Sulfolobus solfataricus. On the phylogenetic tree of large subunits of aerobic CO-dehydrogenases (CoxLs), these proteins of Sulfolobus representatives formed a compact cluster within one of the branches formed by bacterial form I CoxLs. Thus we argue that the ability to oxidize CO aerobically was acquired by Sulfolobus ancestor from Bacteria relatively late in the evolution, presumably after the formation of the atmosphere with a high oxygen content.

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Hydrated, magnesium-rich minerals and subglacial brines exist on the martian surface, so the habitability of high-Mg2+ environments on Earth has extraterrestrial (as well as terrestrial) implications. Here, we report the discovery of a MgCl2-dominated (4.72 M) brine lake on the floor of the Mediterranean Ridge that underlies a 3500-m water column, and name it Lake Hephaestus. Stable isotope analyses indicated that the Hephaestus brine is derived from interactions between ancient bishofite-enriched evaporites and subsurface fluids. Analyses of sediment pore waters indicated that the Hephaestus depression had contained the MgCl2 brine for a remarkably short period; only 700 years. Lake Hephaestus is, therefore, the youngest among currently known submarine athalassohaline brine lakes on Earth. Due to its biologically hostile properties (low water-activity and extreme chaotropicity), the Hephaestus brine is devoid of life. By contrast, the seawater-Hephaestus brine interface has been shown to act as refuge for extremely halophilic and magnesium-adapted stratified communities of microbes, even at MgCl2 concentrations that approach the water-activity limit for life (0.653).

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’., The success of using marine resources for the development of industrial products and processes depends on the manner they are screened, produced and tested on a larger scale.

Predation by grazing protists in aquatic habitats can influence prokaryotic community structure and provides a source of new, labile organic matter. Due to methodological difficulties associated with studies of deep-sea (below photic zone) microbiota, trophic interactions between eukaryotes and prokaryotes in mesopelagic and bathypelagic realms are largely obscured. Further complicating matters, examinations of trophic interactions using water samples that have been exposed to upwards of hundreds of atmospheres of pressure change prior to initiating experiments can potentially introduce significant artifacts. Here we present results of the first study of protistan grazing in water layers ranging from the euphotic zone to the bathypelagic, utilizing the Microbial Sampler-Submersible Incubation Device (MS-SID) that makes possible in situ studies of microbial activities. Protistan grazing in the mesopelagic and bathypelagic realm of the East Mediterranean Sea was quantified using fluorescently labeled prokaryotes (FLP) prepared from the naturally-occurring prokaryotic assemblages. These studies reveal daily prokaryotic removal due to grazing ranging from 31.3±5.9% at 40 m depth to 0.5±0.3% at 950 m. At 3540 m depth, where a chemocline habitat exists with abundant and active prokaryotes above Urania basin, the daily consumption of prokaryotes by protists was 19.9±6.6% of the in situ abundance.

Obtaining an accurate picture of microbial processes occurring in situ is essential for our understanding of marine biogeochemical cycles of global importance. Water samples are typically collected at depth and returned to the sea surface for processing and downstream experiments. Metatranscriptome analysis is one powerful approach for investigating metabolic activities of microorganisms in their habitat and which can be informative for determining responses of microbiota to disturbances such as the Deepwater Horizon oil spill. For studies of microbial processes occurring in the deep sea, however, sample handling, pressure, and other changes during sample recovery can subject microorganisms to physiological changes that alter the expression profile of labile messenger RNA. Here we report a comparison of gene expression profiles for whole microbial communities in a bathypelagic water column sample collected in the Eastern Mediterranean Sea using Niskin bottle sample collection and a new water column sampler for studies of marine microbial ecology, the Microbial Sampler – In Situ Incubation Device (MS-SID). For some taxa, gene expression profiles from samples collected and preserved in situ were significantly different from potentially more stressful Niskin sampling and preservation on deck. Some categories of transcribed genes also appear to be affected by sample handling more than others. This suggests that for future studies of marine microbial ecology, particularly targeting deep sea samples, an in situ sample collection and preservation approach should be considered.

Marine prokaryotes have evolved a broad repertoire of defence systems to protect their genomes from lateral gene transfer including innate or acquired immune systems and infection-induced programmed cell suicide and dormancy. Here we report on the analysis of multiple defence systems present in the genome of the strain Cycloclasticus sp. 78-ME isolated from petroleum deposits of the tanker 'Amoco Milford Haven'. Cycloclasticus are ubiquitous bacteria globally important in polyaromatic hydrocarbons degradation in marine environments. Two 'defence islands' were identified in 78-ME genome: the first harbouring CRISPR-Cas with toxin-antitoxin system, while the second was composed by an array of genes for toxin-antitoxin and restriction-modification proteins. Among all identified spacers of CRISPR-Cas system only seven spacers match sequences of phages and plasmids. Furthermore, a conjugative plasmid p7ME01, which belongs to a new IncP-1θ ancestral archetype without any accessory mobile elements was found in 78-ME. Our results provide the context to the co-occurrence of diverse defence mechanisms in the genome of Cycloclasticus sp. 78-ME, which protect the genome of this highly specialized PAH-degrader. This study contributes to the further understanding of complex networks established in petroleum-based microbial communities.

One of the main challenges of bioremediation is to define efficient protocols having a low environmental impact. We have investigated the effect of three treatments in oily-seawater after a real oil-spill occurred in the Gulf of Taranto (Italy). Biostimulation with inorganic nutrients allowed the biodegradation of the 73 +/- 2.4% of hydrocarbons, bioaugmentation with a selected hydrocarbonoclastic consortium consisting of Alcanivorax borkumensis, Alcanivorax dieselolei, Marinobacter hydrocarbonoclasticus, Cycloclasticus sp. 78-ME and Thalassolituus oleivorans degraded 79 +/- 3.2%, while the addition of nutrients and a washing agent has allowed the degradation of the 69 +/- 2.6%. On the other hand, microbial community was severely affected by the addition of the washing agent and the same product seemed to inhibit the growth of the majority of strains composing the selected consortium at the tested concentration. The use of dispersant should be accurately evaluated also considering its effect on the principal actors of biodegradation. (C) 2016 Elsevier Ltd. All rights reserved.

Microorganisms inhabiting saline environments are an interesting ecological model for the study of the adaptation of organisms to extreme living conditions and constitute a precious resource of enzymes and bioproducts for biotechnological applications. We analyzed the microbial communities in nine ponds with increasing salt concentrations (salinity range 4.9&ndash, 36.0%) of the Saltern of Margherita di Savoia (Italy), the largest thalassohaline saltern in Europe. A deep-metabarcoding NGS procedure addressing separately the V5-V6 and V3-V4 hypervariable regions of the 16S rRNA gene of Bacteria and Archaea, respectively, and a CARD-FISH (catalyzed reporter deposition fluorescence in situ hybridization) analysis allowed us to profile the dynamics of microbial populations at the different salt concentrations. Both the domains were detected throughout the saltern, even if the low relative abundance of Archaea in the three ponds with the lowest salinities prevented the construction of the relative amplicon libraries. The highest cell counts were recorded at 14.5% salinity for Bacteria and at 24.1% salinity for Archaea. While Bacteria showed the greatest number of genera in the first ponds (salinity range 4.9&ndash, 14.5%), archaeal genera were more numerous in the last ponds of the saltern (salinity 24.1&ndash, 36.0%). Among prokaryotes, Salinibacter was the genus with the maximum abundance (~49% at 34.6% salinity). Other genera detected at high abundance were the archaeal Haloquadratum (~43% at 36.0% salinity) and Natronomonas (~18% at 13.1% salinity) and the bacterial &ldquo, Candidatus Aquiluna&rdquo, (~19% at 14.5% salinity). Interestingly, &ldquo, had not been identified before in thalassohaline waters.

The continuous growth of global plastics production, including polyesters, has resulted in increasing plastic pollution and subsequent negative environmental impacts. Therefore, enzyme-catalyzed depolymerization of synthetic polyesters as a plastics recycling approach has become a focus of research. In this study, we screened over 200 purified uncharacterized hydrolases from environmental metagenomes and sequenced microbial genomes and identified at least 10 proteins with high hydrolytic activity against synthetic polyesters. These include the metagenomic esterases MGS0156 and GEN0105, which hydrolyzed polylactic acid (PLA), polycaprolactone, as well as bis(benzoyloxyethyl)-terephthalate. With solid PLA as a substrate, both enzymes produced a mixture of lactic acid monomers, dimers, and higher oligomers as products. The crystal structure of MGS0156 was determined at 1.95 A resolution and revealed a modified α/β hydrolase fold, with a lid domain and highly hydrophobic active site. Mutational studies of MGS0156 identified the residues critical for hydrolytic activity against both polyester and monoester substrates, with two-times higher polyesterase activity in the MGS0156 L169A mutant protein. Thus, our work identified novel, highly active polyesterases in environmental metagenomes and provided molecular insights into their activity, thereby augmenting our understanding of enzymatic polyester hydrolysis.

The ubiquity of strictly anaerobic sulfur-respiring haloarchaea in hypersaline systems with circumneutral pH has shaken a traditional concept of this group as predominantly aerobic heterotrophs. Here, we demonstrated that this functional group of haloarchaea also has its representatives in hypersaline alkaline lakes. Sediments from various hypersaline soda lakes showed high activity of sulfur reduction only partially inhibited by antibiotics. Eight pure cultures of sulfur-reducing natronoarchaea were isolated from such sediments using formate and butyrate as electron donors and sulfur as an electron acceptor. Unlike strict anaerobic haloarchaea, these novel sulfur-reducing natronoarchaea are facultative anaerobes, whose metabolic capabilities were inferred from cultivation experiments and genomic/proteomic reconstruction. While sharing many physiological traits with strict anaerobic haloarchaea, following metabolic distinctions make these new organisms be successful in both anoxic and aerobic habitats: the recruiting of heme-copper quinol oxidases as terminal electron sink in aerobic respiratory chain and the utilization of formate, hydrogen or short-chain fatty acids as electron donors during anaerobic growth with elemental sulfur. Obtained results significantly advance the emerging concept of halo(natrono)archaea as important players in the anaerobic sulfur and carbon cycling in various salt-saturated habitats.

Covering two-thirds of our planet, the global deep ocean plays a central role in supporting life on Earth. Among other processes, this biggest ecosystem buffers the rise of atmospheric CO2. Despite carbon sequestration in the deep ocean has been known for a long time, microbial activity in the meso- and bathypelagic realm via the "assimilation of bicarbonate in the dark" (ABD) has only recently been described in more details. Based on recent findings, this process seems primarily the result of chemosynthetic and anaplerotic reactions driven by different groups of deep-sea prokaryoplankton. We quantified bicarbonate assimilation in relation to total prokaryotic abundance, prokaryotic heterotrophic production and respiration in the meso- and bathypelagic Mediterranean Sea. The measured ABD values, ranging from 133 to 370 μg C m-3 d-1, were among the highest ones reported worldwide for similar depths, likely due to the elevated temperature of the deep Mediterranean Sea (13-14°C also at abyssal depths). Integrated over the dark water column (≥200 m depth), bicarbonate assimilation in the deep-sea ranged from 396 to 873 mg C m-2 d-1. This quantity of produced de novo organic carbon amounts to about 85-424% of the phytoplankton primary production and covers up to 62% of deep-sea prokaryotic total carbon demand. Hence, the ABD process in the meso- and bathypelagic Mediterranean Sea might substantially contribute to the inorganic and organic pool and significantly sustain the deep-sea microbial food web. To elucidate the ABD key-players, we established three actively nitrifying and CO2-fixing prokaryotic enrichments. Consortia were characterized by the co-occurrence of chemolithoautotrophic Thaumarchaeota and chemoheterotrophic proteobacteria. One of the enrichments, originated from Ionian bathypelagic waters (3,000 m depth) and supplemented with low concentrations of ammonia, was dominated by the Thaumarchaeota "low-ammonia-concentration" deep-sea ecotype, an enigmatic and ecologically important group of organisms, uncultured until this study.

Substrate specificity and selectivity of a biocatalyst are determined by the protein sequence and structure of its active site. Finding versatile biocatalysts acting against multiple substrates while at the same time being chiral selective is of interest for the pharmaceutical and chemical industry. However, the relationships between these two properties in natural microbial enzymes remain underexplored. Here, we performed an experimental analysis of substrate promiscuity and chiral selectivity in a set of 145 purified esterases from phylogenetically and environmentally diverse microorganisms, which were assayed against 96 diverse esters, 20 of which were enantiomers. Our results revealed a negative correlation between substrate promiscuity and chiral selectivity in the evaluated enzymes. Esterases displaying prominent substrate promiscuity and large catalytic environments are characterized by low chiral selectivity, a feature that has limited commercial value. Although a low level of substrate promiscuity does not guarantee high chiral selectivity, the probability that esterases with smaller active sites possess chiral selectivity factors of interest for industry (>25) is significantly higher than for promiscuous enzymes. Together, the present study unambiguously demonstrates that promiscuous and selective esterases appear to be rare in nature and that substrate promiscuity can be used as an indicator of the chiral selectivity level of esterases, and vice versa.