73 results on '"Golyshina OV"'
Search Results
2. Novel hydrolase diversity retrieved from a metagenome library of bovine rumen microflora
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Ferrer M, Golyshina OV, Chernikova TN, Khachane AN, Reyes-Duarte D, Santos VA, Strompl C, Elborough K, Jarvis G, Neef A, Yakimov MM, Timmis KN, and Golyshin PN
- Abstract
A metagenome expression library of bulk DNA extracted from the rumen content of a dairy cow was established in a phage lambda vector and activity-based screening employed to explore the functional diversity of the microbial flora. Twenty-two clones specifying distinct hydrolytic activities (12 esterases, nine endo-beta-1,4-glucanases and one cyclodextrinase) were identified in the library and characterized. Sequence analysis of the retrieved enzymes revealed that eight (36%) were entirely new and formed deep-branched phylogenetic lineages with no close relatives among known ester- and glycosyl-hydrolases. Bioinformatic analyses of the hydrolase gene sequences, and the sequences and contexts of neighbouring genes, suggested tentative phylogenetic assignments of the rumen organisms producing the retrieved enzymes. The phylogenetic novelty of the hydrolases suggests that some of them may have potential for new applications in biocatalysis.
- Published
- 2005
3. Moderately thermostable GH1 β-glucosidases from hyperacidophilic archaeon Cuniculiplasma divulgatum S5.
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Khusnutdinova AN, Tran H, Devlekar S, Distaso MA, Kublanov IV, Skarina T, Stogios P, Savchenko A, Ferrer M, Golyshina OV, Yakunin AF, and Golyshin PN
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- Enzyme Stability, Hydrogen-Ion Concentration, beta-Glucosidase genetics, beta-Glucosidase metabolism, Archaeal Proteins genetics, Archaeal Proteins metabolism, Archaeal Proteins chemistry, Substrate Specificity, Temperature, Phylogeny
- Abstract
Family GH1 glycosyl hydrolases are ubiquitous in prokaryotes and eukaryotes and are utilized in numerous industrial applications, including bioconversion of lignocelluloses. In this study, hyperacidophilic archaeon Cuniculiplasma divulgatum (S5T=JCM 30642T) was explored as a source of novel carbohydrate-active enzymes. The genome of C. divulgatum encodes three GH1 enzyme candidates, from which CIB12 and CIB13 were heterologously expressed and characterized. Phylogenetic analysis of CIB12 and CIB13 clustered them with β-glucosidases from genuinely thermophilic archaea including Thermoplasma acidophilum, Picrophilus torridus, Sulfolobus solfataricus, Pyrococcus furiosus, and Thermococcus kodakarensis. Purified enzymes showed maximal activities at pH 4.5-6.0 (CIB12) and 4.5-5.5 (CIB13) with optimal temperatures at 50°C, suggesting a high-temperature origin of Cuniculiplasma spp. ancestors. Crystal structures of both enzymes revealed a classical (α/β)8 TIM-barrel fold with the active site located inside the barrel close to the C-termini of β-strands including the catalytic residues Glu204 and Glu388 (CIB12), and Glu204 and Glu385 (CIB13). Both enzymes preferred cellobiose over lactose as substrates and were classified as cellobiohydrolases. Cellobiose addition increased the biomass yield of Cuniculiplasma cultures growing on peptides by 50%, suggesting that the cellobiohydrolases expand the carbon substrate range and hence environmental fitness of Cuniculiplasma., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)
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- 2024
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4. Oxyplasma meridianum gen. nov., sp. nov., an extremely acidophilic organotrophic member of the order Thermoplasmatales .
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Golyshina OV, Lunev EA, Distaso MA, Bargiela R, Gaines MC, Daum B, Ferrer M, Bale NJ, Koenen M, Damsté JSS, Yakimov MM, and Golyshin PN
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- Italy, Thermoplasmales genetics, Thermoplasmales classification, Thermoplasmales isolation & purification, Geologic Sediments microbiology, Genome, Archaeal, RNA, Ribosomal, 16S genetics, Phylogeny, Base Composition, Sequence Analysis, DNA, DNA, Archaeal genetics
- Abstract
A mesophilic, hyperacidophilic archaeon, strain M1
T , was isolated from a rock sample from Vulcano Island, Italy. Cells of this organism were cocci with an average diameter of 1 µm. Some cells possessed filaments. The strain grew in the range of temperatures between 15 and 52 °C and pH 0.5-4.0 with growth optima at 40 °C and pH 1.0. Strain M1T was aerobic and chemoorganotrophic, growing on complex substrates, such as casamino acids, trypticase, tryptone, yeast and beef extracts. No growth at expenses of oxidation of elemental sulphur or reduced sulphur compounds, pyrite, or ferrous sulphate was observed. The core lipids were glycerol dibiphytanyl glycerol tetraether lipids (membrane spanning) with 0 to 4 cyclopentane moieties and archaeol, with trace amounts of hydroxy archaeol. The dominant quinone was MK-7 : 7. The genome size of M1T was 1.67 Mbp with a G+C content of 39.76 mol%, and both characteristics were well within the common range for Thermoplasmatales . The phylogenetic analysis based on 16S rRNA gene sequence placed the strain M1T within the order Thermoplasmatales with sequence identities of 90.9, 90.3 and 90.5% to the closest SSU rRNA gene sequences from organisms with validly published names, Thermoplasma acidophilum , Thermoplasma volcanium and Thermogymnomonas acidicola , respectively. Based on the results of our genomic, phylogenetic, physiological and chemotaxonomic studies, we propose that strain M1T (=DSM 116605T =JCM 36570T ) represents a new genus and species, Oxyplasma meridianum gen. nov., sp. nov., within the order Thermoplasmatales .- Published
- 2024
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5. Nanohaloarchaea as beneficiaries of xylan degradation by haloarchaea.
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La Cono V, Messina E, Reva O, Smedile F, La Spada G, Crisafi F, Marturano L, Miguez N, Ferrer M, Selivanova EA, Golyshina OV, Golyshin PN, Rohde M, Krupovic M, Merkel AY, Sorokin DY, Hallsworth JE, and Yakimov MM
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- Ecosystem, Xylans, Haloferax
- Abstract
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., (© 2023 The Authors. Microbial Biotechnology published by Applied Microbiology International and John Wiley & Sons Ltd.)
- Published
- 2023
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6. Evolutionary patterns of archaea predominant in acidic environment.
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Bargiela R, Korzhenkov AA, McIntosh OA, Toshchakov SV, Yakimov MM, Golyshin PN, and Golyshina OV
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Background: Archaea of the order Thermoplasmatales are widely distributed in natural acidic areas and are amongst the most acidophilic prokaryotic organisms known so far. These organisms are difficult to culture, with currently only six genera validly published since the discovery of Thermoplasma acidophilum in 1970. Moreover, known great diversity of uncultured Thermoplasmatales represents microbial dark matter and underlines the necessity of efforts in cultivation and study of these archaea. Organisms from the order Thermoplasmatales affiliated with the so-called "alphabet-plasmas", and collectively dubbed "E-plasma", were the focus of this study. These archaea were found predominantly in the hyperacidic site PM4 of Parys Mountain, Wales, UK, making up to 58% of total metagenomic reads. However, these archaea escaped all cultivation attempts., Results: Their genome-based metabolism revealed its peptidolytic potential, in line with the physiology of the previously studied Thermoplasmatales isolates. Analyses of the genome and evolutionary history reconstruction have shown both the gain and loss of genes, that may have contributed to the success of the "E-plasma" in hyperacidic environment compared to their community neighbours. Notable genes among them are involved in the following molecular processes: signal transduction, stress response and glyoxylate shunt, as well as multiple copies of genes associated with various cellular functions; from energy production and conversion, replication, recombination, and repair, to cell wall/membrane/envelope biogenesis and archaella production. History events reconstruction shows that these genes, acquired by putative common ancestors, may determine the evolutionary and functional divergences of "E-plasma", which is much more developed than other representatives of the order Thermoplasmatales. In addition, the ancestral hereditary reconstruction strongly indicates the placement of Thermogymnomonas acidicola close to the root of the Thermoplasmatales., Conclusions: This study has analysed the metagenome-assembled genome of "E-plasma", which denotes the basis of their predominance in Parys Mountain environmental microbiome, their global ubiquity, and points into the right direction of further cultivation attempts. The results suggest distinct evolutionary trajectories of organisms comprising the order Thermoplasmatales, which is important for the understanding of their evolution and lifestyle., (© 2023. The Author(s).)
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- 2023
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7. Thermophilic Carboxylesterases from Hydrothermal Vents of the Volcanic Island of Ischia Active on Synthetic and Biobased Polymers and Mycotoxins.
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Distaso MA, Chernikova TN, Bargiela R, Coscolín C, Stogios P, Gonzalez-Alfonso JL, Lemak S, Khusnutdinova AN, Plou FJ, Evdokimova E, Savchenko A, Lunev EA, Yakimov MM, Golyshina OV, Ferrer M, Yakunin AF, and Golyshin PN
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- Polymers, Hydrolases metabolism, Polyesters, Plastics, Substrate Specificity, Carboxylic Ester Hydrolases metabolism, Hydrothermal Vents
- Abstract
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/β-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 β-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.
- Published
- 2023
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8. Microbial Diversity of a Disused Copper Mine Site (Parys Mountain, UK), Dominated by Intensive Eukaryotic Filamentous Growth.
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Distaso MA, Bargiela R, Johnson B, McIntosh OA, Williams GB, Jones DL, Golyshin PN, and Golyshina OV
- Abstract
The Parys Mountain copper mine (Wales, UK) contains a wide range of discrete environmental microniches with various physicochemical conditions that shape microbial community composition. Our aim was to assess the microbial community in the sediments and overlying water column in an acidic mine drainage (AMD) site containing abundant filamentous biogenic growth via application of a combination of chemical analysis and taxonomic profiling using 16S rRNA gene amplicon sequencing. Our results were then compared to previously studied sites at Parys Mt. Overall, the sediment microbiome showed a dominance of bacteria over archaea, particularly those belonging to Proteobacteria (genera Acidiphilium and Acidisphaera ), Acidobacteriota (subgroup 1), Chloroflexota (AD3 cluster), Nitrospirota ( Leptospirillum ) and the uncultured Planctomycetota/CPIa-3 termite group. Archaea were only present in the sediment in small quantities, being represented by the Terrestrial Miscellaneous Euryarchaeota Group (TMEG), Thermoplasmatales and Ca . Micrarchaeota ( Ca . Micracaldota). Bacteria, mostly of the genera Acidiphilium and Leptospirillum , also dominated within the filamentous streamers while archaea were largely absent. This study found pH and dissolved solutes to be the most important parameters correlating with relative proportions of bacteria to archaea in an AMD environment and revealed the abundance patterns of native acidophilic prokaryotes inhabiting Parys Mt sites and their niche specificities.
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- 2022
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9. Metagenomic Mining for Esterases in the Microbial Community of Los Rueldos Acid Mine Drainage Formation.
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Vidal P, Martínez-Martínez M, Fernandez-Lopez L, Roda S, Méndez-García C, Golyshina OV, Guallar V, Peláez AI, and Ferrer M
- Abstract
Acid mine drainage (AMD) systems are extremely acidic and are metal-rich formations inhabited by relatively low-complexity communities of acidophiles whose enzymes remain mostly uncharacterized. Indeed, enzymes from only a few AMD sites have been studied. The low number of available cultured representatives and genome sequences of acidophiles inhabiting AMDs makes it difficult to assess the potential of these environments for enzyme bioprospecting. In this study, using naïve and in silico metagenomic approaches, we retrieved 16 esterases from the α/β-hydrolase fold superfamily with the closest match from uncultured acidophilic Acidobacteria, Actinobacteria (Acidithrix, Acidimicrobium , and Ferrimicrobium), Acidiphilium , and other Proteobacteria inhabiting the Los Rueldos site, which is a unique AMD formation in northwestern Spain with a pH of ∼2. Within this set, only two polypeptides showed high homology (99.4%), while for the rest, the pairwise identities ranged between 4 and 44.9%, suggesting that the diversity of active polypeptides was dominated not by a particular type of protein or highly similar clusters of proteins, but by diverse non-redundant sequences. The enzymes exhibited amino acid sequence identities ranging from 39 to 99% relative to homologous proteins in public databases, including those from other AMDs, thus indicating the potential novelty of proteins associated with a specialized acidophilic community. Ten of the 16 hydrolases were successfully expressed in Escherichia coli . The pH for optimal activity ranged from 7.0 to 9.0, with the enzymes retaining 33-68% of their activities at pH 5.5, which was consistent with the relative frequencies of acid residues (from 54 to 67%). The enzymes were the most active at 30-65°C, retaining 20-61% of their activity under the thermal conditions characterizing Los Rueldos (13.8 ± 0.6°C). The analysis of the substrate specificity revealed the capacity of six hydrolases to efficiently degrade (up to 1,652 ± 75 U/g at pH 8.0 and 30°C) acrylic- and terephthalic-like [including bis(2-hydroxyethyl)-terephthalate, BHET] esters, and these enzymes could potentially be of use for developing plastic degradation strategies yet to be explored. Our assessment uncovers the novelty and potential biotechnological interest of enzymes present in the microbial populations that inhibit the Los Rueldos AMD system., 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 © 2022 Vidal, Martínez-Martínez, Fernandez-Lopez, Roda, Méndez-García, Golyshina, Guallar, Peláez and Ferrer.)
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- 2022
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10. Utilization of low-molecular-weight organic compounds by the filterable fraction of a lotic microbiome.
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Ghuneim LJ, Distaso MA, Chernikova TN, Bargiela R, Lunev EA, Korzhenkov AA, Toshchakov SV, Rojo D, Barbas C, Ferrer M, Golyshina OV, Golyshin PN, and Jones DL
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- Carbon, Fresh Water, RNA, Ribosomal, 16S genetics, Rivers, Microbiota, Organic Chemicals
- Abstract
Filterable microorganisms participate in dissolved organic carbon (DOC) cycling in freshwater systems, however their exact functional role remains unknown. We determined the taxonomic identity and community dynamics of prokaryotic microbiomes in the 0.22 µm-filtered fraction and unfiltered freshwater from the Conwy River (North Wales, UK) in microcosms and, using targeted metabolomics and 14C-labelling, examined their role in the utilization of amino acids, organic acids and sugars spiked at environmentally-relevant (nanomolar) concentrations. To identify changes in community structure, we used 16S rRNA amplicon and shotgun sequencing. Unlike the unfiltered water samples where the consumption of DOC was rapid, the filtered fraction showed a 3-day lag phase before the consumption started. Analysis of functional categories of clusters of orthologous groups of proteins (COGs) showed that COGs associated with energy production increased in number in both fractions with substrate addition. The filtered fraction utilized low-molecular-weight (LMW) DOC at much slower rates than the whole community. Addition of nanomolar concentrations of LMW DOC did not measurably influence the composition of the microbial community nor the rate of consumption across all substrate types in either fraction. We conclude that due to their low activity, filterable microorganisms play a minor role in LMW DOC processing within a short residence time of lotic freshwater systems., (© The Author(s) 2020. Published by Oxford University Press on behalf of FEMS.)
- Published
- 2021
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11. Corrigendum: High Representation of Archaea Across All Depths in Oxic and Low-pH Sediment Layers Underlying an Acidic Stream.
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Distaso MA, Bargiela R, Brailsford FL, Williams GB, Wright S, Lunev EA, Toshchakov SV, Yakimov MM, Jones DL, Golyshin PN, and Golyshina OV
- Abstract
[This corrects the article DOI: 10.3389/fmicb.2020.576520.]., (Copyright © 2021 Distaso, Bargiela, Brailsford, Williams, Wright, Lunev, Toshchakov, Yakimov, Jones, Golyshin and Golyshina.)
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- 2021
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12. High Representation of Archaea Across All Depths in Oxic and Low-pH Sediment Layers Underlying an Acidic Stream.
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Distaso MA, Bargiela R, Brailsford FL, Williams GB, Wright S, Lunev EA, Toshchakov SV, Yakimov MM, Jones DL, Golyshin PN, and Golyshina OV
- Abstract
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., (Copyright © 2020 Distaso, Bargiela, Brailsford, Williams, Wright, Lunev, Toshchakov, Yakimov, Jones, Golyshin and Golyshina.)
- Published
- 2020
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13. Symbiosis between nanohaloarchaeon and haloarchaeon is based on utilization of different polysaccharides.
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La Cono V, Messina E, Rohde M, Arcadi E, Ciordia S, Crisafi F, Denaro R, Ferrer M, Giuliano L, Golyshin PN, Golyshina OV, Hallsworth JE, La Spada G, Mena MC, Merkel AY, Shevchenko MA, Smedile F, Sorokin DY, Toshchakov SV, and Yakimov MM
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- Archaeal Proteins genetics, Archaeal Proteins metabolism, Coculture Techniques, Gene Expression Regulation, Archaeal, Genome, Archaeal, Genomics, Phylogeny, Halobacteriaceae physiology, Nanoarchaeota physiology, Polysaccharides metabolism, Symbiosis physiology
- Abstract
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., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)
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- 2020
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14. Proteome Cold-Shock Response in the Extremely Acidophilic Archaeon, Cuniculiplasma divulgatum .
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Bargiela R, Lanthaler K, Potter CM, Ferrer M, Yakunin AF, Paizs B, Golyshin PN, and Golyshina OV
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The archaeon Cuniculiplasma divulgatum is ubiquitous in acidic environments with low-to-moderate temperatures. However, molecular mechanisms underlying its ability to thrive at lower temperatures remain unexplored. Using mass spectrometry (MS)-based proteomics, we analysed the effect of short-term (3 h) exposure to cold. The C. divulgatum genome encodes 2016 protein-coding genes, from which 819 proteins were identified in the cells grown under optimal conditions. In line with the peptidolytic lifestyle of C. divulgatum , its intracellular proteome revealed the abundance of proteases, ABC transporters and cytochrome C oxidase. From 747 quantifiable polypeptides, the levels of 582 proteins showed no change after the cold shock, whereas 104 proteins were upregulated suggesting that they might be contributing to cold adaptation. The highest increase in expression appeared in low-abundance (0.001-0.005 fmol%) proteins for polypeptides' hydrolysis (metal-dependent hydrolase), oxidation of amino acids (FAD-dependent oxidoreductase), pyrimidine biosynthesis (aspartate carbamoyltransferase regulatory chain proteins), citrate cycle (2-oxoacid ferredoxin oxidoreductase) and ATP production (V type ATP synthase). Importantly, the cold shock induced a substantial increase (6% and 9%) in expression of the most-abundant proteins, thermosome beta subunit and glutamate dehydrogenase. This study has outlined potential mechanisms of environmental fitness of Cuniculiplasma spp. allowing them to colonise acidic settings at low/moderate temperatures.
- Published
- 2020
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15. Microbial Communities of Polymetallic Deposits' Acidic Ecosystems of Continental Climatic Zone With High Temperature Contrasts.
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Gavrilov SN, Korzhenkov AA, Kublanov IV, Bargiela R, Zamana LV, Popova AA, Toshchakov SV, Golyshin PN, and Golyshina OV
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Acid mine drainage (AMD) systems are globally widespread and are an important source of metal pollution in riverine and coastal systems. Microbial AMD communities have been extensively studied for their ability to thrive under extremely acidic conditions and for their immense contribution to the dissolution of metal ores. However, little is known on microbial inhabitants of AMD systems subjected to extremely contrasting continental seasonal temperature patterns as opposed to maritime climate zones, experiencing much weaker annual temperature variations. Here, we investigated three types of AMD sites in Eastern Transbaikalia (Russia). In this region, all surface water bodies undergo a deep and long (up to 6 months) freezing, with seasonal temperatures varying between -33 and +24°C, which starkly contrasts the common well-studied AMD environments. We sampled acidic pit lake (Sherlovaya Gora site) located in the area of a polymetallic deposit, acidic drainage water from Bugdaya gold-molybdenum-tungsten deposit and Ulan-Bulak natural acidic spring. These systems showed the abundance of bacteria-derived reads mostly affiliated with Actinobacteria, Acidobacteria, Alpha- and Gammaproteobacteria , chloroplasts, Chloroflexi, Bacteroidetes , and Firmicutes . Furthermore, candidate taxa " Ca . Saccharibacteria" (previously known as TM7), " Ca . Parcubacteria" (OD1) and WPS-2 were represented in substantial quantities (10-20%). Heterotrophy and iron redox cycling can be considered as central processes of carbon and energy flow for majority of detected bacterial taxa. Archaea were detected in low numbers, with Terrestrial Miscellaneous Euryarchaeal Group (TMEG), to be most abundant (3%) in acidic spring Ulan-Bulak. Composition of these communities was found to be typical in comparison to other AMD sites; however, certain groups (as Ignavibacteriae ) could be specifically associated with this area. This study provides insight into the microbial diversity patterns in acidic ecosystems formed in areas of polymetallic deposits in extreme continental climate zone with contrasting temperature parameters.
- Published
- 2019
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16. Diversity of " Ca . Micrarchaeota" in Two Distinct Types of Acidic Environments and Their Associations with Thermoplasmatales .
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Golyshina OV, Bargiela R, Toshchakov SV, Chernyh NA, Ramayah S, Korzhenkov AA, Kublanov IV, and Golyshin PN
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- Archaea chemistry, Archaea classification, Ecosystem, Fresh Water microbiology, Genome, Archaeal genetics, Hot Springs, Phylogeny, RNA, Ribosomal, 16S genetics, Rivers microbiology, Soil chemistry, Thermoplasmales chemistry, Wales, Acids chemistry, Archaea genetics, Soil Microbiology, Thermoplasmales genetics
- Abstract
" Candidatus Micrarchaeota" are widely distributed in acidic environments; however, their cultivability and our understanding of their interactions with potential hosts are very limited. Their habitats were so far attributed with acidic sites, soils, peats, freshwater systems, and hypersaline mats. Using cultivation and culture-independent approaches (16S rRNA gene clonal libraries, high-throughput amplicon sequencing of V3-V4 region of 16S rRNA genes), we surveyed the occurrence of these archaea in geothermal areas on Kamchatka Peninsula and Kunashir Island and assessed their taxonomic diversity in relation with another type of low-pH environment, acid mine drainage stream (Wales, UK). We detected " Ca . Micrarchaeota" in thermophilic heterotrophic enrichment cultures of Kunashir and Kamchatka that appeared as two different phylotypes, namely " Ca . Mancarchaeum acidiphilum"-, and ARMAN-2-related, alongside their potential hosts, Cuniculiplasma spp. and other Thermoplasmatales archaea without defined taxonomic position. These clusters of " Ca . Micrarchaeota" together with three other groups were also present in mesophilic acid mine drainage community. Present work expands our knowledge on the diversity of " Ca . Micrarchaeota" in thermophilic and mesophilic acidic environments, suggests cultivability patterns of acidophilic archaea and establishes potential links between low-abundance species of thermophilic " Ca . Micrarchaeota" and certain Thermoplasmatales , such as Cuniculiplasma spp. in situ .
- Published
- 2019
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17. Archaea dominate the microbial community in an ecosystem with low-to-moderate temperature and extreme acidity.
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Korzhenkov AA, Toshchakov SV, Bargiela R, Gibbard H, Ferrer M, Teplyuk AV, Jones DL, Kublanov IV, Golyshin PN, and Golyshina OV
- Subjects
- Acids metabolism, Archaea genetics, Bacteria classification, Bacteria genetics, Cold Temperature, Ecosystem, Metagenome genetics, Microbiota physiology, RNA, Ribosomal, 16S genetics, Wales, Archaea classification, Archaea isolation & purification, Bacteria isolation & purification, Geologic Sediments microbiology
- Abstract
Background: The current view suggests that in low-temperature acidic environments, archaea are significantly less abundant than bacteria. Thus, this study of the microbiome of Parys Mountain (Anglesey, UK) sheds light on the generality of this current assumption. Parys Mountain is a historically important copper mine and its acid mine drainage (AMD) water streams are characterised by constant moderate temperatures (8-18 °C), extremely low pH (1.7) and high concentrations of soluble iron and other metal cations., Results: Metagenomic and SSU rRNA amplicon sequencing of DNA from Parys Mountain revealed a significant proportion of archaea affiliated with Euryarchaeota, which accounted for ca. 67% of the community. Within this phylum, potentially new clades of Thermoplasmata were overrepresented (58%), with the most predominant group being "E-plasma", alongside low-abundant Cuniculiplasmataceae, 'Ca. Micrarchaeota' and 'Terrestrial Miscellaneous Euryarchaeal Group' (TMEG) archaea, which were phylogenetically close to Methanomassilicoccales and clustered with counterparts from acidic/moderately acidic settings. In the sediment, archaea and Thermoplasmata contributed the highest numbers in V3-V4 amplicon reads, in contrast with the water body community, where Proteobacteria, Nitrospirae, Acidobacteria and Actinobacteria outnumbered archaea. Cultivation efforts revealed the abundance of archaeal sequences closely related to Cuniculiplasma divulgatum in an enrichment culture established from the filterable fraction of the water sample. Enrichment cultures with unfiltered samples showed the presence of Ferrimicrobium acidiphilum, C. divulgatum, 'Ca. Mancarchaeum acidiphilum Mia14', 'Ca. Micrarchaeota'-related and diverse minor (< 2%) bacterial metagenomic reads., Conclusion: Contrary to expectation, our study showed a high abundance of archaea in this extremely acidic mine-impacted environment. Further, archaeal populations were dominated by one particular group, suggesting that they are functionally important. The prevalence of archaea over bacteria in these microbiomes and their spatial distribution patterns represents a novel and important advance in our understanding of acidophile ecology. We also demonstrated a procedure for the specific enrichment of cell wall-deficient members of the archaeal component of this community, although the large fraction of archaeal taxa remained unculturable. Lastly, we identified a separate clustering of globally occurring acidophilic members of TMEG that collectively belong to a distinct order within Thermoplasmata with yet unclear functional roles in the ecosystem.
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- 2019
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18. Bioprospecting Reveals Class III ω-Transaminases Converting Bulky Ketones and Environmentally Relevant Polyamines.
- Author
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Coscolín C, Katzke N, García-Moyano A, Navarro-Fernández J, Almendral D, Martínez-Martínez M, Bollinger A, Bargiela R, Gertler C, Chernikova TN, Rojo D, Barbas C, Tran H, Golyshina OV, Koch R, Yakimov MM, Bjerga GEK, Golyshin PN, Jaeger KE, and Ferrer M
- Subjects
- Amino Acid Sequence, Bacterial Proteins chemistry, Bacterial Proteins metabolism, Pseudomonas oleovorans enzymology, Pseudomonas oleovorans metabolism, Sequence Alignment, Transaminases metabolism, Bacterial Proteins genetics, Bioprospecting, Genes, Bacterial, Ketones metabolism, Polyamines metabolism, Pseudomonas oleovorans genetics, Transaminases genetics
- Abstract
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., (Copyright © 2019 Coscolín et al.)
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- 2019
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19. Decoding the ocean's microbiological secrets for marine enzyme biodiscovery.
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Ferrer M, Méndez-García C, Bargiela R, Chow J, Alonso S, García-Moyano A, Bjerga GEK, Steen IH, Schwabe T, Blom C, Vester J, Weckbecker A, Shahgaldian P, de Carvalho CCCR, Meskys R, Zanaroli G, Glöckner FO, Fernández-Guerra A, Thambisetty S, de la Calle F, Golyshina OV, Yakimov MM, Jaeger KE, Yakunin AF, Streit WR, McMeel O, Calewaert JB, Tonné N, and Golyshin PN
- Subjects
- Bacteria enzymology, Biodiversity, Aquatic Organisms enzymology, Oceans and Seas, Water Microbiology
- Abstract
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'.
- Published
- 2019
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20. Cuniculiplasmataceae, their ecogenomic and metabolic patterns, and interactions with 'ARMAN'.
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Golyshina OV, Bargiela R, and Golyshin PN
- Subjects
- Metabolome, Thermoplasmales classification, Thermoplasmales metabolism, Genome, Archaeal, Phylogeny, Thermoplasmales genetics
- Abstract
Recently, the order Thermoplasmatales was expanded through the cultivation and description of species Cuniculiplasma divulgatum and corresponding family Cuniculiplasmataceae. Initially isolated from acidic streamers, signatures of these archaea were ubiquitously found in various low-pH settings. Eight genomes with various levels of completeness are currently available, all of which exhibit very high sequence identities and genomic conservation. Co-existence of Cuniculiplasmataceae with archaeal Richmond Mine acidophilic nanoorganisms ('ARMAN')-related archaea representing an intriguing group within the "microbial dark matter" suggests their common fundamental environmental strategy and metabolic networking. The specific case of "Candidatus Mancarchaeum acidiphilum" Mia14 phylogenetically affiliated with "Ca. Micrarchaeota" from the superphylum "Ca. Diapherotrites" along with the presence of other representatives of 'DPANN' with significantly reduced genomes points at a high probability of close interactions between the latter and various Thermoplasmatales abundant in situ. This review critically assesses our knowledge on specific functional role and potential of the members of Cuniculiplasmataceae abundant in acidophilic microbiomes through the analysis of distribution, physiological and genomic patterns, and their interactions with 'ARMAN'-related archaea.
- Published
- 2019
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21. Screening and Characterization of Novel Polyesterases from Environmental Metagenomes with High Hydrolytic Activity against Synthetic Polyesters.
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Hajighasemi M, Tchigvintsev A, Nocek B, Flick R, Popovic A, Hai T, Khusnutdinova AN, Brown G, Xu X, Cui H, Anstett J, Chernikova TN, Brüls T, Le Paslier D, Yakimov MM, Joachimiak A, Golyshina OV, Savchenko A, Golyshin PN, Edwards EA, and Yakunin AF
- Subjects
- Esterases, Hydrolases, Hydrolysis, Metagenome, Polyesters
- Abstract
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 Å 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.
- Published
- 2018
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22. Nano-Sized and Filterable Bacteria and Archaea: Biodiversity and Function.
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Ghuneim LJ, Jones DL, Golyshin PN, and Golyshina OV
- Abstract
Nano-sized and filterable microorganisms are thought to represent the smallest living organisms on earth and are characterized by their small size (50-400 nm) and their ability to physically pass through <0.45 μm pore size filters. They appear to be ubiquitous in the biosphere and are present at high abundance across a diverse range of habitats including oceans, rivers, soils, and subterranean bedrock. Small-sized organisms are detected by culture-independent and culture-dependent approaches, with most remaining uncultured and uncharacterized at both metabolic and taxonomic levels. Consequently, their significance in ecological roles remain largely unknown. Successful isolation, however, has been achieved for some species (e.g., Nanoarchaeum equitans and " Candidatus Pelagibacter ubique"). In many instances, small-sized organisms exhibit a significant genome reduction and loss of essential metabolic pathways required for a free-living lifestyle, making their survival reliant on other microbial community members. In these cases, the nano-sized prokaryotes can only be co-cultured with their 'hosts.' This paper analyses the recent data on small-sized microorganisms in the context of their taxonomic diversity and potential functions in the environment.
- Published
- 2018
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23. Determinants and Prediction of Esterase Substrate Promiscuity Patterns.
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Martínez-Martínez M, Coscolín C, Santiago G, Chow J, Stogios PJ, Bargiela R, Gertler C, Navarro-Fernández J, Bollinger A, Thies S, Méndez-García C, Popovic A, Brown G, Chernikova TN, García-Moyano A, Bjerga GEK, Pérez-García P, Hai T, Del Pozo MV, Stokke R, Steen IH, Cui H, Xu X, Nocek BP, Alcaide M, Distaso M, Mesa V, Peláez AI, Sánchez J, Buchholz PCF, Pleiss J, Fernández-Guerra A, Glöckner FO, Golyshina OV, Yakimov MM, Savchenko A, Jaeger KE, Yakunin AF, Streit WR, Golyshin PN, Guallar V, Ferrer M, and The Inmare Consortium
- Subjects
- Catalytic Domain, Substrate Specificity, Esterases chemistry, Esterases metabolism, Phylogeny
- Abstract
Esterases receive special attention because of their wide distribution in biological systems and environments and their importance for physiology and chemical synthesis. The prediction of esterases' substrate promiscuity level from sequence data and the molecular reasons why certain such enzymes are more promiscuous than others remain to be elucidated. This limits the surveillance of the sequence space for esterases potentially leading to new versatile biocatalysts and new insights into their role in cellular function. Here, we performed an extensive analysis of the substrate spectra of 145 phylogenetically and environmentally diverse microbial esterases, when tested with 96 diverse esters. We determined the primary factors shaping their substrate range by analyzing substrate range patterns in combination with structural analysis and protein-ligand simulations. We found a structural parameter that helps rank (classify) the promiscuity level of esterases from sequence data at 94% accuracy. This parameter, the active site effective volume, exemplifies the topology of the catalytic environment by measuring the active site cavity volume corrected by the relative solvent accessible surface area (SASA) of the catalytic triad. Sequences encoding esterases with active site effective volumes (cavity volume/SASA) above a threshold show greater substrate spectra, which can be further extended in combination with phylogenetic data. This measure provides also a valuable tool for interrogating substrates capable of being converted. This measure, found to be transferred to phosphatases of the haloalkanoic acid dehalogenase superfamily and possibly other enzymatic systems, represents a powerful tool for low-cost bioprospecting for esterases with broad substrate ranges, in large scale sequence data sets.
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- 2018
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24. The rumen microbiome: an underexplored resource for novel antimicrobial discovery.
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Oyama LB, Girdwood SE, Cookson AR, Fernandez-Fuentes N, Privé F, Vallin HE, Wilkinson TJ, Golyshin PN, Golyshina OV, Mikut R, Hilpert K, Richards J, Wootton M, Edwards JE, Maresca M, Perrier J, Lundy FT, Luo Y, Zhou M, Hess M, Mantovani HC, Creevey CJ, and Huws SA
- Abstract
Antimicrobial peptides (AMPs) are promising drug candidates to target multi-drug resistant bacteria. The rumen microbiome presents an underexplored resource for the discovery of novel microbial enzymes and metabolites, including AMPs. Using functional screening and computational approaches, we identified 181 potentially novel AMPs from a rumen bacterial metagenome. Here, we show that three of the selected AMPs (Lynronne-1, Lynronne-2 and Lynronne-3) were effective against numerous bacterial pathogens, including methicillin-resistant Staphylococcus aureus (MRSA). No decrease in MRSA susceptibility was observed after 25 days of sub-lethal exposure to these AMPs. The AMPs bound preferentially to bacterial membrane lipids and induced membrane permeability leading to cytoplasmic leakage. Topical administration of Lynronne-1 (10% w/v) to a mouse model of MRSA wound infection elicited a significant reduction in bacterial counts, which was comparable to treatment with 2% mupirocin ointment. Our findings indicate that the rumen microbiome may provide viable alternative antimicrobials for future therapeutic application.
- Published
- 2017
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25. The genome analysis of Oleiphilus messinensis ME102 (DSM 13489 T ) reveals backgrounds of its obligate alkane-devouring marine lifestyle.
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Toshchakov SV, Korzhenkov AA, Chernikova TN, Ferrer M, Golyshina OV, Yakimov MM, and Golyshin PN
- Subjects
- Biodegradation, Environmental, Gammaproteobacteria physiology, Geologic Sediments microbiology, Italy, Phylogeny, Alkanes metabolism, Gammaproteobacteria genetics, Genome, Bacterial
- Abstract
Marine bacterium Oleiphilus messinensis ME102 (DSM 13489
T ) 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 (6.379.281bp) 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., (Copyright © 2017. Published by Elsevier B.V.)- Published
- 2017
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26. Buwchitin: A Ruminal Peptide with Antimicrobial Potential against Enterococcus faecalis .
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Oyama LB, Crochet JA, Edwards JE, Girdwood SE, Cookson AR, Fernandez-Fuentes N, Hilpert K, Golyshin PN, Golyshina OV, Privé F, Hess M, Mantovani HC, Creevey CJ, and Huws SA
- Abstract
Antimicrobial peptides (AMPs) are gaining popularity as alternatives for treatment of bacterial infections and recent advances in omics technologies provide new platforms for AMP discovery. We sought to determine the antibacterial activity of a novel antimicrobial peptide, buwchitin, against Enterococcus faecalis . Buwchitin was identified from a rumen bacterial metagenome library, cloned, expressed and purified. The antimicrobial activity of the recombinant peptide was assessed using a broth microdilution susceptibility assay to determine the peptide's killing kinetics against selected bacterial strains. The killing mechanism of buwchitin was investigated further by monitoring its ability to cause membrane depolarization (diSC
3 (5) method) and morphological changes in E. faecalis cells. Transmission electron micrographs of buwchitin treated E. faecalis cells showed intact outer membranes with blebbing, but no major damaging effects and cell morphology changes. Buwchitin had negligible cytotoxicity against defibrinated sheep erythrocytes. Although no significant membrane leakage and depolarization was observed, buwchitin at minimum inhibitory concentration (MIC) was bacteriostatic against E. faecalis cells and inhibited growth in vitro by 70% when compared to untreated cells. These findings suggest that buwchitin, a rumen derived peptide, has potential for antimicrobial activity against E. faecalis .- Published
- 2017
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27. 'ARMAN' archaea depend on association with euryarchaeal host in culture and in situ.
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Golyshina OV, Toshchakov SV, Makarova KS, Gavrilov SN, Korzhenkov AA, La Cono V, Arcadi E, Nechitaylo TY, Ferrer M, Kublanov IV, Wolf YI, Yakimov MM, and Golyshin PN
- Subjects
- Archaea genetics, Gene Expression Regulation, Archaeal, Genetic Variation, Genome, Archaeal, Microscopy, Fluorescence, Phylogeny, RNA, Archaeal genetics, RNA, Ribosomal, 16S genetics, Species Specificity, Archaea classification, Archaea physiology
- Abstract
Intriguing, yet uncultured 'ARMAN'-like archaea are metabolically dependent on other members of the microbial community. It remains uncertain though which hosts they rely upon, and, because of the lack of complete genomes, to what extent. Here, we report the co-culturing of ARMAN-2-related organism, Mia14, with Cuniculiplasma divulgatum PM4 during the isolation of this strain from acidic streamer in Parys Mountain (Isle of Anglesey, UK). Mia14 is highly enriched in the binary culture (ca. 10% genomic reads) and its ungapped 0.95 Mbp genome points at severe voids in central metabolic pathways, indicating dependence on the host, C. divulgatum PM4. Analysis of C. divulgatum isolates from different sites and shotgun sequence data of Parys Mountain samples suggests an extensive genetic exchange between Mia14 and hosts in situ. Within the subset of organisms with high-quality genomic assemblies representing the 'DPANN' superphylum, the Mia14 lineage has had the largest gene flux, with dozens of genes gained that are implicated in the host interaction.In the absence of complete genomes, the metabolic capabilities of uncultured ARMAN-like archaea have been uncertain. Here, Golyshina et al. apply an enrichment culture technique and find that the ungapped genome of the ARMAN-like archaeon Mia14 has lost key metabolic pathways, suggesting dependence on the host archaeon Cuniculiplasma divulgatum.
- Published
- 2017
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28. Metabolic and evolutionary patterns in the extremely acidophilic archaeon Ferroplasma acidiphilum Y T .
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Golyshina OV, Tran H, Reva ON, Lemak S, Yakunin AF, Goesmann A, Nechitaylo TY, LaCono V, Smedile F, Slesarev A, Rojo D, Barbas C, Ferrer M, Yakimov MM, and Golyshin PN
- Subjects
- Amino Acids metabolism, Archaea genetics, Carbon metabolism, Carbon Dioxide metabolism, Clustered Regularly Interspaced Short Palindromic Repeats, DNA Mismatch Repair, Evolution, Molecular, Gene Expression Regulation, Genome, Archaeal, Genomics methods, Metabolic Networks and Pathways, Mutation, Recombination, Genetic, Archaea metabolism, Biological Evolution, Energy Metabolism
- Abstract
Ferroplasmaceae represent ubiquitous iron-oxidising extreme acidophiles with a number of unique physiological traits. In a genome-based study of Ferroplasma acidiphilum Y
T , the only species of the genus Ferroplasma with a validly published name, we assessed its central metabolism and genome stability during a long-term cultivation experiment. Consistently with physiology, the genome analysis points to F. acidiphilum YT having an obligate peptidolytic oligotrophic lifestyle alongside with anaplerotic carbon assimilation. This narrow trophic specialisation abridges the sugar uptake, although all genes for glycolysis and gluconeogenesis, including bifunctional unidirectional fructose 1,6-bisphosphate aldolase/phosphatase, have been identified. Pyruvate and 2-oxoglutarate dehydrogenases are substituted by 'ancient' CoA-dependent pyruvate and alpha-ketoglutarate ferredoxin oxidoreductases. In the lab culture, after ~550 generations, the strain exhibited the mutation rate of ≥1.3 × 10-8 single nucleotide substitutions per site per generation, which is among the highest values recorded for unicellular organisms. All but one base substitutions were G:C to A:T, their distribution between coding and non-coding regions and synonymous-to-non-synonymous mutation ratios suggest the neutral drift being a prevalent mode in genome evolution in the lab culture. Mutations in nature seem to occur with lower frequencies, as suggested by a remarkable genomic conservation in F. acidiphilum YT variants from geographically distant populations.- Published
- 2017
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29. Activity screening of environmental metagenomic libraries reveals novel carboxylesterase families.
- Author
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Popovic A, Hai T, Tchigvintsev A, Hajighasemi M, Nocek B, Khusnutdinova AN, Brown G, Glinos J, Flick R, Skarina T, Chernikova TN, Yim V, Brüls T, Paslier DL, Yakimov MM, Joachimiak A, Ferrer M, Golyshina OV, Savchenko A, Golyshin PN, and Yakunin AF
- Subjects
- Carboxylic Ester Hydrolases chemistry, Escherichia coli genetics, Gene Library, Metagenomics, Carboxylic Ester Hydrolases genetics, Environmental Microbiology, Metagenome
- Abstract
Metagenomics has made accessible an enormous reserve of global biochemical diversity. To tap into this vast resource of novel enzymes, we have screened over one million clones from metagenome DNA libraries derived from sixteen different environments for carboxylesterase activity and identified 714 positive hits. We have validated the esterase activity of 80 selected genes, which belong to 17 different protein families including unknown and cyclase-like proteins. Three metagenomic enzymes exhibited lipase activity, and seven proteins showed polyester depolymerization activity against polylactic acid and polycaprolactone. Detailed biochemical characterization of four new enzymes revealed their substrate preference, whereas their catalytic residues were identified using site-directed mutagenesis. The crystal structure of the metal-ion dependent esterase MGS0169 from the amidohydrolase superfamily revealed a novel active site with a bound unknown ligand. Thus, activity-centered metagenomics has revealed diverse enzymes and novel families of microbial carboxylesterases, whose activity could not have been predicted using bioinformatics tools.
- Published
- 2017
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30. Biology of archaea from a novel family Cuniculiplasmataceae (Thermoplasmata) ubiquitous in hyperacidic environments.
- Author
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Golyshina OV, Kublanov IV, Tran H, Korzhenkov AA, Lünsdorf H, Nechitaylo TY, Gavrilov SN, Toshchakov SV, and Golyshin PN
- Subjects
- Archaeal Proteins genetics, Archaeal Proteins metabolism, California, Euryarchaeota classification, Euryarchaeota metabolism, Genomics methods, Geography, Hydrogen-Ion Concentration, Microscopy, Electron, Phylogeny, RNA, Ribosomal, 16S genetics, Sequence Analysis, DNA, Spain, Thermoplasmales metabolism, Thermoplasmales ultrastructure, United Kingdom, Acids chemistry, Ecosystem, Euryarchaeota genetics, Genome, Archaeal genetics, Thermoplasmales genetics
- Abstract
The order Thermoplasmatales (Euryarchaeota) is represented by the most acidophilic organisms known so far that are poorly amenable to cultivation. Earlier culture-independent studies in Iron Mountain (California) pointed at an abundant archaeal group, dubbed 'G-plasma'. We examined the genomes and physiology of two cultured representatives of a Family Cuniculiplasmataceae, recently isolated from acidic (pH 1-1.5) sites in Spain and UK that are 16S rRNA gene sequence-identical with 'G-plasma'. Organisms had largest genomes among Thermoplasmatales (1.87-1.94 Mbp), that shared 98.7-98.8% average nucleotide identities between themselves and 'G-plasma' and exhibited a high genome conservation even within their genomic islands, despite their remote geographical localisations. Facultatively anaerobic heterotrophs, they possess an ancestral form of A-type terminal oxygen reductase from a distinct parental clade. The lack of complete pathways for biosynthesis of histidine, valine, leucine, isoleucine, lysine and proline pre-determines the reliance on external sources of amino acids and hence the lifestyle of these organisms as scavengers of proteinaceous compounds from surrounding microbial community members. In contrast to earlier metagenomics-based assumptions, isolates were S-layer-deficient, non-motile, non-methylotrophic and devoid of iron-oxidation despite the abundance of methylotrophy substrates and ferrous iron in situ, which underlines the essentiality of experimental validation of bioinformatic predictions.
- Published
- 2016
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31. Estimating the success of enzyme bioprospecting through metagenomics: current status and future trends.
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Ferrer M, Martínez-Martínez M, Bargiela R, Streit WR, Golyshina OV, and Golyshin PN
- Subjects
- Bacteria chemistry, Bacteria genetics, Bioprospecting methods, Enzymes metabolism, Metagenomics methods, Bacteria enzymology, Bioprospecting trends, Enzymes chemistry, Enzymes genetics, Metagenomics trends
- Abstract
Recent reports have suggested that the establishment of industrially relevant enzyme collections from environmental genomes has become a routine procedure. Across the studies assessed, a mean number of approximately 44 active clones were obtained in an average size of approximately 53,000 clones tested using naïve screening protocols. This number could be significantly increased in shorter times when novel metagenome enzyme sequences obtained by direct sequencing are selected and subjected to high-throughput expression for subsequent production and characterization. The pre-screening of clone libraries by naïve screens followed by the pyrosequencing of the inserts allowed for a 106-fold increase in the success rate of identifying genes encoding enzymes of interest. However, a much longer time, usually on the order of years, is needed from the time of enzyme identification to the establishment of an industrial process. If the hit frequency for the identification of enzymes performing at high turnover rates under real application conditions could be increased while still covering a high natural diversity, the very expensive and time-consuming enzyme optimization phase would likely be significantly shortened. At this point, it is important to review the current knowledge about the success of fine-tuned naïve- and sequence-based screening protocols for enzyme selection and to describe the environments worldwide that have already been subjected to enzyme screen programmes through metagenomic tools. Here, we provide such estimations and suggest the current challenges and future actions needed before environmental enzymes can be successfully introduced into the market., (© 2015 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)
- Published
- 2016
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32. The novel extremely acidophilic, cell-wall-deficient archaeon Cuniculiplasma divulgatum gen. nov., sp. nov. represents a new family, Cuniculiplasmataceae fam. nov., of the order Thermoplasmatales.
- Author
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Golyshina OV, Lünsdorf H, Kublanov IV, Goldenstein NI, Hinrichs KU, and Golyshin PN
- Subjects
- Base Composition, Cell Wall chemistry, DNA, Archaeal genetics, Lipids chemistry, Mining, Molecular Sequence Data, RNA, Ribosomal, 16S genetics, Sequence Analysis, DNA, Spain, Thermoplasmales genetics, Thermoplasmales isolation & purification, United Kingdom, Vitamin K 2 chemistry, Phylogeny, Thermoplasmales classification, Water Microbiology
- Abstract
Two novel cell-wall-less, acidophilic, mesophilic, organotrophic and facultatively anaerobic archaeal strains were isolated from acidic streamers formed on the surfaces of copper-ore-containing sulfidic deposits in south-west Spain and North Wales, UK. Cells of the strains varied from 0.1 to 2 μm in size and were pleomorphic, with a tendency to form filamentous structures. The optimal pH and temperature for growth for both strains were 1.0-1.2 and 37-40 °C, with the optimal substrates for growth being beef extract (3 g l- 1) for strain S5T and beef extract with tryptone (3 and 1 g l- 1, respectively) for strain PM4. The lipid composition was dominated by intact polar lipids consisting of a glycerol dibiphytanyl glycerol tetraether (GDGT) core attached to predominantly glycosidic polar headgroups. In addition, free GDGT and small relative amounts of intact and core diether lipids were present. Strains S5T and PM4 possessed mainly menaquinones with minor fractions of thermoplasmaquinones. The DNA G+C content was 37.3 mol% in strain S5T and 37.16 mol% for strain PM4. A similarity matrix of 16S rRNA gene sequences (identical for both strains) showed their affiliation to the order Thermoplasmatales, with 73.9-86.3 % identity with sequences from members of the order with validly published names. The average nucleotide identity between genomes of the strains determined in silico was 98.75 %, suggesting, together with the 16S rRNA gene-based phylogenetic analysis, that the strains belong to the same species. A novel family, Cuniculiplasmataceae fam. nov., genus Cuniculiplasma gen. nov. and species Cuniculiplasma divulgatum sp. nov. are proposed based on the phylogenetic, chemotaxonomic analyses and physiological properties of the two isolates, S5T and PM4 ( = JCM 30641 = VKM B-2940). The type strain of Cuniculiplasma divulgatum is S5T ( = JCM 30642T = VKM B-2941T).
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- 2016
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33. Diversity of hydrolases from hydrothermal vent sediments of the Levante Bay, Vulcano Island (Aeolian archipelago) identified by activity-based metagenomics and biochemical characterization of new esterases and an arabinopyranosidase.
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Placido A, Hai T, Ferrer M, Chernikova TN, Distaso M, Armstrong D, Yakunin AF, Toshchakov SV, Yakimov MM, Kublanov IV, Golyshina OV, Pesole G, Ceci LR, and Golyshin PN
- Subjects
- Escherichia coli genetics, Gene Library, Genetic Testing, Hydrolases genetics, Islands, Italy, Substrate Specificity, Genetic Variation, Geologic Sediments microbiology, Hydrolases classification, Hydrolases metabolism, Hydrothermal Vents, Metagenome
- Abstract
A metagenomic fosmid expression library established from environmental DNA (eDNA) from the shallow hot vent sediment sample collected from the Levante Bay, Vulcano Island (Aeolian archipelago) was established in Escherichia coli. Using activity-based screening assays, we have assessed 9600 fosmid clones corresponding to approximately 350 Mbp of the cloned eDNA, for the lipases/esterases/lactamases, haloalkane and haloacid dehalogenases, and glycoside hydrolases. Thirty-four positive fosmid clones were selected from the total of 120 positive hits and sequenced to yield ca. 1360 kbp of high-quality assemblies. Fosmid inserts were attributed to the members of ten bacterial phyla, including Proteobacteria, Bacteroidetes, Acidobateria, Firmicutes, Verrucomicrobia, Chloroflexi, Spirochaetes, Thermotogae, Armatimonadetes, and Planctomycetes. Of ca. 200 proteins with high biotechnological potential identified therein, we have characterized in detail three distinct α/β-hydrolases (LIPESV12_9, LIPESV12_24, LIPESV12_26) and one new α-arabinopyranosidase (GLV12_5). All LIPESV12 enzymes revealed distinct substrate specificities tested against 43 structurally diverse esters and 4 p-nitrophenol carboxyl esters. Of 16 different glycosides tested, the GLV12_5 hydrolysed only p-nitrophenol-α-(L)-arabinopyranose with a high specific activity of about 2.7 kU/mg protein. Most of the α/β-hydrolases were thermophilic and revealed a high tolerance to, and high activities in the presence of, numerous heavy metal ions. Among them, the LIPESV12_24 was the best temperature-adapted, retaining its activity after 40 min of incubation at 90 °C. Furthermore, enzymes were active in organic solvents (e.g., >30% methanol). Both LIPESV12_24 and LIPESV12_26 had the GXSXG pentapeptides and the catalytic triads Ser-Asp-His typical to the representatives of carboxylesterases of EC 3.1.1.1.
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- 2015
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34. The aerobic respiratory chain of the acidophilic archaeon Ferroplasma acidiphilum: A membrane-bound complex oxidizing ferrous iron.
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Castelle CJ, Roger M, Bauzan M, Brugna M, Lignon S, Nimtz M, Golyshina OV, Giudici-Orticoni MT, and Guiral M
- Subjects
- Acids chemistry, Aerobiosis physiology, Archaeal Proteins chemistry, Cell Membrane chemistry, Electron Transport, Electron Transport Complex IV chemistry, Ferrous Compounds metabolism, Multiprotein Complexes chemistry, Operon, Oxidation-Reduction, Thermoplasmales growth & development, Thermoplasmales metabolism, Archaeal Proteins metabolism, Cell Membrane metabolism, Electron Transport Complex IV metabolism, Ferrous Compounds chemistry, Multiprotein Complexes metabolism, Oxygen metabolism, Thermoplasmales classification
- Abstract
The extremely acidophilic archaeon Ferroplasma acidiphilum is found in iron-rich biomining environments and is an important micro-organism in naturally occurring microbial communities in acid mine drainage. F. acidiphilum is an iron oxidizer that belongs to the order Thermoplasmatales (Euryarchaeota), which harbors the most extremely acidophilic micro-organisms known so far. At present, little is known about the nature or the structural and functional organization of the proteins in F. acidiphilum that impact the iron biogeochemical cycle. We combine here biochemical and biophysical techniques such as enzyme purification, activity measurements, proteomics and spectroscopy to characterize the iron oxidation pathway(s) in F. acidiphilum. We isolated two respiratory membrane protein complexes: a 850 kDa complex containing an aa3-type cytochrome oxidase and a blue copper protein, which directly oxidizes ferrous iron and reduces molecular oxygen, and a 150 kDa cytochrome ba complex likely composed of a di-heme cytochrome and a Rieske protein. We tentatively propose that both of these complexes are involved in iron oxidation respiratory chains, functioning in the so-called uphill and downhill electron flow pathways, consistent with autotrophic life. The cytochrome ba complex could possibly play a role in regenerating reducing equivalents by a reverse ('uphill') electron flow. This study constitutes the first detailed biochemical investigation of the metalloproteins that are potentially directly involved in iron-mediated energy conservation in a member of the acidophilic archaea of the genus Ferroplasma., (Copyright © 2015 Elsevier B.V. All rights reserved.)
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- 2015
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35. Isolation and characterization of novel lipases/esterases from a bovine rumen metagenome.
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Privé F, Newbold CJ, Kaderbhai NN, Girdwood SG, Golyshina OV, Golyshin PN, Scollan ND, and Huws SA
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- Animals, Cattle, Escherichia coli genetics, Esterases isolation & purification, Gene Expression, Hydrogen-Ion Concentration, Hydrolysis, Molecular Sequence Data, Sequence Analysis, DNA, Temperature, Transformation, Bacterial, Triglycerides metabolism, Esterases genetics, Esterases metabolism, Metagenome, Rumen microbiology
- Abstract
Improving the health beneficial fatty acid content of meat and milk is a major challenge requiring an increased understanding of rumen lipid metabolism. In this study, we isolated and characterized rumen bacterial lipases/esterases using functional metagenomics. Metagenomic libraries were constructed from DNA extracted from strained rumen fluid (SRF), solid-attached bacteria (SAB) and liquid-associated rumen bacteria (LAB), ligated into a fosmid vector and subsequently transformed into an Escherichia coli host. Fosmid libraries consisted of 7,744; 8,448; and 7,680 clones with an average insert size of 30 to 35 kbp for SRF, SAB and LAB, respectively. Transformants were screened on spirit blue agar plates containing tributyrin for lipase/esterase activity. Five SAB and four LAB clones exhibited lipolytic activity, and no positive clones were found in the SRF library. Fosmids from positive clones were pyrosequenced and twelve putative lipase/esterase genes and two phospholipase genes retrieved. Although the derived proteins clustered into diverse esterase and lipase families, a degree of novelty was seen, with homology ranging from 40 to 78% following BlastP searches. Isolated lipases/esterases exhibited activity against mostly short- to medium-chain substrates across a range of temperatures and pH. The function of these novel enzymes recovered in ruminal metabolism needs further investigation, alongside their potential industrial uses.
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- 2015
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36. Bacterial population and biodegradation potential in chronically crude oil-contaminated marine sediments are strongly linked to temperature.
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Bargiela R, Mapelli F, Rojo D, Chouaia B, Tornés J, Borin S, Richter M, Del Pozo MV, Cappello S, Gertler C, Genovese M, Denaro R, Martínez-Martínez M, Fodelianakis S, Amer RA, Bigazzi D, Han X, Chen J, Chernikova TN, Golyshina OV, Mahjoubi M, Jaouanil A, Benzha F, Magagnini M, Hussein E, Al-Horani F, Cherif A, Blaghen M, Abdel-Fattah YR, Kalogerakis N, Barbas C, Malkawi HI, Golyshin PN, Yakimov MM, Daffonchio D, and Ferrer M
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- Aerobiosis, Anaerobiosis, Bacteria genetics, Biodegradation, Environmental, Computer Simulation, Genes, Bacterial, Mediterranean Region, Metabolome, Metabolomics, Principal Component Analysis, RNA, Ribosomal, 16S genetics, Reproducibility of Results, Bacteria growth & development, Geologic Sediments microbiology, Petroleum microbiology, Petroleum Pollution, Temperature
- Abstract
Two of the largest crude oil-polluted areas in the world are the semi-enclosed Mediterranean and Red Seas, but the effect of chronic pollution remains incompletely understood on a large scale. We compared the influence of environmental and geographical constraints and anthropogenic forces (hydrocarbon input) on bacterial communities in eight geographically separated oil-polluted sites along the coastlines of the Mediterranean and Red Seas. The differences in community compositions and their biodegradation potential were primarily associated (P < 0.05) with both temperature and chemical diversity. Furthermore, we observed a link between temperature and chemical and biological diversity that was stronger in chronically polluted sites than in pristine ones where accidental oil spills occurred. We propose that low temperature increases bacterial richness while decreasing catabolic diversity and that chronic pollution promotes catabolic diversification. Our results further suggest that the bacterial populations in chronically polluted sites may respond more promptly in degrading petroleum after accidental oil spills.
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- 2015
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37. Microbial diversity and metabolic networks in acid mine drainage habitats.
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Méndez-García C, Peláez AI, Mesa V, Sánchez J, Golyshina OV, and Ferrer M
- Abstract
Acid mine drainage (AMD) emplacements are low-complexity natural systems. Low-pH conditions appear to be the main factor underlying the limited diversity of the microbial populations thriving in these environments, although temperature, ionic composition, total organic carbon, and dissolved oxygen are also considered to significantly influence their microbial life. This natural reduction in diversity driven by extreme conditions was reflected in several studies on the microbial populations inhabiting the various micro-environments present in such ecosystems. Early studies based on the physiology of the autochthonous microbiota and the growing success of omics-based methodologies have enabled a better understanding of microbial ecology and function in low-pH mine outflows; however, complementary omics-derived data should be included to completely describe their microbial ecology. Furthermore, recent updates on the distribution of eukaryotes and archaea recovered through sterile filtering (herein referred to as filterable fraction) in these environments demand their inclusion in the microbial characterization of AMD systems. In this review, we present a complete overview of the bacterial, archaeal (including filterable fraction), and eukaryotic diversity in these ecosystems, and include a thorough depiction of the metabolism and element cycling in AMD habitats. We also review different metabolic network structures at the organismal level, which is necessary to disentangle the role of each member of the AMD communities described thus far.
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- 2015
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38. Identification and characterization of carboxyl esterases of gill chamber-associated microbiota in the deep-sea shrimp Rimicaris exoculata by using functional metagenomics.
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Alcaide M, Tchigvintsev A, Martínez-Martínez M, Popovic A, Reva ON, Lafraya Á, Bargiela R, Nechitaylo TY, Matesanz R, Cambon-Bonavita MA, Jebbar M, Yakimov MM, Savchenko A, Golyshina OV, Yakunin AF, Golyshin PN, and Ferrer M
- Subjects
- Animals, Atlantic Ocean, Carboxylic Ester Hydrolases genetics, Carboxylic Ester Hydrolases metabolism, Enzyme Activators metabolism, Enzyme Inhibitors metabolism, Enzyme Stability, Hydrothermal Vents, Metagenomics, Molecular Sequence Data, Salts metabolism, Sequence Analysis, DNA, Sequence Homology, Amino Acid, Substrate Specificity, Temperature, Carboxylic Ester Hydrolases isolation & purification, Decapoda microbiology, Gills microbiology, Metagenome, Microbiota
- Abstract
The shrimp Rimicaris exoculata dominates the fauna in deep-sea hydrothermal vent sites along the Mid-Atlantic Ridge (depth, 2,320 m). Here, we identified and biochemically characterized three carboxyl esterases from microbial communities inhabiting the R. exoculata gill that were isolated by naive screens of a gill chamber metagenomic library. These proteins exhibit low to moderate identity to known esterase sequences (≤52%) and to each other (11.9 to 63.7%) and appear to have originated from unknown species or from genera of Proteobacteria related to Thiothrix/Leucothrix (MGS-RG1/RG2) and to the Rhodobacteraceae group (MGS-RG3). A library of 131 esters and 31 additional esterase/lipase preparations was used to evaluate the activity profiles of these enzymes. All 3 of these enzymes had greater esterase than lipase activity and exhibited specific activities with ester substrates (≤356 U mg(-1)) in the range of similar enzymes. MGS-RG3 was inhibited by salts and pressure and had a low optimal temperature (30°C), and its substrate profile clustered within a group of low-activity and substrate-restricted marine enzymes. In contrast, MGS-RG1 and MGS-RG2 were most active at 45 to 50°C and were salt activated and barotolerant. They also exhibited wider substrate profiles that were close to those of highly active promiscuous enzymes from a marine hydrothermal vent (MGS-RG2) and from a cold brackish lake (MGS-RG1). The data presented are discussed in the context of promoting the examination of enzyme activities of taxa found in habitats that have been neglected for enzyme prospecting; the enzymes found in these taxa may reflect distinct habitat-specific adaptations and may constitute new sources of rare reaction specificities., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)
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- 2015
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39. The environment shapes microbial enzymes: five cold-active and salt-resistant carboxylesterases from marine metagenomes.
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Tchigvintsev A, Tran H, Popovic A, Kovacic F, Brown G, Flick R, Hajighasemi M, Egorova O, Somody JC, Tchigvintsev D, Khusnutdinova A, Chernikova TN, Golyshina OV, Yakimov MM, Savchenko A, Golyshin PN, Jaeger KE, and Yakunin AF
- Subjects
- Aquatic Organisms genetics, Carboxylic Ester Hydrolases genetics, Enzyme Activators metabolism, Molecular Sequence Data, Potassium Chloride metabolism, Sequence Analysis, DNA, Sodium Chloride metabolism, Substrate Specificity, Aquatic Organisms enzymology, Carboxylic Ester Hydrolases isolation & purification, Carboxylic Ester Hydrolases metabolism, Cold Temperature, Metagenome
- Abstract
Most of the Earth's biosphere is cold and is populated by cold-adapted microorganisms. To explore the natural enzyme diversity of these environments and identify new carboxylesterases, we have screened three marine metagenome gene libraries for esterase activity. The screens identified 23 unique active clones, from which five highly active esterases were selected for biochemical characterization. The purified metagenomic esterases exhibited high activity against α-naphthyl and p-nitrophenyl esters with different chain lengths. All five esterases retained high activity at 5 °C indicating that they are cold-adapted enzymes. The activity of MGS0010 increased more than two times in the presence of up to 3.5 M NaCl or KCl, whereas the other four metagenomic esterases were inhibited to various degrees by these salts. The purified enzymes showed different sensitivities to inhibition by solvents and detergents, and the activities of MGS0010, MGS0105 and MGS0109 were stimulated three to five times by the addition of glycerol. Screening of purified esterases against 89 monoester substrates revealed broad substrate profiles with a preference for different esters. The metagenomic esterases also hydrolyzed several polyester substrates including polylactic acid suggesting that they can be used for polyester depolymerization. Thus, esterases from marine metagenomes are cold-adapted enzymes exhibiting broad biochemical diversity reflecting the environmental conditions where they evolved.
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- 2015
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40. Pressure adaptation is linked to thermal adaptation in salt-saturated marine habitats.
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Alcaide M, Stogios PJ, Lafraya Á, Tchigvintsev A, Flick R, Bargiela R, Chernikova TN, Reva ON, Hai T, Leggewie CC, Katzke N, La Cono V, Matesanz R, Jebbar M, Jaeger KE, Yakimov MM, Yakunin AF, Golyshin PN, Golyshina OV, Savchenko A, and Ferrer M
- Subjects
- Adaptation, Physiological, Ecosystem, Lakes, Mediterranean Sea, Salinity, Salts, Acclimatization, Aquatic Organisms enzymology, Bacteria enzymology, Hydrostatic Pressure, Seawater microbiology
- Abstract
The present study provides a deeper view of protein functionality as a function of temperature, salt and pressure in deep-sea habitats. A set of eight different enzymes from five distinct deep-sea (3040-4908 m depth), moderately warm (14.0-16.5°C) biotopes, characterized by a wide range of salinities (39-348 practical salinity units), were investigated for this purpose. An enzyme from a 'superficial' marine hydrothermal habitat (65°C) was isolated and characterized for comparative purposes. We report here the first experimental evidence suggesting that in salt-saturated deep-sea habitats, the adaptation to high pressure is linked to high thermal resistance (P value = 0.0036). Salinity might therefore increase the temperature window for enzyme activity, and possibly microbial growth, in deep-sea habitats. As an example, Lake Medee, the largest hypersaline deep-sea anoxic lake of the Eastern Mediterranean Sea, where the water temperature is never higher than 16°C, was shown to contain halopiezophilic-like enzymes that are most active at 70°C and with denaturing temperatures of 71.4°C. The determination of the crystal structures of five proteins revealed unknown molecular mechanisms involved in protein adaptation to poly-extremes as well as distinct active site architectures and substrate preferences relative to other structurally characterized enzymes., (© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.)
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- 2015
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41. Metagenomics as a Tool for Enzyme Discovery: Hydrolytic Enzymes from Marine-Related Metagenomes.
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Popovic A, Tchigvintsev A, Tran H, Chernikova TN, Golyshina OV, Yakimov MM, Golyshin PN, and Yakunin AF
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- Esterases isolation & purification, Gene Library, Lipase isolation & purification, Peptide Hydrolases isolation & purification, Metagenome, Metagenomics methods
- Abstract
This chapter discusses metagenomics and its application for enzyme discovery, with a focus on hydrolytic enzymes from marine metagenomic libraries. With less than one percent of culturable microorganisms in the environment, metagenomics, or the collective study of community genetics, has opened up a rich pool of uncharacterized metabolic pathways, enzymes, and adaptations. This great untapped pool of genes provides the particularly exciting potential to mine for new biochemical activities or novel enzymes with activities tailored to peculiar sets of environmental conditions. Metagenomes also represent a huge reservoir of novel enzymes for applications in biocatalysis, biofuels, and bioremediation. Here we present the results of enzyme discovery for four enzyme activities, of particular industrial or environmental interest, including esterase/lipase, glycosyl hydrolase, protease and dehalogenase.
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- 2015
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42. The gill chamber epibiosis of deep-sea shrimp Rimicaris exoculata: an in-depth metagenomic investigation and discovery of Zetaproteobacteria.
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Jan C, Petersen JM, Werner J, Teeling H, Huang S, Glöckner FO, Golyshina OV, Dubilier N, Golyshin PN, Jebbar M, and Cambon-Bonavita MA
- Subjects
- Animals, Carbon Cycle, Chemoautotrophic Growth, DNA, Bacterial genetics, Epsilonproteobacteria genetics, Gammaproteobacteria genetics, Hydrothermal Vents, In Situ Hybridization, Fluorescence, Photosynthesis, RNA, Ribosomal, 16S genetics, Sequence Analysis, DNA, Symbiosis, Decapoda microbiology, Epsilonproteobacteria metabolism, Gammaproteobacteria metabolism, Gills microbiology, Metagenomics
- Abstract
The gill chamber of deep-sea hydrothermal vent shrimp Rimicaris exoculata hosts a dense community of epibiotic bacteria dominated by filamentous Epsilonproteobacteria and Gammaproteobacteria. Using metagenomics on shrimp from the Rainbow hydrothermal vent field, we showed that both epibiont groups have the potential to grow autotrophically and oxidize reduced sulfur compounds or hydrogen with oxygen or nitrate. For carbon fixation, the Epsilonproteobacteria use the reductive tricarboxylic acid cycle, whereas the Gammaproteobacteria use the Calvin-Benson-Bassham cycle. Only the epsilonproteobacterial epibionts had the genes necessary for producing ammonium. This ability likely minimizes direct competition between epibionts and also broadens the spectrum of environmental conditions that the shrimp may successfully inhabit. We identified genes likely to be involved in shrimp-epibiont interactions, as well as genes for nutritional and detoxification processes that might benefit the host. Shrimp epibionts at Rainbow are often coated with iron oxyhydroxides, whose origin is intensely debated. We identified 16S rRNA sequences and functional genes affiliated with iron-oxidizing Zetaproteobacteria, which indicates that biological iron oxidation might play a role in forming these deposits. Fluorescence in situ hybridizations confirmed the presence of active Zetaproteobacteria in the R. exoculata gill chamber, thus providing the first evidence for a Zetaproteobacteria-invertebrate association., (© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.)
- Published
- 2014
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43. Halorhabdus tiamatea: proteogenomics and glycosidase activity measurements identify the first cultivated euryarchaeon from a deep-sea anoxic brine lake as potential polysaccharide degrader.
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Werner J, Ferrer M, Michel G, Mann AJ, Huang S, Juarez S, Ciordia S, Albar JP, Alcaide M, La Cono V, Yakimov MM, Antunes A, Taborda M, da Costa MS, Hai T, Glöckner FO, Golyshina OV, Golyshin PN, and Teeling H
- Subjects
- Adaptation, Physiological, Anaerobiosis physiology, Biological Evolution, Ecosystem, Enzyme Assays, Glycoside Hydrolases genetics, Glycoside Hydrolases metabolism, Halobacteriaceae classification, Halobacteriaceae enzymology, Indian Ocean, Lakes microbiology, Oxygen metabolism, Oxygen pharmacology, Phylogeny, Sodium Chloride, Utah, Genome, Archaeal, Halobacteriaceae genetics, Metagenomics, Polysaccharides metabolism, Salt Tolerance genetics, Water Microbiology
- Abstract
Euryarchaea from the genus Halorhabdus have been found in hypersaline habitats worldwide, yet are represented by only two isolates: Halorhabdus utahensis AX-2(T) from the shallow Great Salt Lake of Utah, and Halorhabdus tiamatea SARL4B(T) from the Shaban deep-sea hypersaline anoxic lake (DHAL) in the Red Sea. We sequenced the H. tiamatea genome to elucidate its niche adaptations. Among sequenced archaea, H. tiamatea features the highest number of glycoside hydrolases, the majority of which were expressed in proteome experiments. Annotations and glycosidase activity measurements suggested an adaptation towards recalcitrant algal and plant-derived hemicelluloses. Glycosidase activities were higher at 2% than at 0% or 5% oxygen, supporting a preference for low-oxygen conditions. Likewise, proteomics indicated quinone-mediated electron transport at 2% oxygen, but a notable stress response at 5% oxygen. Halorhabdus tiamatea furthermore encodes proteins characteristic for thermophiles and light-dependent enzymes (e.g. bacteriorhodopsin), suggesting that H. tiamatea evolution was mostly not governed by a cold, dark, anoxic deep-sea habitat. Using enrichment and metagenomics, we could demonstrate presence of similar glycoside hydrolase-rich Halorhabdus members in the Mediterranean DHAL Medee, which supports that Halorhabdus species can occupy a distinct niche as polysaccharide degraders in hypersaline environments., (© 2014 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)
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- 2014
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44. Microbial stratification in low pH oxic and suboxic macroscopic growths along an acid mine drainage.
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Méndez-García C, Mesa V, Sprenger RR, Richter M, Diez MS, Solano J, Bargiela R, Golyshina OV, Manteca Á, Ramos JL, Gallego JR, Llorente I, Martins dos Santos VA, Jensen ON, Peláez AI, Sánchez J, and Ferrer M
- Subjects
- Archaea genetics, Archaea growth & development, Archaea metabolism, Bacteria genetics, Bacteria growth & development, Bacteria metabolism, Biofilms, Genomics, Hydrogen-Ion Concentration, Iron analysis, Proteome, Rivers, Sulfides analysis, Archaea classification, Bacteria classification, Mining
- Abstract
Macroscopic growths at geographically separated acid mine drainages (AMDs) exhibit distinct populations. Yet, local heterogeneities are poorly understood. To gain novel mechanistic insights into this, we used OMICs tools to profile microbial populations coexisting in a single pyrite gallery AMD (pH ∼2) in three distinct compartments: two from a stratified streamer (uppermost oxic and lowermost anoxic sediment-attached strata) and one from a submerged anoxic non-stratified mat biofilm. The communities colonising pyrite and those in the mature formations appear to be populated by the greatest diversity of bacteria and archaea (including 'ARMAN' (archaeal Richmond Mine acidophilic nano-organisms)-related), as compared with the known AMD, with ∼44.9% unclassified sequences. We propose that the thick polymeric matrix may provide a safety shield against the prevailing extreme condition and also a massive carbon source, enabling non-typical acidophiles to develop more easily. Only 1 of 39 species were shared, suggesting a high metabolic heterogeneity in local microenvironments, defined by the O2 concentration, spatial location and biofilm architecture. The suboxic mats, compositionally most similar to each other, are more diverse and active for S, CO2, CH4, fatty acid and lipopolysaccharide metabolism. The oxic stratum of the streamer, displaying a higher diversity of the so-called 'ARMAN'-related Euryarchaeota, shows a higher expression level of proteins involved in signal transduction, cell growth and N, H2, Fe, aromatic amino acids, sphingolipid and peptidoglycan metabolism. Our study is the first to highlight profound taxonomic and functional shifts in single AMD formations, as well as new microbial species and the importance of H2 in acidic suboxic macroscopic growths.
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- 2014
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45. Microbial life in the Lake Medee, the largest deep-sea salt-saturated formation.
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Yakimov MM, La Cono V, Slepak VZ, La Spada G, Arcadi E, Messina E, Borghini M, Monticelli LS, Rojo D, Barbas C, Golyshina OV, Ferrer M, Golyshin PN, and Giuliano L
- Subjects
- Betaine metabolism, Betaine pharmacology, Bicarbonates chemistry, Biodiversity, Ecosystem, Epsilonproteobacteria, Mediterranean Region, Molecular Sequence Data, RNA, Ribosomal, 16S genetics, Saline Solution, Hypertonic, Salt Tolerance, Seawater chemistry, Sodium Chloride, Water Microbiology, Alphaproteobacteria classification, Alphaproteobacteria genetics, Alphaproteobacteria metabolism, Gammaproteobacteria classification, Gammaproteobacteria genetics, Gammaproteobacteria metabolism, Halobacteriales classification, Halobacteriales genetics, Halobacteriales metabolism, Lakes microbiology
- Abstract
Deep-sea hypersaline anoxic lakes (DHALs) of the Eastern Mediterranean represent some of the most hostile environments on our planet. We investigated microbial life in the recently discovered Lake Medee, the largest DHAL found to-date. Medee has two unique features: a complex geobiochemical stratification and an absence of chemolithoautotrophic Epsilonproteobacteria, which usually play the primary role in dark bicarbonate assimilation in DHALs interfaces. Presumably because of these features, Medee is less productive and exhibits reduced diversity of autochthonous prokaryotes in its interior. Indeed, the brine community almost exclusively consists of the members of euryarchaeal MSBL1 and bacterial KB1 candidate divisions. Our experiments utilizing cultivation and [(14)C]-assimilation, showed that these organisms at least partially rely on reductive cleavage of osmoprotectant glycine betaine and are engaged in trophic cooperation. These findings provide novel insights into how prokaryotic communities can adapt to salt-saturated conditions and sustain active metabolism at the thermodynamic edge of life.
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- 2013
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46. Single residues dictate the co-evolution of dual esterases: MCP hydrolases from the α/β hydrolase family.
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Alcaide M, Tornés J, Stogios PJ, Xu X, Gertler C, Di Leo R, Bargiela R, Lafraya A, Guazzaroni ME, López-Cortés N, Chernikova TN, Golyshina OV, Nechitaylo TY, Plumeier I, Pieper DH, Yakimov MM, Savchenko A, Golyshin PN, and Ferrer M
- Subjects
- Amino Acid Sequence, Bacterial Proteins genetics, Burkholderia chemistry, Crystallography, X-Ray, Esterases genetics, Hydrolases genetics, Molecular Sequence Data, Proteobacteria chemistry, Pseudomonas chemistry, Pseudomonas genetics, Sphingomonas chemistry, Sphingomonas genetics, Bacterial Proteins chemistry, Esterases chemistry, Evolution, Molecular, Hydrolases chemistry
- Abstract
Several members of the C-C MCP (meta-cleavage product) hydrolase family demonstrate an unusual ability to hydrolyse esters as well as the MCPs (including those from mono- and bi-cyclic aromatics). Although the molecular mechanisms responsible for such substrate promiscuity are starting to emerge, the full understanding of these complex enzymes is far from complete. In the present paper, we describe six distinct α/β hydrolases identified through genomic approaches, four of which demonstrate the unprecedented characteristic of activity towards a broad spectrum of substrates, including p-nitrophenyl, halogenated, fatty acyl, aryl, glycerol, cinnamoyl and carbohydrate esters, lactones, 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate and 2-hydroxy-6-oxohepta-2,4-dienoate. Using structural analysis and site-directed mutagenesis we have identified the three residues (Ser32, Val130 and Trp144) that determine the unusual substrate specificity of one of these proteins, CCSP0084. The results may open up new research avenues into comparative catalytic models, structural and mechanistic studies, and biotechnological applications of MCP hydrolases.
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- 2013
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47. Genome Sequence of Thalassolituus oleivorans MIL-1 (DSM 14913T).
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Golyshin PN, Werner J, Chernikova TN, Tran H, Ferrer M, Yakimov MM, Teeling H, and Golyshina OV
- Abstract
Thalassolituus oleivorans is one of the most prevalent marine gammaproteobacteria in microbial communities, emerging after oil spills in coastal, estuarine, and surface seawaters. Here, we present the assembled genome of strain T. oleivorans MIL-1 (DSM 14913(T)), which is 3,920,328 bp with a G+C content of 46.6%.
- Published
- 2013
- Full Text
- View/download PDF
48. Genome sequence and functional genomic analysis of the oil-degrading bacterium Oleispira antarctica.
- Author
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Kube M, Chernikova TN, Al-Ramahi Y, Beloqui A, Lopez-Cortez N, Guazzaroni ME, Heipieper HJ, Klages S, Kotsyurbenko OR, Langer I, Nechitaylo TY, Lünsdorf H, Fernández M, Juárez S, Ciordia S, Singer A, Kagan O, Egorova O, Petit PA, Stogios P, Kim Y, Tchigvintsev A, Flick R, Denaro R, Genovese M, Albar JP, Reva ON, Martínez-Gomariz M, Tran H, Ferrer M, Savchenko A, Yakunin AF, Yakimov MM, Golyshina OV, Reinhardt R, and Golyshin PN
- Subjects
- Alcanivoraceae genetics, Alcanivoraceae metabolism, Amino Acid Sequence, Bacterial Proteins genetics, Biodegradation, Environmental, Chromosome Mapping, Cold Temperature, Gammaproteobacteria classification, Gammaproteobacteria metabolism, Gene Transfer, Horizontal, Genome Size, Industrial Oils, Molecular Chaperones genetics, Molecular Sequence Data, Phylogeny, Protein Folding, Salinity, Sequence Analysis, DNA, Adaptation, Physiological genetics, Bacterial Proteins chemistry, Gammaproteobacteria genetics, Genome, Bacterial, Molecular Chaperones chemistry
- Abstract
Ubiquitous bacteria from the genus Oleispira drive oil degradation in the largest environment on Earth, the cold and deep sea. Here we report the genome sequence of Oleispira antarctica and show that compared with Alcanivorax borkumensis--the paradigm of mesophilic hydrocarbonoclastic bacteria--O. antarctica has a larger genome that has witnessed massive gene-transfer events. We identify an array of alkane monooxygenases, osmoprotectants, siderophores and micronutrient-scavenging pathways. We also show that at low temperatures, the main protein-folding machine Cpn60 functions as a single heptameric barrel that uses larger proteins as substrates compared with the classical double-barrel structure observed at higher temperatures. With 11 protein crystal structures, we further report the largest set of structures from one psychrotolerant organism. The most common structural feature is an increased content of surface-exposed negatively charged residues compared to their mesophilic counterparts. Our findings are relevant in the context of microbial cold-adaptation mechanisms and the development of strategies for oil-spill mitigation in cold environments.
- Published
- 2013
- Full Text
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49. Microbial β-glucosidases from cow rumen metagenome enhance the saccharification of lignocellulose in combination with commercial cellulase cocktail.
- Author
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Del Pozo MV, Fernández-Arrojo L, Gil-Martínez J, Montesinos A, Chernikova TN, Nechitaylo TY, Waliszek A, Tortajada M, Rojas A, Huws SA, Golyshina OV, Newbold CJ, Polaina J, Ferrer M, and Golyshin PN
- Abstract
Background: A complete saccharification of plant polymers is the critical step in the efficient production of bio-alcohols. Beta-glucosidases acting in the degradation of intermediate gluco-oligosaccharides produced by cellulases limit the yield of the final product., Results: In the present work, we have identified and then successfully cloned, expressed, purified and characterised 4 highly active beta-glucosidases from fibre-adherent microbial community from the cow rumen. The enzymes were most active at temperatures 45-55°C and pH 4.0-7.0 and exhibited high affinity and activity towards synthetic substrates such as p-nitrophenyl-beta-D-glucopyranoside (pNPbetaG) and pNP-beta-cellobiose, as well as to natural cello-oligosaccharides ranging from cellobiose to cellopentaose. The apparent capability of the most active beta-glucosidase, herein named LAB25g2, was tested for its ability to improve, at low dosage (31.25 units g-1 dry biomass, using pNPbetaG as substrate), the hydrolysis of pre-treated corn stover (dry matter content of 20%; 350 g glucan kg-1 dry biomass) in combination with a beta-glucosidase-deficient commercial Trichoderma reseei cellulase cocktail (5 units g-1 dry biomass in the basis of pNPbetaG). LAB25g2 increased the final hydrolysis yield by a factor of 20% (44.5 ± 1.7% vs. 34.5 ± 1.5% in control conditions) after 96-120 h as compared to control reactions in its absence or in the presence of other commercial beta-glucosidase preparations. The high stability (half-life higher than 5 days at 50°C and pH 5.2) and 2-38000 fold higher (as compared with reported beta-glucosidases) activity towards cello-oligosaccharides may account for its performance in supplementation assays., Conclusions: The results suggest that beta-glucosidases from yet uncultured bacteria from animal digestomes may be of a potential interest for biotechnological processes related to the effective bio-ethanol production in combination with low dosage of commercial cellulases.
- Published
- 2012
- Full Text
- View/download PDF
50. Functional metagenomics unveils a multifunctional glycosyl hydrolase from the family 43 catalysing the breakdown of plant polymers in the calf rumen.
- Author
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Ferrer M, Ghazi A, Beloqui A, Vieites JM, López-Cortés N, Marín-Navarro J, Nechitaylo TY, Guazzaroni ME, Polaina J, Waliczek A, Chernikova TN, Reva ON, Golyshina OV, and Golyshin PN
- Subjects
- Animals, Cattle, Female, Glycoside Hydrolases metabolism, Models, Molecular, Phylogeny, Protein Conformation, Glycoside Hydrolases genetics, Lignin metabolism, Metagenomics, Plants metabolism, Polymers metabolism, Rumen enzymology
- Abstract
Microbial communities from cow rumen are known for their ability to degrade diverse plant polymers at high rates. In this work, we identified 15 hydrolases through an activity-centred metagenome analysis of a fibre-adherent microbial community from dairy cow rumen. Among them, 7 glycosyl hydrolases (GHs) and 1 feruloyl esterase were successfully cloned, expressed, purified and characterised. The most striking result was a protein of GH family 43 (GHF43), hereinafter designated as R_09-02, which had characteristics very distinct from the other proteins in this family with mono-functional β-xylosidase, α-xylanase, α-L-arabinase and α-L-arabinofuranosidase activities. R_09-02 is the first multifunctional enzyme to exhibit β-1,4 xylosidase, α-1,5 arabinofur(pyr)anosidase, β-1,4 lactase, α-1,6 raffinase, α-1,6 stachyase, β-galactosidase and α-1,4 glucosidase activities. The R_09-02 protein appears to originate from the chromosome of a member of Clostridia, a class of phylum Firmicutes, members of which are highly abundant in ruminal environment. The evolution of R_09-02 is suggested to be driven from the xylose- and arabinose-specific activities, typical for GHF43 members, toward a broader specificity to the glucose- and galactose-containing components of lignocellulose. The apparent capability of enzymes from the GHF43 family to utilise xylose-, arabinose-, glucose- and galactose-containing oligosaccharides has thus far been neglected by, or could not be predicted from, genome and metagenome sequencing data analyses. Taking into account the abundance of GHF43-encoding gene sequences in the rumen (up to 7% of all GH-genes) and the multifunctional phenotype herein described, our findings suggest that the ecological role of this GH family in the digestion of ligno-cellulosic matter should be significantly reconsidered.
- Published
- 2012
- Full Text
- View/download PDF
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