Discovery and Characterization of a New Cold-Active Protease From an Extremophilic Bacterium via Comparative Genome Analysis and in vitro Expression

Following a screening of Antarctic glacier forefield-bacteria for novel cold-active enzymes, a psychrophilic strain Psychrobacter sp. 94-6PB was selected for further characterization of enzymatic activities. The strain produced lipases and proteases in the temperature range of 4–18°C. The coding sequence of an extracellular serine-protease was then identified via comparative analysis across Psychrobacter sp. genomes, PCR-amplified in our strain 94-6PB and expressed in the heterologous host E. coli. The purified enzyme (80 kDa) resulted to be a cold-active alkaline protease, performing best at temperatures of 20–30°C and pH 7-9. It was stable in presence of common inhibitors [β-mercaptoethanol (β-ME), dithiothreitol (DTT), urea, phenylmethylsulfonyl fluoride (PMSF) and ethylenediaminetetraacetic acid (EDTA)] and compatible with detergents and surfactants (Tween 20, Tween 80, hydrogen peroxide and Triton X-100). Because of these properties, the P94-6PB protease may be suitable for use in a new generation of laundry products for cold washing. Furthermore, we assessed the microdiversity of this enzyme in Psychrobacter organisms from different cold habitats and found several gene clusters that correlated with specific ecological niches. We then discussed the role of habitat specialization in shaping the biodiversity of proteins and enzymes and anticipate far-reaching implications for the search of novel variants of biotechnological products

Characterisation of East Siberian paleodiversity based on ancient DNA analyses of the Batagay megaslump exposure

With the ongoing Arctic warming, permafrost thaw accelerated during the last decade as much as it is now a global concern for biodiversity loss, food webs and biogeochemical cycling. This rapid permafrost degradation forms features such as massive retrogressive thaw slumps that give access to exceptional records for Quaternary biodiversity change investigations. The Batagay megaslump located in northern Yakutia, East Siberia, is the world’s largest thawslump known to date, and along its ~55m high headwall it gives access to Late and Mid Pleistocene permafrost deposits up to more than 500 kyrs in age. During an expedition to this unique site in 2017, sediment samples were collected with ages from more than 500 kyrs to modern time for the analysis of ancient DNA (aDNA). Our aim is to characterise the biodiversity and changes over geological timescales of this region in East Siberia. Using the aDNA extracted from these ancient environmental samples, we first performed a metabarcoding analysis (chloroplast trnL) to investigate past vegetation composition. We then performed a shotgun metagenomic analysis, which enabled a much higher depth of sequence data and allowed us to access the entire biodiversity, from Eukaryotes to Prokaryotes, Archaea and Viruses. This approach opened up new horizons, making it possible not only to investigate biodiversity composition and changes but also to infer on potential interactions across taxa and kingdoms. Both methods together allowed comparison and ensured robustness of the results obtained. We present here one of the very first studies done on the global, past and modern, biodiversity of permafrost regions which holds an enormous potential to reveal new insights into the evolution of this fragile ecosystem

The microbiome of ancient ice wedges in the Muostakh ‘disappearing island’

Climate change-driven thermal erosion makes Muostakh Island in the southern Laptev Sea (70°35´ N, 130° 0´ E) a very fragile ecosystem of the Arctic. Thus, understanding its biodiversity, the changes and loss in response to climate is a timely and pressing scientific objective. Here, we characterize the microbiome associated with several ice wedges covering the past ~45,000 years of climate/ecosystem history. Ice wedges are a specific feature in the northern permafrost landscapes. They develop seasonally by spring-melting of snow that runs through permafrost contraction cracks, accumulates and creates ice formations in the wintertime through congelifraction. Such environment offers ideal conditions for the preservation of microbial cells and DNA over geological time. Our work tackles four main research aspects, requiring an interdisciplinary approach with synergies between microbial ecology, geo- and paleo-sciences. First, we characterize the ice wedge mineral composition as an environmental micro-niche. Second, we analyze the biodiversity of the microbial communities via shotgun metagenomics of the ancient DNA (aDNA) extracted from the ice wedges. Third, we investigate the biomass content by recovering and enumerating microbial cells present in the ice wedges. In addition, we apply infrared spectroscopy to obtain cellular fingerprints that can serve as biomarkers. Finally, we assess the physiological state of microorganisms using stable isotope probing (SIP) experiments in microcosms that reproduce the environmental conditions (subzero temperature and anoxic conditions). By integrating microbial biodiversity with activity and environmental context, this study will provide valuable new insights into Muostakh’s ice wedge microbiome and the dynamics underlying its changes over time and climatic conditions

Discovery and Characterization of a New Cold-Active Protease From an Extremophilic Bacterium via Comparative Genome Analysis and in vitro Expression

Following a screening of Antarctic glacier forefield-bacteria for novel cold-active enzymes, a psychrophilic strain Psychrobacter sp. 94-6PB was selected for further characterization of enzymatic activities. The strain produced lipases and proteases in the temperature range of 4–18°C. The coding sequence of an extracellular serine-protease was then identified via comparative analysis across Psychrobacter sp. genomes, PCR-amplified in our strain 94-6PB and expressed in the heterologous host E. coli. The purified enzyme (80 kDa) resulted to be a cold-active alkaline protease, performing best at temperatures of 20–30°C and pH 7-9. It was stable in presence of common inhibitors [β-mercaptoethanol (β-ME), dithiothreitol (DTT), urea, phenylmethylsulfonyl fluoride (PMSF) and ethylenediaminetetraacetic acid (EDTA)] and compatible with detergents and surfactants (Tween 20, Tween 80, hydrogen peroxide and Triton X-100). Because of these properties, the P94-6PB protease may be suitable for use in a new generation of laundry products for cold washing. Furthermore, we assessed the microdiversity of this enzyme in Psychrobacter organisms from different cold habitats and found several gene clusters that correlated with specific ecological niches. We then discussed the role of habitat specialization in shaping the biodiversity of proteins and enzymes and anticipate far-reaching implications for the search of novel variants of biotechnological products.EC/H2020/657473/EU/Life at its Extremes: Biodiversity and Activity of Microorganisms in deep Permafrost/BioFrostEC/H2020/764591/EU/Synthetic Circuits for Robust Orthogonal Production/SynCropDFG, 5472008, SPP 1158: Bereich Infrastruktur - Antarktisforschung mit vergleichenden Untersuchungen in arktischen Eisgebiete

Investigation of bacterial biosurfactant production for industrial use

This study presents part of a multifaceted study of microbial biosurfactants and their industrial potential. The first step was the assembly of a microbial culture collection of a variety of biosurfactant-producing organisms with the choice of biosurfactant types informed by the industrial partner. The choice was based on two main criteria, the ease of product recovery and its final yield. Two groups of glycolipid biosurfactants were selected for further study: rhamnolipids produced by Pseudomonas aeruginosa and sophorolipids by Candida spp. Efforts to manipulate the biosurfactant chemical profiles by changing the cultivation media (carbon source in particular) and conditions in shake-flasks, demonstrated that there is only a limited possibility for changing the biosurfactant composition. This raised the question of the extent to which biosurfactant production is constrained by genetic determinants? To overcome the limitations of the flask-scale production, selected P. aeruginosa and Candida strains were used in bioreactors, and rhamnolipids and sophorolipids were synthesised in quantities sufficient for extraction and purification. The purified biosurfactants were used by the project partners for further characterisation and formulation in trial industrial products. Rhamnolipid yields were approximately 10 g/L whilst sophorolipid production exceeded 100 g/L. Detailed examination of the orcinol assay, which is widely used for the determination of rhamnolipid yields, showed that the method is flawed and provides an overestimate of yield when compared to quantification following extraction and purification. The culture approach had demonstrated the restricted possibilities for manipulating rhamnolipid production profile in P. aeruginosa and therefore a wider range of strains from different environmental niches were selected for genetic analysis. The aim of this part of the investigation was to establish the extent of natural gene variation which could be exploited for customised biosurfactant production. The comparative analysis of the rhl genes, coding the factors involved in rhamnolipid biosynthesis, was carried out on different P. aeruginosa strains isolated from water, soil and including pathogenic strains infecting cystic fibrosis patients. The extent of the gene sequence diversity resulted < 5%, which indicated that the rhl genes are conserved and are part of the core genome of P. aeruginosa. The single polymorphisms that occurred on the gene sequences, which gave rise to several variants, revealed no clear effect on the phenotype but appeared rather random. These variants showed also no specific correlation with different habitats. The analysis of the codon usage further supported the confidence in the highly conservative nature of the rhl genes. Most amino acids were encoded by highly preferred codons, some of which were selected upon the optimal translational efficiency, some others were instead determined by the high GC content of P. aeruginosa genome. On the whole, our data correlated well in showing that rhamnolipid biosurfactants produced by P. aeruginosa are conserved in their structure and profile as well in the genetic makeup. As involved in many important biological activities under a variety of environmental conditions, rhamnolipids developed as highly fit molecules. In the light of this, it appears that the likelihood that isolates of P. aeruginosa displaying unusual rhamnolipid profiles and features occurring naturally would be limited. Alternative routes for the development of industrially customised rhamnolipid biosurfactants are suggested as either the genetic manipulation of P. aeruginosa or the screening of rhamnolipid-producing organisms closely related.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Paleometagenomics reveals environmental microbiome response to vegetation changes in northern Siberia over the millennia

The study of environmental ancient DNA provides us with the unique opportunity to link environmental with ecosystem change over a millennial timescale. Paleorecords such as lake sediments contain genetic pools of past living organisms that are a valuable source of information to reconstruct how ecosystems were and how they changed in response to climate in the past. Here, we report on paleometagenomics of a sedimentary record in northern Siberia covering the past 6700 years. We integrated taxonomic with functional gene analysis, which enabled to shed light not only on community compositions but also on eco-physiological adaptations and ecosystem functioning. We reconstructed the presence of an open boreal forest 6700 years ago that over time was gradually replaced by tundra. This vegetation change had major consequences on the environmental microbiome, primarily enriching bacterial and archaeal ammonia oxidizers (e.g., Nitrospira, Nitrosopumilus, and Ca. Nitrosocosmicus) in the tundra ecosystem. We identified a core microbiome conserved through time and largely consisting of heterotrophic bacteria of the Bacteroidetes phylum (e.g., Mucilaginibacter) harboring numerous functional genes for degradation of plant-biomass and abiotic and biotic stress resistance. Archaea were also a key functional guild, involved in nitrogen and carbon cycling, not only methanogenesis but possibly also degradation of plant material via enzymes such as cellulases and amylases. Overall, the paleo-perspective offered by our study can have a profound impact on modern climate change biology, by helping to explain and predict the ecological interplay among multiple ecosystem levels based on past experiences

Pleistocene glacial and interglacial ecosystems inferred from ancient DNA analyses of permafrost sediments from Batagay megaslump, East Siberia

Pronounced glacial and interglacial climate cycles characterized northern ecosystems during the Pleistocene. Our understanding of the resultant community transformations and past ecological interactions strongly depends on the taxa found in fossil assemblages. Here, we present a shotgun metagenomic analysis of sedimentary ancient DNA (sedaDNA) to infer past ecosystem-wide biotic composition (from viruses to megaherbivores) from the Middle and Late Pleistocene at the Batagay megaslump, East Siberia. The shotgun DNA records of past vegetation composition largely agree with pollen and plant metabarcoding data from the same samples. Interglacial ecosystems at Batagay attributed to Marine Isotope Stage (MIS) 17 and MIS 7 were characterized by forested vegetation (Pinus, Betula, Alnus) and open grassland. The microbial and fungal communities indicate strong activity related to soil decomposition, especially during MIS17. The local landscape likely featured more open, herb-dominated areas, and the vegetation mosaic supported birds and small omnivorous mammals. Parts of the area were intermittently/partially flooded as suggested by the presence of water-dependent taxa. During MIS 3, the sampled ecosystems are identified as cold-temperate, periodically flooded grassland. Diverse megafauna (Mammuthus, Equus, Coelodonta) coexisted with small mammals (rodents). The MIS 2 ecosystems existed under harsher conditions, as suggested by the presence of cold-adapted herbaceous taxa. Typical Pleistocene megafauna still inhabited the area. The new approach, in which shotgun sequencing is supported by metabarcoding and pollen data, enables the investigation of community composition changes across a broad range of taxonomic groups and inferences about trophic interactions and aspects of soil microbial ecology

Pleistocene glacial and interglacial ecosystems inferred from ancient DNA analyses of permafrost sediments from Batagay megaslump, East Siberia

Pronounced glacial and interglacial climate cycles characterized northern ecosystems during the Pleistocene. Our understanding of the resultant community transformations and past ecological interactions strongly depends on the taxa found in fossil assemblages. Here, we present a shotgun metagenomic analysis of sedimentary ancient DNA (sedaDNA) to infer past ecosystem-wide biotic composition (from viruses to megaherbivores) from the Middle and Late Pleistocene at the Batagay megaslump, East Siberia. The shotgun DNA records of past vegetation composition largely agree with pollen and plant metabarcoding data from the same samples. Interglacial ecosystems at Batagay attributed to Marine Isotope Stage (MIS) 17 and MIS 7 were characterized by forested vegetation (Pinus, Betula, Alnus) and open grassland. The microbial and fungal communities indicate strong activity related to soil decomposition, especially during MIS17. The local landscape likely featured more open, herb-dominated areas, and the vegetation mosaic supported birds and small omnivorous mammals. Parts of the area were intermittently/partially flooded as suggested by the presence of water-dependent taxa. During MIS 3, the sampled ecosystems are identified as cold-temperate, periodically flooded grassland. Diverse megafauna (Mammuthus, Equus, Coelodonta) coexisted with small mammals (rodents). The MIS 2 ecosystems existed under harsher conditions, as suggested by the presence of cold-adapted herbaceous taxa. Typical Pleistocene megafauna still inhabited the area. The new approach, in which shotgun sequencing is supported by metabarcoding and pollen data, enables the investigation of community composition changes across a broad range of taxonomic groups and inferences about trophic interactions and aspects of soil microbial ecology.</p

Directed microbial biosynthesis of deuterated biosurfactants and potential future application to other bioactive molecules

Deuterated rhamnolipids were produced using strain AD7 of Pseudomonas aeruginosa, which was progressively adapted to increasing levels of deuterium in D(2)O and carbon substrates. Fourteen different deuterated rhamnolipid structures, including structural isomers, were produced which is similar to normal protonated structures. There were two main products monorhamnolipid Rha-C(10)-C(10) and dirhamnolipid Rha(2)-C(10)-C(10). The levels of deuteration varied from 16% with 25% D(2)O + h-glycerol to 90% with 100% D(2)O + d-glycerol. When d-tetradecane was used with H(2)O, virtually all the deuterium appeared in the lipid chains while using h-tetradecane + D(2)O led to the majority of deuterium in the sugars. The adaptation to growth in deuterium appeared to be metabolic since no genetic changes could be found in the key rhamnolipid biosynthetic genes, the rhamnosyl transferases RhlB and RhlC. Deuterated sophorolipids were similarly produced using Candida bombicola and Candida apicola although in this case, no adaptation process was necessary. Up to 40 different sophorolipids were produced by these yeasts. However, unlike the rhamnolipids, use of D(2)O did not lead to any deuteration of the lipid chains, but direct incorporation into the lipid was achieved using d-isostearic acid. The results from these experiments show the feasibility of producing deuterated bioactive compounds from microorganisms coupled with the possibility of manipulating the pattern of labelling through judicious use of different deuterated substrates