Polysphaeroides filiformis, a proterozoic cyanobacterial microfossil and implications for cyanobacteria evolution.

Deciphering the fossil record of cyanobacteria is crucial to understand their role in the chemical and biological evolution of the early Earth. They profoundly modified the redox conditions of early ecosystems more than 2.4 Ga ago, the age of the Great Oxidation Event (GOE), and provided the ancestor of the chloroplast by endosymbiosis, leading the diversification of photosynthetic eukaryotes. Here, we analyze the morphology, ultrastructure, chemical composition, and metals distribution of Polysphaeroides filiformis from the 1040-1006 Ma Mbuji-Mayi Supergroup (DR Congo). We evidence trilaminar and bilayered ultrastructures for the sheath and the cell wall, respectively, and the preservation of Ni-tetrapyrrole moieties derived from chlorophyll in intracellular inclusions. This approach allows an unambiguous interpretation of P. filiformis as a branched and multiseriate photosynthetic cyanobacterium belonging to the family of Stigonemataceae. It also provides a possible minimum age for the emergence of multiseriate true branching nitrogen-fixing and probably heterocytous cyanobacteria.

Oldest thylakoids in fossil cells directly evidence oxygenic photosynthesis.

Today oxygenic photosynthesis is unique to cyanobacteria and their plastid relatives within eukaryotes. Although its origin before the Great Oxidation Event is still debated1-4, the accumulation of O2 profoundly modified the redox chemistry of the Earth and the evolution of the biosphere, including complex life. Understanding the diversification of cyanobacteria is thus crucial to grasping the coevolution of our planet and life, but their early fossil record remains ambiguous5. Extant cyanobacteria include the thylakoid-less Gloeobacter-like group and the remainder of cyanobacteria that acquired thylakoid membranes6,7. The timing of this divergence is indirectly estimated at between 2.7 and 2.0 billion years ago (Ga) based on molecular clocks and phylogenies8-11 and inferred from the earliest undisputed fossil record of Eoentophysalis belcherensis, a 2.018-1.854 Ga pleurocapsalean cyanobacterium preserved in silicified stromatolites12,13. Here we report the oldest direct evidence of thylakoid membranes in a parallel-to-contorted arrangement within the enigmatic cylindrical microfossils Navifusa majensis from the McDermott Formation, Tawallah Group, Australia (1.78-1.73 Ga), and in a parietal arrangement in specimens from the Grassy Bay Formation, Shaler Supergroup, Canada (1.01-0.9 Ga). This discovery extends their fossil record by at least 1.2 Ga and provides a minimum age for the divergence of thylakoid-bearing cyanobacteria at roughly 1.75 Ga. It allows the unambiguous identification of early oxygenic photosynthesizers and a new redox proxy for probing early Earth ecosystems, highlighting the importance of examining the ultrastructure of fossil cells to decipher their palaeobiology and early evolution.

Is There Such a Thing as a Biosignature?

The concept of a biosignature is widely used in astrobiology to suggest a link between some observation and a biological cause, given some context. The term itself has been defined and used in several ways in different parts of the scientific community involved in the search for past or present life on Earth and beyond. With the ongoing acceleration in the search for life in distant time and/or deep space, there is a need for clarity and accuracy in the formulation and reporting of claims. Here, we critically review the biosignature concept(s) and the associated nomenclature in light of several problems and ambiguities emphasized by recent works. One worry is that these terms and concepts may imply greater certainty than is usually justified by a rational interpretation of the data. A related worry is that terms such as "biosignature" may be inherently misleading, for example, because the divide between life and non-life-and their observable effects-is fuzzy. Another worry is that different parts of the multidisciplinary community may use non-equivalent or conflicting definitions and conceptions, leading to avoidable confusion. This review leads us to identify a number of pitfalls and to suggest how they can be circumvented. In general, we conclude that astrobiologists should exercise particular caution in deciding whether and how to use the concept of biosignature when thinking and communicating about habitability or life. Concepts and terms should be selected carefully and defined explicitly where appropriate. This would improve clarity and accuracy in the formulation of claims and subsequent technical and public communication about some of the most profound and important questions in science and society. With this objective in mind, we provide a checklist of questions that scientists and other interested parties should ask when assessing any reported detection of a "biosignature" to better understand exactly what is being claimed.

Novel diversity of polar Cyanobacteria revealed by genome-resolved metagenomics.

Benthic microbial mats dominated by Cyanobacteria are important features of polar lakes. Although culture-independent studies have provided important insights into the diversity of polar Cyanobacteria, only a handful of genomes have been sequenced to date. Here, we applied a genome-resolved metagenomics approach to data obtained from Arctic, sub-Antarctic and Antarctic microbial mats. We recovered 37 metagenome-assembled genomes (MAGs) of Cyanobacteria representing 17 distinct species, most of which are only distantly related to genomes that have been sequenced so far. These include (i) lineages that are common in polar microbial mats such as the filamentous taxa Pseudanabaena, Leptolyngbya, Microcoleus/Tychonema and Phormidium; (ii) the less common taxa Crinalium and Chamaesiphon; (iii) an enigmatic Chroococcales lineage only distantly related to Microcystis; and (iv) an early branching lineage in the order Gloeobacterales that is distributed across the cold biosphere, for which we propose the name Candidatus Sivonenia alaskensis. Our results show that genome-resolved metagenomics is a powerful tool for expanding our understanding of the diversity of Cyanobacteria, especially in understudied remote and extreme environments.

Characterization of the Halochromic Gloeocapsin Pigment, a Cyanobacterial Biosignature for Paleobiology and Astrobiology.

Ultraviolet (UV)-screening compounds represent a substantial asset for the survival of cyanobacteria in extreme environments exposed to high doses of UV radiations on modern and early Earth. Among these molecules, the halochromic pigment gloeocapsin remains poorly characterized and studied. In this study, we identified a gloeocapsin-producing cultivable cyanobacteria: the strain Phormidesmis nigrescens ULC007. We succeeded to extract, to partially purify, and to compare the dark blue pigment from both the ULC007 culture and an environmental Gloeocapsa alpina dominated sample. FT-IR and Raman spectra of G. alpina and P. nigrescens ULC007 pigment extracts strongly suggested a common backbone structure. The high-pressure liquid chromatography-UV-MS/MS analysis of the ULC007 pigment extract allowed to narrow down the molecular formula of gloeocapsin to potentially five candidates within three classes of halochromic molecules: anthraquinone derivatives, coumarin derivatives, and flavonoids. With the discovery of gloeocapsin in P. nigrescens, the production of this pigment is now established for three lineages of cyanobacteria (including G. alpina, P. nigrescens, and Solentia paulocellulare) that belong to three distinct orders (Chroococcales, Pleurocapsales, Synechoccocales), inhabiting very diverse environments. This suggests that gloeocapsin production was a trait of their common ancestor or was acquired by lateral gene transfer. This work represents an important step toward the elucidation of the structure of this enigmatic pigment and its biosynthesis, and it potentially provides a new biosignature for ancient cyanobacteria. It also gives a glimpse on the evolution of UV protection strategies, which are relevant for early phototrophic life on Earth and possibly beyond.

Intracellular bound chlorophyll residues identify 1 Gyr-old fossils as eukaryotic algae.

The acquisition of photosynthesis is a fundamental step in the evolution of eukaryotes. However, few phototrophic organisms are unambiguously recognized in the Precambrian record. The in situ detection of metabolic byproducts in individual microfossils is the key for the direct identification of their metabolisms. Here, we report a new integrative methodology using synchrotron-based X-ray fluorescence and absorption. We evidence bound nickel-geoporphyrins moieties in low-grade metamorphic rocks, preserved in situ within cells of a ~1 Gyr-old multicellular eukaryote, Arctacellularia tetragonala. We identify these moieties as chlorophyll derivatives, indicating that A. tetragonala was a phototrophic eukaryote, one of the first unambiguous algae. This new approach, applicable to overmature rocks, creates a strong new proxy to understand the evolution of phototrophy and diversification of early ecosystems.

Surfactin Stimulated by Pectin Molecular Patterns and Root Exudates Acts as a Key Driver of the Bacillus-Plant Mutualistic Interaction.

Bacillus velezensis is considered as a model species belonging to the so-called Bacillus subtilis complex that evolved typically to dwell in the soil rhizosphere niche and establish an intimate association with plant roots. This bacterium provides protection to its natural host against diseases and represents one of the most promising biocontrol agents. However, the molecular basis of the cross talk that this bacterium establishes with its natural host has been poorly investigated. We show here that these plant-associated bacteria have evolved a polymer-sensing system to perceive their host and that, in response, they increase the production of the surfactin-type lipopeptide. Furthermore, we demonstrate that surfactin synthesis is favored upon growth on root exudates and that this lipopeptide is a key component used by the bacterium to optimize biofilm formation, motility, and early root colonization. In this specific nutritional context, the bacterium also modulates qualitatively the pattern of surfactin homologues coproduced in planta and forms mainly variants that are the most active at triggering plant immunity. Surfactin represents a shared good as it reinforces the defensive capacity of the host. IMPORTANCE Within the plant-associated microbiome, some bacterial species are of particular interest due to the disease protective effect they provide via direct pathogen suppression and/or stimulation of host immunity. While these biocontrol mechanisms are quite well characterized, we still poorly understand the molecular basis of the cross talk these beneficial bacteria initiate with their host. Here, we show that the model species Bacillus velezensis stimulates the production of the surfactin lipopeptide upon sensing pectin as a cell surface molecular pattern and upon feeding on root exudates. Surfactin favors bacterial rhizosphere fitness on one hand and primes the plant immune system on the other hand. Our data therefore illustrate how both partners use this multifunctional compound as a unique shared good to sustain a mutualistic interaction.

Characterization of a nontoxic pyomelanin pigment produced by the yeast Yarrowia lipolytica.

In this study, we report on the ability of the yeast Yarrowia lipolytica W29 to produce an extracellular melanin-like brown pigment at high yield (0.5 mg/ml) in culture medium supplemented with L-tyrosine. This pigment has been characterized as pyomelanin and its synthesis was found to occur by the so-called HGA-melanin pathway. The purified pyomelanin was found embedded with antioxidant properties as it exhibited a radical scavenging activity toward 1,1-diphenyl-2-picrylhydrazyl (DPPH) with IC50 of 230 µg/ml. It was also characterized as noncytotoxic toward two mammalian cell lines, namely the mouse fibroblast NIH3T3 and human keratinocytes HaCaT. When blended with different commercial sunscreens, the purified pyomelanin increased significantly the sun protection factor (SPF) value, highlighting its potential utilization as UV-filter in cosmetic preparations.

Cyanobacteria evolution: Insight from the fossil record.

Cyanobacteria played an important role in the evolution of Early Earth and the biosphere. They are responsible for the oxygenation of the atmosphere and oceans since the Great Oxidation Event around 2.4 Ga, debatably earlier. They are also major primary producers in past and present oceans, and the ancestors of the chloroplast. Nevertheless, the identification of cyanobacteria in the early fossil record remains ambiguous because the morphological criteria commonly used are not always reliable for microfossil interpretation. Recently, new biosignatures specific to cyanobacteria were proposed. Here, we review the classic and new cyanobacterial biosignatures. We also assess the reliability of the previously described cyanobacteria fossil record and the challenges of molecular approaches on modern cyanobacteria. Finally, we suggest possible new calibration points for molecular clocks, and strategies to improve our understanding of the timing and pattern of the evolution of cyanobacteria and oxygenic photosynthesis.

Marked Succession of Cyanobacterial Communities Following Glacier Retreat in the High Arctic.

Cyanobacteria are important colonizers of recently deglaciated proglacial soil but an in-depth investigation of cyanobacterial succession following glacier retreat has not yet been carried out. Here, we report on the successional trajectories of cyanobacterial communities in biological soil crusts (BSCs) along a 100-year deglaciation gradient in three glacier forefields in central Svalbard, High Arctic. Distance from the glacier terminus was used as a proxy for soil age (years since deglaciation), and cyanobacterial abundance and community composition were evaluated by epifluorescence microscopy and pyrosequencing of partial 16S rRNA gene sequences, respectively. Succession was characterized by a decrease in phylotype richness and a marked shift in community structure, resulting in a clear separation between early (10-20 years since deglaciation), mid (30-50 years), and late (80-100 years) communities. Changes in cyanobacterial community structure were mainly connected with soil age and associated shifts in soil chemical composition (mainly moisture, SOC, SMN, K, and Na concentrations). Phylotypes associated with early communities were related either to potentially novel lineages (< 97.5% similar to sequences currently available in GenBank) or lineages predominantly restricted to polar and alpine biotopes, suggesting that the initial colonization of proglacial soil is accomplished by cyanobacteria transported from nearby glacial environments. Late communities, on the other hand, included more widely distributed genotypes, which appear to establish only after the microenvironment has been modified by the pioneering taxa.

Consensus assessment of the contamination level of publicly available cyanobacterial genomes.

Publicly available genomes are crucial for phylogenetic and metagenomic studies, in which contaminating sequences can be the cause of major problems. This issue is expected to be especially important for Cyanobacteria because axenic strains are notoriously difficult to obtain and keep in culture. Yet, despite their great scientific interest, no data are currently available concerning the quality of publicly available cyanobacterial genomes. As reliably detecting contaminants is a complex task, we designed a pipeline combining six methods in a consensus strategy to assess the contamination level of 440 genome assemblies of Cyanobacteria. Two methods are based on published reference databases of ribosomal genes (SSU rRNA 16S and ribosomal proteins), one is indirectly based on a reference database of marker genes (CheckM), and three are based on complete genome analysis. Among those genome-wide methods, Kraken and DIAMOND blastx share the same reference database that we derived from Ensembl Bacteria, whereas CONCOCT does not require any reference database, instead relying on differences in DNA tetramer frequencies. Given that all the six methods appear to have their own strengths and limitations, we used the consensus of their rankings to infer that >5% of cyanobacterial genome assemblies are highly contaminated by foreign DNA (i.e., contaminants were detected by 5 or 6 methods). Our results will help researchers to check the quality of publicly available genomic data before use in their own analyses. Moreover, we argue that journals should make mandatory the submission of raw read data along with genome assemblies in order to facilitate the detection of contaminants in sequence databases.

Community structure and distribution of benthic cyanobacteria in Antarctic lacustrine microbial mats.

The terrestrial Antarctic Realm has recently been divided into 16 Antarctic Conservation Biogeographic Regions (ACBRs) based on environmental properties and the distribution of biota. Despite their prominent role in the primary production and nutrient cycling in Antarctic lakes, cyanobacteria were only poorly represented in the biological dataset used to delineate these ACBRs. Here, we provide a first high-throughput sequencing insight into the spatial distribution of benthic cyanobacterial communities in Antarctic lakes located in four distinct, geographically distant ACBRs and covering a range of limnological conditions. Cyanobacterial community structure differed between saline and freshwater lakes. No clear bioregionalization was observed, as clusters of community similarity encompassed lakes from distinct ACBRs. Most phylotypes (77.0%) were related to cyanobacterial lineages (defined at ≥99.0% 16S rRNA gene sequence similarity) restricted to the cold biosphere, including lineages potentially endemic to Antarctica (55.4%). The latter were generally rare and restricted to a small number of lakes, while more ubiquitous phylotypes were generally abundant and present in different ACBRs. These results point to a widespread distribution of some cosmopolitan cyanobacterial phylotypes across the different Antarctic ice-free regions, but also suggest the existence of dispersal barriers both within and between Antarctica and the other continents.

Draft Genome Sequence of the Axenic Strain Phormidesmispriestleyi ULC007, a Cyanobacterium Isolated from Lake Bruehwiler (Larsemann Hills, Antarctica).

Phormidesmis priestleyi ULC007 is an Antarctic freshwater cyanobacterium. Its draft genome is 5,684,389 bp long. It contains a total of 5,604 protein-encoding genes, of which 22.2% have no clear homologues in known genomes. To date, this draft genome is the first one ever determined for an axenic cyanobacterium from Antarctica.

Evolution of the Northern Rockweed, Fucus distichus, in a Regime of Glacial Cycling: Implications for Benthic Algal Phylogenetics.

Northern hemisphere rockweeds (Fucus) are thought to have evolved in the North Pacific and then spread to the North Atlantic following the opening of the Bering Strait. They have dispersed and widely speciated in the North Atlantic and its tributary seas. Fucus distichus is likely near the ancestral member of this genus, and studies have shown that there are several species/subspecies in this complex (i.e. F. evanescens and F. gardneri). We used phylogenetic and haplotype analyses to test the phylogenetic relationships and biogeography of F. distichus. Our data and subsequent analyses demonstrate that, unlike previous studies that lacked samples from an extensive geographical area of the Arctic and Subarctic, there is a distinct Arctic haplotype that is the source of subspecies in both the North Pacific and North Atlantic. Fucus distichus occupies a low tide zone habitat, and in Arctic/Subarctic regions it is adapted to the severe stress of sea ice coverage and disturbance during many months per year. We hypothesize that the very large geographic area of Arctic and Subarctic rocky shores available to this species during interglacials, supported by large Arctic/Subarctic fringe areas as well as unglaciated refugia during glacial cycles, provided a robust population and gene pool (described by the Thermogeographic Model). This gene pool dilutes that of the more fragmented and area-limited Temperate/Boreal area populations when they are brought together during glacial cycles. We suggest that similar subspecies complexes for a variety of Arctic/Subarctic shore biota should be examined further in this context, rather than arbitrarily being split up into numerous species.

Phylogenetic analysis of cultivation-resistant terrestrial cyanobacteria with massive sheaths (Stigonema spp. and Petalonema alatum, Nostocales, Cyanobacteria) using single-cell and filament sequencing of environmental samples.

Molecular assessment of a large portion of traditional cyanobacterial taxa has been hindered by the failure to isolate and grow them in culture. In this study, we developed an optimized protocol for single cell/filament isolation and 16S rRNA gene sequencing of terrestrial cyanobacteria with large mucilaginous sheaths, and applied it to determine the phylogenetic position of typical members of the genera Petalonema and Stigonema. A methodology based on a glass-capillary isolation technique and a semi-nested PCR protocol enabled reliable sequencing of the 16S rRNA gene from all samples analyzed. Ten samples covering seven species of Stigonema from Europe, North and Central America, and Hawaii, and the type species of Petalonema from Slovakia were sequenced. Contrary to some previous studies, which proposed a relationship with heteropolar nostocalean cyanobacteria, Petalonema appeared to belong to the family Scytonemataceae. Analysis of Stigonema specimens recovered a unique coherent phylogenetic cluster, substantially broadening our knowledge of the molecular diversity within this genus. Neither the uni- to biseriate species nor the multiseriate species formed monophyletic subclusters within the genus. Typical multiseriate species of Stigonema clustered in a phylogenetic branch derived from uni- to biseriate S. ocellatum Thuret ex Bornet & Flahault in our analysis, suggesting that species with more complex thalli may have evolved from the more simple ones. We propose the technique tested in this study as a promising tool for a future revision of the molecular taxonomy in cyanobacteria.

Novel organic solvent-tolerant esterase isolated by metagenomics: insights into the lipase/esterase classification.

in order to isolate novel organic solvent-tolerant (OST) lipases, a metagenomic library was built using DNA derived from a temperate forest soil sample. A two-step activity-based screening allowed the isolation of a lipolytic clone active in the presence of organic solvents. Sequencing of the plasmid pRBest recovered from the positive clone revealed the presence of a putative lipase/esterase encoding gene. The deduced amino acid sequence (RBest1) contains the conserved lipolytic enzyme signature and is related to the previously described OST lipase from Lysinibacillus sphaericus 205y, which is the sole studied prokaryotic enzyme belonging to the 4.4 α/ß hydrolase subgroup (abH04.04). Both in vivo and in vitro studies of the substrate specificity of RBest1, using triacylglycerols or nitrophenyl-esters, respectively, revealed that the enzyme is highly specific for butyrate (C4) compounds, behaving as an esterase rather than a lipase. The RBest1 esterase was purified and biochemically characterized. The optimal esterase activity was observed at pH 6.5 and at temperatures ranging from 38 to 45 °C. Enzymatic activity, determined by hydrolysis of p-nitrophenyl esters, was found to be affected by the presence of different miscible and non-miscible organic solvents, and salts. Noteworthy, RBest1 remains significantly active at high ionic strength. These findings suggest that RBest1 possesses the ability of OST enzymes to molecular adaptation in the presence of organic compounds and resistance of halophilic proteins.

Novel organic solvent-tolerant esterase isolated by metagenomics: insights into the lipase/esterase classification / Nueva esterasa tolerante a los solventes orgánicos aislada por metagenómica: ideas sobre la clasificación de las esterasas/lipasas

In order to isolate novel organic solvent-tolerant (OST) lipases, a metagenomic library was built using DNA derived from a temperate forest soil sample. A two-step activity-based screening allowed the isolation of a lipolytic clone active in the presence of organic solvents. Sequencing of the plasmid pRBest recovered from the positive clone revealed the presence of a putative lipase/esterase encoding gene. The deduced amino acid sequence (RBest1) contains the conserved lipolytic enzyme signature and is related to the previously described OST lipase from Lysinibacillus sphaericus 205y, which is the sole studied prokaryotic enzyme belonging to the 4.4 a/ß hydrolase subgroup (abH04.04). Both in vivo and in vitro studies of the substrate specificity of RBest1, using triacylglycerols or nitrophenyl-esters, respectively, revealed that the enzyme is highly specific for butyrate (C4) compounds, behaving as an esterase rather than a lipase. The RBest1 esterase was purified and biochemically characterized. The optimal esterase activity was observed at pH 6.5 and at temperatures ranging from 38 to 45 °C. Enzymatic activity, determined by hydrolysis of p-nitrophenyl esters, was found to be affected by the presence of different miscible and non-miscible organic solvents, and salts. Noteworthy, RBest1 remains significantly active at high ionic strength. These findings suggest that RBest1 possesses the ability of OST enzymes to molecular adaptation in the presence of organic compounds and resistance of halophilic proteins.

Con el fin de aislar nuevas variantes de lipasas tolerantes a solventes organicos (OST), se construyo una libreria metagenomica a partir de ADN obtenido de una muestra de suelo de bosque templado. A traves de un monitoreo en dos etapas, basado en la deteccion de actividades, se aislo un clon con actividad lipolitica en presencia de solventes organicos. La secuenciacion del plasmido pRBest recuperado del clon positivo revelo la presencia de un gen codificante de una hipotetica lipasa/esterasa. La secuencia deducida de amino acidos (RBest1) contiene los motivos conservados de enzimas lipoliticas y esta relacionada con la lipasa OST previamente descrita de Lysinibacillus sphaericus 205y, que es la unica enzima procariota estudiada perteneciente al subgrupo 4.4 de a/ß hidrolasas (abH4.04). Estudios in vivo e in vitro sobre la especificidad de sustratos de RBest1, utilizando triacil-gliceroles o p-nitrofenil-esteres, respectivamente, revelaron que la enzima es altamente especifica para compuestos butiricos (C4), comportandose como una esterasa y no como una lipasa. La esterasa RBest1 fue purificada y caracterizada bioquimicamente. La actividad optima de esterasa fue observada a pH 6,5 y las temperaturas optimas fueron entre 38 y 45 °C. Se establecio que la actividad enzimatica, determinada por hidrolisis de p-nitrofenil esteres, es afectada en presencia de diferentes solventes organicos miscibles y no miscibles, y tambien sales. Notoriamente, RBest1 permanece significativamente activa a elevadas fuerzas ionicas. Estos hallazgos sugieren que RBest1 posee la capacidad de las enzimas OST de la adaptacion molecular en presencia de compuestos organicos, asi como la resistencia de las proteinas halofilas.

Novel organic solvent-tolerant esterase isolated by metagenomics: insights into the lipase/esterase classification / Nueva esterasa tolerante a los solventes orgánicos aislada por metagenómica: ideas sobre la clasificación de las esterasas/lipasas

In order to isolate novel organic solvent-tolerant (OST) lipases, a metagenomic library was built using DNA derived from a temperate forest soil sample. A two-step activity-based screening allowed the isolation of a lipolytic clone active in the presence of organic solvents. Sequencing of the plasmid pRBest recovered from the positive clone revealed the presence of a putative lipase/esterase encoding gene. The deduced amino acid sequence (RBest1) contains the conserved lipolytic enzyme signature and is related to the previously described OST lipase from Lysinibacillus sphaericus 205y, which is the sole studied prokaryotic enzyme belonging to the 4.4 a/ß hydrolase subgroup (abH04.04). Both in vivo and in vitro studies of the substrate specificity of RBest1, using triacylglycerols or nitrophenyl-esters, respectively, revealed that the enzyme is highly specific for butyrate (C4) compounds, behaving as an esterase rather than a lipase. The RBest1 esterase was purified and biochemically characterized. The optimal esterase activity was observed at pH 6.5 and at temperatures ranging from 38 to 45 °C. Enzymatic activity, determined by hydrolysis of p-nitrophenyl esters, was found to be affected by the presence of different miscible and non-miscible organic solvents, and salts. Noteworthy, RBest1 remains significantly active at high ionic strength. These findings suggest that RBest1 possesses the ability of OST enzymes to molecular adaptation in the presence of organic compounds and resistance of halophilic proteins.(AU)

Con el fin de aislar nuevas variantes de lipasas tolerantes a solventes organicos (OST), se construyo una libreria metagenomica a partir de ADN obtenido de una muestra de suelo de bosque templado. A traves de un monitoreo en dos etapas, basado en la deteccion de actividades, se aislo un clon con actividad lipolitica en presencia de solventes organicos. La secuenciacion del plasmido pRBest recuperado del clon positivo revelo la presencia de un gen codificante de una hipotetica lipasa/esterasa. La secuencia deducida de amino acidos (RBest1) contiene los motivos conservados de enzimas lipoliticas y esta relacionada con la lipasa OST previamente descrita de Lysinibacillus sphaericus 205y, que es la unica enzima procariota estudiada perteneciente al subgrupo 4.4 de a/ß hidrolasas (abH4.04). Estudios in vivo e in vitro sobre la especificidad de sustratos de RBest1, utilizando triacil-gliceroles o p-nitrofenil-esteres, respectivamente, revelaron que la enzima es altamente especifica para compuestos butiricos (C4), comportandose como una esterasa y no como una lipasa. La esterasa RBest1 fue purificada y caracterizada bioquimicamente. La actividad optima de esterasa fue observada a pH 6,5 y las temperaturas optimas fueron entre 38 y 45 °C. Se establecio que la actividad enzimatica, determinada por hidrolisis de p-nitrofenil esteres, es afectada en presencia de diferentes solventes organicos miscibles y no miscibles, y tambien sales. Notoriamente, RBest1 permanece significativamente activa a elevadas fuerzas ionicas. Estos hallazgos sugieren que RBest1 posee la capacidad de las enzimas OST de la adaptacion molecular en presencia de compuestos organicos, asi como la resistencia de las proteinas halofilas.(AU)

Novel organic solvent-tolerant esterase isolated by metagenomics: insights into the lipase/esterase classification. / Novel organic solvent-tolerant esterase isolated by metagenomics: insights into the lipase/esterase classification.

in order to isolate novel organic solvent-tolerant (OST) lipases, a metagenomic library was built using DNA derived from a temperate forest soil sample. A two-step activity-based screening allowed the isolation of a lipolytic clone active in the presence of organic solvents. Sequencing of the plasmid pRBest recovered from the positive clone revealed the presence of a putative lipase/esterase encoding gene. The deduced amino acid sequence (RBest1) contains the conserved lipolytic enzyme signature and is related to the previously described OST lipase from Lysinibacillus sphaericus 205y, which is the sole studied prokaryotic enzyme belonging to the 4.4 α/ß hydrolase subgroup (abH04.04). Both in vivo and in vitro studies of the substrate specificity of RBest1, using triacylglycerols or nitrophenyl-esters, respectively, revealed that the enzyme is highly specific for butyrate (C4) compounds, behaving as an esterase rather than a lipase. The RBest1 esterase was purified and biochemically characterized. The optimal esterase activity was observed at pH 6.5 and at temperatures ranging from 38 to 45 °C. Enzymatic activity, determined by hydrolysis of p-nitrophenyl esters, was found to be affected by the presence of different miscible and non-miscible organic solvents, and salts. Noteworthy, RBest1 remains significantly active at high ionic strength. These findings suggest that RBest1 possesses the ability of OST enzymes to molecular adaptation in the presence of organic compounds and resistance of halophilic proteins.

Bacillus amyloliquefaciens GA1 as a source of potent antibiotics and other secondary metabolites for biocontrol of plant pathogens.

BACKGROUND: Phytopathogenic fungi affecting crop and post-harvested vegetables are a major threat to food production and food storage. To face these drawbacks, producers have become increasingly dependent on agrochemicals. However, intensive use of these compounds has led to the emergence of pathogen resistance and severe negative environmental impacts. There are also a number of plant diseases for which chemical solutions are ineffective or non-existent as well as an increasing demand by consumers for pesticide-free food. Thus, biological control through the use of natural antagonistic microorganisms has emerged as a promising alternative to chemical pesticides for more rational and safe crop management. RESULTS: The genome of the plant-associated B. amyloliquefaciens GA1 was sample sequenced. Several gene clusters involved in the synthesis of biocontrol agents were detected. Four gene clusters were shown to direct the synthesis of the cyclic lipopeptides surfactin, iturin A and fengycin as well as the iron-siderophore bacillibactin. Beside these non-ribosomaly synthetised peptides, three additional gene clusters directing the synthesis of the antibacterial polyketides macrolactin, bacillaene and difficidin were identified. Mass spectrometry analysis of culture supernatants led to the identification of these secondary metabolites, hence demonstrating that the corresponding biosynthetic gene clusters are functional in strain GA1. In addition, genes encoding enzymes involved in synthesis and export of the dipeptide antibiotic bacilysin were highlighted. However, only its chlorinated derivative, chlorotetaine, could be detected in culture supernatants. On the contrary, genes involved in ribosome-dependent synthesis of bacteriocin and other antibiotic peptides were not detected as compared to the reference strain B. amyloliquefaciens FZB42. CONCLUSION: The production of all of these antibiotic compounds highlights B. amyloliquefaciens GA1 as a good candidate for the development of biocontrol agents.