Zobellia roscoffensis sp. nov. and Zobellia nedashkovskayae sp. nov., two flavobacteria from the epiphytic microbiota of the brown alga Ascophyllum nodosum, and emended description of the genus Zobellia.

Four marine bacterial strains were isolated from a thallus of the brown alga Ascophyllum nodosum collected in Roscoff, France. Cells were Gram-stain-negative, strictly aerobic, non-flagellated, gliding, rod-shaped and grew optimally at 25-30 °C, at pH 7-8 and with 2-4 % NaCl. Phylogenetic analyses of their 16S rRNA gene sequences showed that the bacteria were affiliated to the genus Zobellia (family Flavobacteriaceae, phylum Bacteroidetes). The four strains exhibited 97.8-100 % 16S rRNA gene sequence similarity values among themselves, 97.9-99.1 % to the type strains of Zobellia amurskyensis KMM 3526T and Zobellia laminariae KMM 3676T, and less than 99 % to other species of the genus Zobellia. The DNA G+C content of the four strains ranged from 36.7 to 37.7 mol%. Average nucleotide identity and digital DNA-DNA hybridization calculations between the new strains and other members of the genus Zobellia resulted in values of 76.4-88.9 % and below 38.5 %, respectively. Phenotypic, phylogenetic and genomic analyses showed that the four strains are distinct from species of the genus Zobellia with validly published names. They represent two novel species of the genus Zobellia, for which the names Zobellia roscoffensis sp. nov. and Zobellia nedashkovskayae sp. nov. are proposed with Asnod1-F08T (RCC6906T=KMM 6823T=CIP 111902T) and Asnod2-B07-BT (RCC6908T=KMM 6825T=CIP 111904T), respectively, as the type strains.

Nonribosomal peptides in fungal cell factories: from genome mining to optimized heterologous production.

Fungi are notoriously prolific producers of secondary metabolites including nonribosomal peptides (NRPs). The structural complexity of NRPs grants them interesting activities such as antibiotic, anti-cancer, and anti-inflammatory properties. The discovery of these compounds with attractive activities can be achieved by using two approaches: either by screening samples originating from various environments for their biological activities, or by identifying the related clusters in genomic sequences thanks to bioinformatics tools. This genome mining approach has grown tremendously due to recent advances in genome sequencing, which have provided an incredible amount of genomic data from hundreds of microbial species. Regarding fungal organisms, the genomic data have revealed the presence of an unexpected number of putative NRP-related gene clusters. This highlights fungi as a goldmine for the discovery of putative novel bioactive compounds. Recent development of NRP dedicated bioinformatics tools have increased the capacity to identify these gene clusters and to deduce NRPs structures, speeding-up the screening process for novel metabolites discovery. Unfortunately, the newly identified compound is frequently not or poorly produced by native producers due to a lack of expression of the related genes cluster. A frequently employed strategy to increase production rates consists in transferring the related biosynthetic pathway in heterologous hosts. This review aims to provide a comprehensive overview about the topic of NRPs discovery, from gene cluster identification by genome mining to the heterologous production in fungal hosts. The main computational tools and methods for genome mining are herein presented with an emphasis on the particularities of the fungal systems. The different steps of the reconstitution of NRP biosynthetic pathway in heterologous fungal cell factories will be discussed, as well as the key factors to consider for maximizing productivity. Several examples will be developed to illustrate the potential of heterologous production to both discover uncharacterized novel compounds predicted in silico by genome mining, and to enhance the productivity of interesting bio-active natural products.

Astin C Production by the Endophytic Fungus Cyanodermella asteris in Planktonic and Immobilized Culture Conditions.

The fungal endophyte Cyanodermella asteris (C. asteris) has been recently isolated from the medicinal plant Aster tataricus (A. tataricus). This fungus produces astin C, a cyclic pentapeptide with anticancer and anti-inflammatory properties. The production of this secondary metabolite is compared in immobilized and planktonic conditions. For immobilized cultures, a stainless steel packing immersed in the culture broth is used as a support. In these conditions, the fungus exclusively grows on the packing, which provides a considerable advantage for astin C recovery and purification. C. asteris metabolism is different according to the culture conditions in terms of substrate consumption rate, cell growth, and astin C production. Immobilized-cell cultures yield a 30% increase of astin C production, associated with a 39% increase in biomass. The inoculum type as spores rather than hyphae, and a pre-inoculation washing procedure with sodium hydroxide, turns out to be beneficial both for astin C production and fungus development onto the support. Finally, the influence of culture parameters such as pH and medium composition on astin C production is evaluated. With optimized culture conditions, astin C yield is further improved reaching a five times higher final specific yield compared to the value reported with astin C extraction from A. tataricus (0.89 mg g-1 and 0.16 mg g-1 respectively).

Temporal Dynamics of Soil Microbial Communities below the Seedbed under Two Contrasting Tillage Regimes.

Agricultural productivity relies on a wide range of ecosystem services provided by the soil biota. Plowing is a fundamental component of conventional farming, but long-term detrimental effects such as soil erosion and loss of soil organic matter have been recognized. Moving towards more sustainable management practices such as reduced tillage or crop residue retention can reduce these detrimental effects, but will also influence structure and function of the soil microbiota with direct consequences for the associated ecosystem services. Although there is increasing evidence that different tillage regimes alter the soil microbiome, we have a limited understanding of the temporal dynamics of these effects. Here, we used high-throughput sequencing of bacterial and fungal ribosomal markers to explore changes in soil microbial community structure under two contrasting tillage regimes (conventional and reduced tillage) either with or without crop residue retention. Soil samples were collected over the growing season of two crops (Vicia faba and Triticum aestivum) below the seedbed (15-20 cm). Tillage, crop and growing stage were significant determinants of microbial community structure, but the impact of tillage showed only moderate temporal dependency. Whereas the tillage effect on soil bacteria showed some temporal dependency and became less strong at later growing stages, the tillage effect on soil fungi was more consistent over time. Crop residue retention had only a minor influence on the community. Six years after the conversion from conventional to reduced tillage, soil moisture contents and nutrient levels were significantly lower under reduced than under conventional tillage. These changes in edaphic properties were related to specific shifts in microbial community structure. Notably, bacterial groups featuring copiotrophic lifestyles or potentially carrying the ability to degrade more recalcitrant compounds were favored under conventional tillage, whereas taxa featuring more oligotrophic lifestyles were more abundant under reduced tillage. Our study found that, under the specific edaphic and climatic context of central Belgium, different tillage regimes created different ecological niches that select for different microbial lifestyles with potential consequences for the ecosystem services provided to the plants and their environment.

Discovering novel enzymes by functional screening of plurigenomic libraries from alga-associated Flavobacteriia and Gammaproteobacteria.

Alga-associated microorganisms, in the context of their numerous interactions with the host and the complexity of the marine environment, are known to produce diverse hydrolytic enzymes with original biochemistry. We recently isolated several macroalgal-polysaccharide-degrading bacteria from the surface of the brown alga Ascophyllum nodosum. These active isolates belong to two classes: the Flavobacteriia and the Gammaproteobacteria. In the present study, we constructed two "plurigenomic" (with multiple bacterial genomes) libraries with the 5 most interesting isolates (regarding their phylogeny and their enzymatic activities) of each class (Fv and Gm libraries). Both libraries were screened for diverse hydrolytic activities. Five activities, out of the 48 previously identified in the natural polysaccharolytic isolates, were recovered by functional screening: a xylanase (GmXyl7), a beta-glucosidase (GmBg1), an esterase (GmEst7) and two iota-carrageenases (Fvi2.5 and Gmi1.3). We discuss here the potential role of the used host-cell, the average DNA insert-sizes and the used restriction enzymes on the divergent screening yields obtained for both libraries and get deeper inside the "great screen anomaly". Interestingly, the discovered esterase probably stands for a novel family of homoserine o-acetyltransferase-like-esterases, while the two iota-carrageenases represent new members of the poorly known GH82 family (containing only 19 proteins since its description in 2000). These original results demonstrate the efficiency of our uncommon "plurigenomic" library approach and the underexplored potential of alga-associated cultivable microbiota for the identification of novel and algal-specific enzymes.

Isolation and cultivation of xylanolytic and cellulolytic Sarocladium kiliense and Trichoderma virens from the gut of the termite Reticulitermes santonensis.

The purpose of this work was the isolation and cultivation of cellulolytic and xylanolytic microorganisms extracted from the gut of the lower termite Reticulitermes santonensis. Microcrystalline cellulose (with and without lignin) and beech wood xylan were used as diets instead of poplar wood in order to select cellulose and hemicellulose-degrading fungi. The strain Sarocladium kiliense (Acremonium kiliense) CTGxxyl was isolated from the termites fed on xylan, while the strain Trichoderma virens CTGxAviL was isolated from the termites fed on cellulose (with and without lignin). Both molds were cultivated in liquid media containing different substrates: agro-residues or purified polymers. S. kiliense produced maximal ß-glucosidase, endo-1,4-ß-D-glucanase, exo-1,4-ß-D-glucanase and endo-1,4-ß-D-xylanase activities of 0.103, 3.99, 0.53, and 40.8 IU/ml, respectively. T. virens produced maximal ß-xylosidase, endo-1,4-ß-D-glucanase, exo-1,4-ß-D-glucanase, and endo-1,4-ß-D-xylanase activities of 0.38, 1.48, 0.69, and 426 IU/ml. The cellulase and the xylanase of S. kiliense, less common than T. virens, were further investigated. The optimal activity of the xylanase was observed at pH 9-10 at 60 °C. The cellulase showed its maximal activity at pH 10, 70 °C. Zymography identified different xylanases produced by both molds, and some fragment sizes were highlighted: 35, 100, and 170 kDa for S. kiliense and 20, 40, 80, and 170 kDa for T. virens. In both cases, endo-1,4-ß-D-xylanase activities were confirmed through mass spectrometry.

The Cultivable Surface Microbiota of the Brown Alga Ascophyllum nodosum is Enriched in Macroalgal-Polysaccharide-Degrading Bacteria.

Bacteria degrading algal polysaccharides are key players in the global carbon cycle and in algal biomass recycling. Yet the water column, which has been studied largely by metagenomic approaches, is poor in such bacteria and their algal-polysaccharide-degrading enzymes. Even more surprisingly, the few published studies on seaweed-associated microbiomes have revealed low abundances of such bacteria and their specific enzymes. However, as macroalgal cell-wall polysaccharides do not accumulate in nature, these bacteria and their unique polysaccharidases must not be that uncommon. We, therefore, looked at the polysaccharide-degrading activity of the cultivable bacterial subpopulation associated with Ascophyllum nodosum. From A. nodosum triplicates, 324 bacteria were isolated and taxonomically identified. Out of these isolates, 78 (~25%) were found to act on at least one tested algal polysaccharide (agar, ι- or κ-carrageenan, or alginate). The isolates "active" on algal-polysaccharides belong to 11 genera: Cellulophaga, Maribacter, Algibacter, and Zobellia in the class Flavobacteriia (41) and Pseudoalteromonas, Vibrio, Cobetia, Shewanella, Colwellia, Marinomonas, and Paraglaceciola in the class Gammaproteobacteria (37). A major part represents likely novel species. Different proportions of bacterial phyla and classes were observed between the isolated cultivable subpopulation and the total microbial community previously identified on other brown algae. Here, Bacteroidetes and Gammaproteobacteria were found to be the most abundant and some phyla (as Planctomycetes and Cyanobacteria) frequently encountered on brown algae weren't identified. At a lower taxonomic level, twelve genera, well-known to be associated with algae (with the exception for Colwellia), were consistently found on all three A. nosodum samples. Even more interesting, 9 of the 11 above mentioned genera containing polysaccharolytic isolates were predominant in this common core. The cultivable fraction of the bacterial community associated with A. nodosum is, thus, significantly enriched in macroalgal-polysaccharide-degrading bacteria and these bacteria seem important for the seaweed holobiont even though they are under-represented in alga-associated microbiome studies.

Diversity of bacterial communities in a profile of a winter wheat field: known and unknown members.

In soils, bacteria are very abundant and diverse. They are involved in various agro-ecosystem processes such as the nitrogen cycle, organic matter degradation, and soil formation. Yet, little is known about the distribution and composition of bacterial communities through the soil profile, particularly in agricultural soils, as most studies have focused only on topsoils or forest and grassland soils. In the present work, we have used bar-coded pyrosequencing analysis of the V3 region of the 16S rRNA gene to analyze bacterial diversity in a profile (depths 10, 25, and 45 cm) of a well-characterized field of winter wheat. Taxonomic assignment was carried out with the Ribosomal Database Project (RDP) Classifier program with three bootstrap scores: a main run at 0.80, a confirmation run at 0.99, and a run at 0 to gain information on the unknown bacteria. Our results show that biomass and bacterial quantity and diversity decreased greatly with depth. Depth also had an impact, in terms of relative sequence abundance, on 81 % of the most represented taxonomic ranks, notably the ranks Proteobacteria, Bacteroidetes, Actinobacteridae, and Acidobacteria. Bacterial community composition differed more strongly between the topsoil (10 and 25 cm) and subsoil (45 cm) than between levels in the topsoil, mainly because of shifts in the carbon, nitrogen, and potassium contents. The subsoil also contained more unknown bacteria, 53.96 % on the average, than did the topsoil, with 42.06 % at 10 cm and 45.59 % at 25 cm. Most of these unknown bacteria seem to belong to Deltaproteobacteria, Actinobacteria, Rhizobiales, and Acidobacteria.

Identification and characterization of a halotolerant, cold-active marine endo-ß-1,4-glucanase by using functional metagenomics of seaweed-associated microbiota.

A metagenomic library was constructed from microorganisms associated with the brown alga Ascophyllum nodosum. Functional screening of this library revealed 13 novel putative esterase loci and two glycoside hydrolase loci. Sequence and gene cluster analysis showed the wide diversity of the identified enzymes and gave an idea of the microbial populations present during the sample collection period. Lastly, an endo-ß-1,4-glucanase having less than 50% identity to sequences of known cellulases was purified and partially characterized, showing activity at low temperature and after prolonged incubation in concentrated salt solutions.

Widespread occurrence of expressed fungal secretory peroxidases in forest soils.

Fungal secretory peroxidases mediate fundamental ecological functions in the conversion and degradation of plant biomass. Many of these enzymes have strong oxidizing activities towards aromatic compounds and are involved in the degradation of plant cell wall (lignin) and humus. They comprise three major groups: class II peroxidases (including lignin peroxidase, manganese peroxidase, versatile peroxidase and generic peroxidase), dye-decolorizing peroxidases, and heme-thiolate peroxidases (e.g. unspecific/aromatic peroxygenase, chloroperoxidase). Here, we have repeatedly observed a widespread expression of all major peroxidase groups in leaf and needle litter across a range of forest ecosystems (e.g. Fagus, Picea, Acer, Quercus, and Populus spp.), which are widespread in Europe and North America. Manganese peroxidases and unspecific peroxygenases were found expressed in all nine investigated forest sites, and dye-decolorizing peroxidases were observed in five of the nine sites, thereby indicating biological significance of these enzymes for fungal physiology and ecosystem processes. Transcripts of selected secretory peroxidase genes were also analyzed in pure cultures of several litter-decomposing species and other fungi. Using this information, we were able to match, in environmental litter samples, two manganese peroxidase sequences to Mycena galopus and Mycena epipterygia and one unspecific peroxygenase transcript to Mycena galopus, suggesting an important role of this litter- and coarse woody debris-dwelling genus in the disintegration and transformation of litter aromatics and organic matter formation.

MALDI-TOF MS analysis of cellodextrins and xylo-oligosaccharides produced by hindgut homogenates of Reticulitermes santonensis.

Hindgut homogenates of the termite Reticulitermes santonensis were incubated with carboxymethyl cellulose (CMC), crystalline celluloses or xylan substrates. Hydrolysates were analyzed with matrix-assisted laser desorption/ionization coupled to time-of-flight mass spectrometry (MALDI-TOF MS). The method was first set up using acid hydrolysis analysis to characterize non-enzymatic profiles. Commercial enzymes of Trichoderma reesei or T. longibrachiatum were also tested to validate the enzymatic hydrolysis analysis. For CMC hydrolysis, data processing and visual display were optimized to obtain comprehensive profiles and allow rapid comparison and evaluation of enzymatic selectivity, according to the number of substituents of each hydrolysis product. Oligosaccharides with degrees of polymerization (DPs) ranging from three to 12 were measured from CMC and the enzymatic selectivity was demonstrated. Neutral and acidic xylo-oligosaccharides with DPs ranging from three to 11 were measured from xylan substrate. These results are of interest for lignocellulose biomass valorization and demonstrated the potential of termites and their symbiotic microbiota as a source of interesting enzymes for oligosaccharides production.

Microorganisms living on macroalgae: diversity, interactions, and biotechnological applications.

Marine microorganisms play key roles in every marine ecological process, hence the growing interest in studying their populations and functions. Microbial communities on algae remain underexplored, however, despite their huge biodiversity and the fact that they differ markedly from those living freely in seawater. The study of this microbiota and of its relationships with algal hosts should provide crucial information for ecological investigations on algae and aquatic ecosystems. Furthermore, because these microorganisms interact with algae in multiple, complex ways, they constitute an interesting source of novel bioactive compounds with biotechnological potential, such as dehalogenases, antimicrobials, and alga-specific polysaccharidases (e.g., agarases, carrageenases, and alginate lyases). Here, to demonstrate the huge potential of alga-associated organisms and their metabolites in developing future biotechnological applications, we first describe the immense diversity and density of these microbial biofilms. We further describe their complex interactions with algae, leading to the production of specific bioactive compounds and hydrolytic enzymes of biotechnological interest. We end with a glance at their potential use in medical and industrial applications.

Two promising alkaline ß-glucosidases isolated by functional metagenomics from agricultural soil, including one showing high tolerance towards harsh detergents, oxidants and glucose.

New ß-glucosidase activities were identified by screening metagenomic libraries constructed with DNA isolated from the topsoil of a winter wheat field. Two of the corresponding proteins, displaying an unusual preference for alkaline conditions, were selected for purification by Ni-NTA chromatography. AS-Esc6, a 762-amino-acid enzyme belonging to glycoside hydrolase family 3, proved to be a mesophilic aryl-ß-glucosidase with maximal activity around pH 8 and 40 °C. A similar pH optimum was found for AS-Esc10, a 475-amino-acid GH1-family enzyme, but this enzyme remained significantly active across a wider pH range and was also markedly more stable than AS-Esc6 at pH greater than 10. AS-Esc10 was found to degrade cellobiose and diverse aryl glycosides, with an optimal temperature of 60 °C and good stability up to 50 °C. Unlike AS-Esc6, which showed a classically low inhibitory constant for glucose (14 mM), AS-Esc10 showed enhanced activity in the presence of molar concentrations of glucose. AS-Esc10 was highly tolerant to hydrogen peroxide and also to sodium dodecyl sulfate, this being indicative of kinetic stability. This unique combination of properties makes AS-Esc10 a particularly promising candidate whose potential in biotechnological applications is worth exploring further.

Isolation of amylolytic, xylanolytic, and cellulolytic microorganisms extracted from the gut of the termite Reticulitermes santonensis by means of a micro-aerobic atmosphere.

The aim of this work was to isolate enzyme-producing microorganisms from the tract of the termite Reticulitermes santonensis. The microorganisms were extracted from the guts and anaerobic (CO2 or CO2/H2) and micro-aerobic atmospheres were used to stimulate growth. Three different strategies were tried out. First, the sample was spread on Petri dishes containing solid media with carboxymethylcellulose, microcrystalline cellulose or cellobiose. This technique allowed us to isolate two bacteria: Streptomyces sp. strain ABGxAviA1 and Pseudomonas sp. strain ABGxCellA. The second strategy consisted in inoculating a specific liquid medium containing carboxymethylcellulose, microcrystalline cellulose, or cellobiose. The samples were then spread on Petri dishes with the same specific medium containing carboxymethylcellulose, microcrystalline cellulose, or cellobiose. This led to the isolation of the mold Aspergillus sp. strain ABGxAviA2. Finally, the third strategy consisted in heating the first culture and spreading samples on agar plates containing rich medium. This led to the isolation of the bacterium Bacillus subtilis strain ABGx. All those steps were achieved in controlled atmospheres. The four enzyme-producing strains which were isolated were obtained by using a micro-aerobic atmosphere. Later, enzymatic assays were performed on the four strains. Streptomyces sp. strain ABGxAviA1 was found to produce only amylase, while Pseudomonas sp. strain ABGxCellA was found to produce ß-glucosidase as well. Aspergillus sp. strain ABGxAviA2 showed ß-glucosidase, amylase, cellulase, and xylanase activities. Finally, B. subtilis strain ABGx produced xylanase and amylase.

Symbiont diversity in Reticulitermes santonensis (Isoptera: Rhinotermitidae): investigation strategy through proteomics.

The complex microbial community living in the hindgut of lower termites includes prokaryotes, flagellates, yeasts, and filamentous fungi. Many microorganisms are found in the termite gut, but only a few are thought to be involved in symbiotic association to participate in cellulose digestion. Proteomics provides analyses from both taxonomical and functional perspectives. We aimed to identify symbiont diversity in the gut of Reticulitermes santonensis (Feytaud), via complementary electrospray ionization associated to ion trap tandem mass spectrometry (LC-MS/MS) and two-dimensional gel electrophoresis associated to matrix-assisted laser desorption-ionization-time-of-flight mass spectrometry analysis. One specific challenge to the study of lower termites is the relatively few data available on abundant symbiotic flagellates. Analysis based on LC-MS/MS revealed few protein families showing assignments to eukaryotes and the taxonomic origin of highly represented actins could not be established. Tubulins proved to be the most suitable protein family with which to identify flagellate populations from hindgut samples using LC-MS/MS, compared with other protein families, although this method targeted few prokaryotes in our assay. Similarly, two-dimensional gel electrophoresis associated to matrix-assisted laser desorption-ionization-time-of-flight mass spectrometry did not succeed in identifying flagellate populations, but did permit the identification of most of the prokaryotic components of the symbiotic system. Finally, fungi and yeasts were identified by both methods. Owing to the lack of sequenced genes in flagellates, targeting tubulins for LC-MS/MS could allow fingerprints of flagellate populations to be established. Experimental and technical improvements might increase the efficiency of identification of prokaryotic populations in the near future, based on metaproteomic development.

Characterization of a new oxidant-stable serine protease isolated by functional metagenomics.

A novel serine protease gene, SBcas3.3, was identified by functional screening of a forest-soil metagenomic library on agar plates supplemented with AZCL-casein. Overproduction in Escherichia coli revealed that the enzyme is produced as a 770-amino-acid precursor which is processed to a mature protease of ~55 kDa. The latter was purified by affinity chromatography for characterization with the azocasein substrate. The enzyme proved to be an alkaline protease showing maximal activity between pH 9 and 10 and at 50°C. Treatment with the chelating agent ethylenediaminetetraacetic acid irreversibly denatured the protease, whose stability was found to depend strictly on calcium ions. The enzyme appeared relatively resistant to denaturing and reducing agents, and its activity was enhanced in the presence of 10 ml/l nonionic detergent (Tween 20, Tween 80, or Triton X-100). Moreover, SBcas3.3 displayed oxidant stability, a feature particularly sought in the detergent and bleaching industries. SBcas3.3 was activated by hydrogen peroxide at concentrations up to 10 g/l and it still retained 30% of activity in 50 g/l H2O2.

Isolation and cultivation of a xylanolytic Bacillus subtilis extracted from the gut of the termite Reticulitermes santonensis.

The aim of this work was the isolation of xylanolytic microorganisms from the digestive tract of the termite Reticulitermes santonensis. The reducing sugars released after the hydrolysis of xylans can be further fermented to provide bioethanol. A xylanolytic strain of Bacillus subtilis was isolated from the hindgut of the termite and displayed amylase and xylanase activities. The bacterium was grown on media containing agricultural residues: wheat bran, wheat distiller's grains, and rapeseed oil cake. Wheat bran led to the highest induction of xylanase activity, although the development of the strain was less fast than in the other media. It was possible to reach maximal xylanase activities of 44.3, 33.5, and 29.1 I.U./ml in the media containing wheat bran, wheat distiller's grains, and rapeseed oil cake, respectively. Mass spectrometry identified a wide range of xylose oligomers, highlighting an endoxylanase activity. The enzyme was stable up to 45 °C and displayed an optimal pH close to 8.

Bacillus subtilis as a tool for screening soil metagenomic libraries for antimicrobial activities.

Finding new antimicrobial activities by functional metagenomics has been shown to depend on the heterologous host used to express the foreign DNA. Therefore, efforts are devoted to developing new tools for constructing metagenomic libraries in shuttle vectors replicatable in phylogenetically distinct hosts. Here we evaluated the use of the Escherichia coli-Bacillus subtilis shuttle vector pHT01 to construct a forest-soil metagenomic library. This library was screened in both hosts for antimicrobial activities against four opportunistic bacteria: Proteus vulgaris, Bacillus cereus, Staphylococcus epidermidis, and Micrococcus luteus. A new antibacterial activity against B. cereus was found upon screening in B. subtilis. The new antimicrobial agent, sensitive to proteinase K, was not active when the corresponding DNA fragment was expressed in E. coli. Our results validate the use of pHT01 as a shuttle vector and B. subtilis as a host to isolate new activities by functional metagenomics.

An eYFP reporter gene for the yeast two-hybrid system.

The yeast two-hybrid system is a powerful tool for detecting binary protein interactions, widely used in large-scale interactome mapping. We modified two yeast strains commonly used in yeast two-hybrid experiments by integrating into their genomes a new reporter gene encoding the enhanced yellow fluorescent protein eYFP. The suitability of this reporter gene for interaction screening was evaluated by fluorescence microscopy and fluorescence-activated cell sorting analysis. The gene shows good potential as a two-hybrid reporter gene for detecting strong interactions. Gal4 transcriptional activation gives rise to sufficient fluorescence for detection with a flow cytometer, but the eYFP reporter is not sensitive enough for detecting weak or moderate interactions. This study highlights the advantages of a fluorescent reporter gene in yeast two-hybrid screening.