Manuel Porcar

Effects of Bacillus thuringiensis δ-Endotoxins on the Pea Aphid (Acyrthosiphon pisum)

ABSTRACT Four Bacillus thuringiensis δ-endotoxins, Cry3A, Cry4Aa, Cry11Aa, and Cyt1Aa, were found to exhibit low to moderate toxicity on the pea aphid, Acyrthosiphon pisum, in terms both of mortality and growth rate. Cry1Ab was essentially nontoxic except at high rates. To demonstrate these effects, we had to use exhaustive buffer-based controls.

The ten grand challenges of synthetic life.

The construction of artificial life is one of the main scientific challenges of the Synthetic Biology era. Advances in DNA synthesis and a better understanding of regulatory processes make the goal of constructing the first artificial cell a realistic possibility. This would be both a fundamental scientific milestone and a starting point of a vast range of applications, from biofuel production to drug design. However, several major issues might hamper the objective of achieving an artificial cell. From the bottom-up to the selection-based strategies, this work encompasses the ten grand challenges synthetic biologists will have to be aware of in order to cope with the task of creating life in the lab.

Molecular and Insecticidal Characterization of a Cry1I Protein Toxic to Insects of the Families Noctuidae, Tortricidae, Plutellidae, and Chrysomelidae

ABSTRACT The most notable characteristic of Bacillus thuringiensis is its ability to produce insecticidal proteins. More than 300 different proteins have been described with specific activity against insect species. We report the molecular and insecticidal characterization of a novel cry gene encoding a protein of the Cry1I group with toxic activity towards insects of the families Noctuidae, Tortricidae, Plutellidae, and Chrysomelidae. PCR analysis detected a DNA sequence with an open reading frame of 2.2 kb which encodes a protein with a molecular mass of 80.9 kDa. Trypsin digestion of this protein resulted in a fragment of ca. 60 kDa, typical of activated Cry1 proteins. The deduced sequence of the protein has homologies of 96.1% with Cry1Ia1, 92.8% with Cry1Ib1, and 89.6% with Cry1Ic1. According to the Cry protein classification criteria, this protein was named Cry1Ia7. The expression of the gene in Escherichia coli resulted in a protein that was water soluble and toxic to several insect species. The 50% lethal concentrations for larvae of Earias insulana, Lobesia botrana, Plutella xylostella, and Leptinotarsa decemlineata were 21.1, 8.6, 12.3, and 10.0 μg/ml, respectively. Binding assays with biotinylated toxins to E. insulana and L. botrana midgut membrane vesicles revealed that Cry1Ia7 does not share binding sites with Cry1Ab or Cry1Ac proteins, which are commonly present in B. thuringiensis-treated crops and commercial B. thuringiensis-based bioinsecticides. We discuss the potential of Cry1Ia7 as an active ingredient which can be used in combination with Cry1Ab or Cry1Ac in pest control and the management of resistance to B. thuringiensis toxins.

Microbial Diversity in the Midguts of Field and Lab-Reared Populations of the European Corn Borer Ostrinia nubilalis

Background Insects are associated with microorganisms that contribute to the digestion and processing of nutrients. The European Corn Borer (ECB) is a moth present world-wide, causing severe economical damage as a pest on corn and other crops. In the present work, we give a detailed view of the complexity of the microorganisms forming the ECB midgut microbiota with the objective of comparing the biodiversity of the midgut-associated microbiota and explore their potential as a source of genes and enzymes with biotechnological applications. Methodological/Principal Findings A high-throughput sequencing approach has been used to identify bacterial species, genes and metabolic pathways, particularly those involved in plant-matter degradation, in two different ECB populations (field-collected vs. lab-reared population with artificial diet). Analysis of the resulting sequences revealed the massive presence of Staphylococcus warneri and Weissella paramesenteroides in the lab-reared sample. This enabled us to reconstruct both genomes almost completely. Despite the apparently low diversity, 208 different genera were detected in the sample, although most of them at very low frequency. By contrast, the natural population exhibited an even higher taxonomic diversity along with a wider array of cellulolytic enzyme families. However, in spite of the differences in relative abundance of major taxonomic groups, not only did both metagenomes share a similar functional profile but also a similar distribution of non-redundant genes in different functional categories. Conclusions/Significance Our results reveal a highly diverse pool of bacterial species in both O. nubilalis populations, with major differences: The lab-reared sample is rich in gram-positive species (two of which have almost fully sequenced genomes) while the field sample harbors mainly gram-negative species and has a larger set of cellulolytic enzymes. We have found a clear relationship between the diet and the midgut microbiota, which reveals the selection pressure of food on the community of intestinal bacteria.

Eubacteria and archaea communities in seven mesophile anaerobic digester plants in Germany.

BackgroundOnly a fraction of the microbial species used for anaerobic digestion in biogas production plants are methanogenic archaea. We have analyzed the taxonomic profiles of eubacteria and archaea, a set of chemical key parameters, and biogas production in samples from nine production plants in seven facilities in Thuringia, Germany, including co-digesters, leach-bed, and sewage sludge treatment plants. Reactors were sampled twice, at a 1-week interval, and three biological replicates were taken in each case.ResultsA complex taxonomic composition was found for both eubacteria and archaea, both of which strongly correlated with digester type. Plant-degrading Firmicutes as well as Bacteroidetes dominated eubacteria profiles in high biogas-producing co-digesters; whereas Bacteroidetes and Spirochaetes were the major phyla in leach-bed and sewage sludge digesters. Methanoculleus was the dominant archaea genus in co-digesters, whereas Methanosarcina and Methanosaeta were the most abundant methanogens in leachate from leach-bed and sewage sludge digesters, respectively.ConclusionsThis is one of the most comprehensive characterizations of the microbial communities of biogas-producing facilities. Bacterial profiles exhibited very low variation within replicates, including those of semi-solid samples; and, in general, low variation in time. However, facility type correlated closely with the bacterial profile: each of the three reactor types exhibited a characteristic eubacteria and archaea profile. Digesters operated with solid feedstock, and high biogas production correlated with abundance of plant degraders (Firmicutes) and biofilm-forming methanogens (Methanoculleus spp.). By contrast, low biogas-producing sewage sludge treatment digesters correlated with high titers of volatile fatty acid-adapted Methanosaeta spp.

iGEM 2.0—refoundations for engineering biology

volume 32 NumBeR 5 mAy 2014 nature biotechnology that, absent a duty to disclose, no disclosure is required. The periodic disclosure requirements—such as Form 10-Ks, Form 10-Qs and proxy statements, as well as the Form 8-K mandating current reporting obligations of material events—impose limits on that premise, while still providing meaningful protection to companies in many circumstances. Weakening that approach or mandating that companies report new developments more quickly than currently required would place additional burdens on companies to increase staff for this disclosure framework and could result in liability in more situations than is already the case. Many factors have contributed to our current state of US public company disclosure. The SEC’s current efforts to consider disclosure reform that could reduce the burdens of that disclosure are welcome. Biotech companies and BIO have an opportunity to contribute to the success of that effort and to help shape the reforms in ways that particularly benefit biotech companies and their investors. This is an opportunity to make their voices heard and help shape their disclosure destiny.

Effects of Bacillus thuringiensis Cry1Ab and Cry3Aa endotoxins on predatory Coleoptera tested through artificial diet-incorporation bioassays.

Traditional approaches to studying the effects of genetically modified (GM) crops on beneficial insects involve either field assays, comparing insect population levels between control and GM crops or tritrophic bioassays with contaminated insects - usually larvae or eggs of Lepidoptera - as preys. Here, we report the results of a bioassay using an artificial diet, suitable for predatory Coleoptera, to supply Bacillus thuringiensis (Bt) solubilized Cry1Ab and Cry3Aa as well as trypsin-activated Cry1Ab to Atheta coriaria and Cryptolaemus montrouzieri adults and young larvae of Adalia bipunctata. Water, solubilization buffer and trypsin-treated solubilization buffer were used as controls. In total, 1600 insects were assayed. Assays showed a relatively low mortality rate in the controls, ranging from as low as 7% after 15 days (C. montrouzieri) to about 15-20% after five days (A. bipunctata) or 15 days (A. coriaria). For all three predators, there were no statistical differences between the mortality recorded in any of the treatment groups and the corresponding controls. These results indicate a lack of short- (A. bipunctata) and long-term (A. coriaria and C. montrouzieri) mortality associated with oral ingestion of Cry1Ab and Cry3Aa at the high dose tested (50 microg ml-1). We discuss the relevance of these findings for the ecology of beneficial Coleoptera and compatibility with Bt and GM Bt crops.

Mealybugs nested endosymbiosis: going into the ‘matryoshka’ system in Planococcus citri in depth

BackgroundIn all branches of life there are plenty of symbiotic associations. Insects are particularly well suited to establishing intracellular symbiosis with bacteria, providing them with metabolic capabilities they lack. Essential primary endosymbionts can coexist with facultative secondary symbionts which can, eventually, establish metabolic complementation with the primary endosymbiont, becoming a co-primary. Usually, both endosymbionts maintain their cellular identity. An exception is the endosymbiosis found in mealybugs of the subfamily Pseudoccinae, such as Planococcus citri, with Moranella endobia located inside Tremblaya princeps.ResultsWe report the genome sequencing of M. endobia str. PCVAL and the comparative genomic analyses of the genomes of strains PCVAL and PCIT of both consortium partners. A comprehensive analysis of their functional capabilities and interactions reveals their functional coupling, with many cases of metabolic and informational complementation. Using comparative genomics, we confirm that both genomes have undergone a reductive evolution, although with some unusual genomic features as a consequence of coevolving in an exceptional compartmentalized organization.ConclusionsM. endobia seems to be responsible for the biosynthesis of most cellular components and energy provision, and controls most informational processes for the consortium, while T. princeps appears to be a mere factory for amino acid synthesis, and translating proteins, using the precursors provided by M. endobia. In this scenario, we propose that both entities should be considered part of a composite organism whose compartmentalized scheme (somehow) resembles a eukaryotic cell.

The Generalist Inside the Specialist: Gut Bacterial Communities of Two Insect Species Feeding on Toxic Plants Are Dominated by Enterococcus sp.

Some specialist insects feed on plants rich in secondary compounds, which pose a major selective pressure on both the phytophagous and the gut microbiota. However, microbial communities of toxic plant feeders are still poorly characterized. Here, we show the bacterial communities of the gut of two specialized Lepidoptera, Hyles euphorbiae and Brithys crini, which exclusively feed on latex-rich Euphorbia sp. and alkaloid-rich Pancratium maritimum, respectively. A metagenomic analysis based on high-throughput sequencing of the 16S rRNA gene revealed that the gut microbiota of both insects is dominated by the phylum Firmicutes, and especially by the common gut inhabitant Enterococcus sp. Staphylococcus sp. are also found in H. euphorbiae though to a lesser extent. By scanning electron microscopy, we found a dense ring-shaped bacterial biofilm in the hindgut of H. euphorbiae, and identified the most prominent bacterium in the biofilm as Enterococcus casseliflavus through molecular techniques. Interestingly, this species has previously been reported to contribute to the immobilization of latex-like molecules in the larvae of Spodoptera litura, a highly polyphagous lepidopteran. The E. casseliflavus strain was isolated from the gut and its ability to tolerate natural latex was tested under laboratory conditions. This fact, along with the identification of less frequent bacterial species able to degrade alkaloids and/or latex, suggest a putative role of bacterial communities in the tolerance of specialized insects to their toxic diet.

Confidence, tolerance, and allowance in biological engineering: The nuts and bolts of living things

The emphasis of systems and synthetic biology on quantitative understanding of biological objects and their eventual re‐design has raised the question of whether description and construction standards that are commonplace in electric and mechanical engineering are applicable to live systems. The tuning of genetic devices to deliver a given activity is generally context‐dependent, thereby undermining the re‐usability of parts, and predictability of function, necessary for manufacturing new biological objects. Tolerance (acceptable limits within the unavoidable divergence of a nominal value) and allowance (deviation introduced on purpose for the sake of flexibility and hence modularity, i.e. fitting together with a variety of other components) are key aspects of standardization that need to be brought to biological design. These should endow functional building blocks with a pre‐specified level of confidence for bespoke biosystems engineering. However, in the absence of more fundamental knowledge, fine‐tuning necessarily relies on evolutionary/combinatorial gravitation toward a fixed objective.