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β-lactamases are the main molecules responsible for giving bacterial resistance against β-lactam antibiotics. The study of β-lactamases has allowed the development of antibiotics capable of inhibiting these enzymes. In this context, extended spectrum β-lactamase (ESBL) TLA-1 has spread in Escherichia coli and Enterobacter cloacae clinical isolates during the last 30 years in Mexico. In this research, the 3D structures of ESBL TLA-1 and TLA-1 S70G mutant, both ligand-free and in complex with clavulanic acid were determined by X-ray crystallography. Four clavulanic acid molecules were found in the structure of TLA-1, two of those were intermediaries of the acylation process and were localized covalently bound to two different amino acid residues, Ser70 and Ser237. The coordinates of TLA-1 in complex with clavulanic acid shows the existence of a second acylation site, additional to Ser70, which might be extendable to several members of the subclass A β-lactamases family. This is the first time that two serines involved in binding clavulanic acid has been reported and described to an atomic level.

Next-generation technologies for determination of genomics and transcriptomics composition have a wide range of applications. Moreover, the development of tools for big data set analysis has allowed the identification of molecules and networks involved in metabolism, evolution or behavior. By natural habitats aquatic organisms have implemented molecular strategies for survival, including the production and secretion of toxic compounds for their predators; therefore these organisms are possible sources of proteins or peptides with potential biotechnological application. In the last decade anthozoans, mainly octocorals but also sea anemones, have been proben to be a source of natural products. Members of the genus Anthopleura are one of the best known and most studied sea anemones because they are common constituents of rocky intertidal communities and show interesting ecological and biological phenomena (e.g. intraespecific competition, symbiosis, etc.); however, many aspects of these taxa remain in need to be analyzed. This work describes the transcriptome sequencing of Anthopleura dowii Verrill, 1869 (Cnidaria: Anthozoa: Actiniaria); this is the first report of this kind for these species. The data set used to construct the transcriptome has been deposited on NCBI's database. Illumina sequence reads are available under BioProject accession number PRJNA329297 and Sequence Read Archive under accession number SRP078992.

During X-ray data collection from a multicopper oxidase (MCO) crystal, electrons and protons are mainly released into the system by the radiolysis of water molecules, leading to the X-ray-induced reduction of O2to 2H2O at the trinuclear copper cluster (TNC) of the enzyme. In this work, 12 crystallographic structures ofThermus thermophilusHB27 multicopper oxidase (Tth-MCO) in holo, apo and Hg-bound forms and with different X-ray absorbed doses have been determined. In holoTth-MCO structures with four Cu atoms, the proton-donor residue Glu451 involved in O2reduction was found in a double conformation: Glu451a (∼7 Å from the TNC) and Glu451b (∼4.5 Å from the TNC). A positive peak of electron density above 3.5σ in anFo−Fcmap for Glu451a O∊2indicates the presence of a carboxyl functional group at the side chain, while its significant absence in Glu451b strongly suggests a carboxylate functional group. In contrast, for apoTth-MCO and in Hg-bound structures neither the positive peak nor double conformations were observed. Together, these observations provide the first structural evidence for a proton-relay mechanism in the MCO family and also support previous studies indicating that Asp106 does not provide protons for this mechanism. In addition, eight composite structures (Tth-MCO-C1–8) with different X-ray-absorbed doses allowed the observation of different O2-reduction states, and a total depletion of T2Cu at doses higher than 0.2 MGy showed the high susceptibility of this Cu atom to radiation damage, highlighting the importance of taking radiation effects into account in biochemical interpretations of an MCO structure.

Sea anemone venom contains a complex and diverse arsenal of peptides and proteins of pharmacological and biotechnological interest, however, only venom from a few species has been explored from a global perspective to date. In the present study, we identified the polypeptides present in the venom of the sea anemone Anthopleura dowii Verrill, 1869 through a transcriptomic and proteomic analysis of the tentacles and the proteomic profile of the secreted mucus. In our transcriptomic results, we identified 261 polypeptides related to or predicted to be secreted in the venom, including proteases, neurotoxins that could act as either potassium (K+) or sodium (Na+) channels inhibitors, protease inhibitors, phospholipases A2, and other polypeptides. Our proteomic data allowed the identification of 156 polypeptides&mdash, 48 exclusively identified in the mucus, 20 in the tentacles, and 88 in both protein samples. Only 23 polypeptides identified by tandem mass spectrometry (MS/MS) were related to the venom and 21 exclusively identified in the mucus, most corresponding to neurotoxins and hydrolases. Our data contribute to the knowledge of evolutionary and venomic analyses of cnidarians, particularly of sea anemones.

Cry and Cyt toxins produced by Bacillus thuringiensis are insecticidal proteins used worldwide to control different insect pests, either as spray products or expressed in transgenic Bt-crops. Extensive studies have shown that they are safe to the environment and non-toxic to other organisms. However, still there are concerns regarding to the potential impact of Bt-crops on non-target organisms. One possibility to counteract the action of Cry or Cyt toxins is the use of dominant negative mutants also called anti-toxin that could inhibit the toxicity of their corresponding native toxin. Additionally, the evolution of insect resistance threatens the effectiveness of Bt-crops, since several examples of insects resistant to Bt-crops have been recently documented. The study of the mode of action of Cry and Cyt toxins has shown that oligomerization is an important step to form pores in the target midgut cells to eventually kill the larvae. This knowledge allowed us to develop strategies to produce toxin mutants that act as anti-toxins to counter Cry or Cyt toxins action in specific environments and to construct CryMod toxins that are able to kill insects that developed resistance to native Cry toxins. We will review the constructions of Cry and Cyt anti-toxins. The non-toxic helix α-4 mutants of Cry1Ab are able to oligomerize and to interact with native toxin forming inactive hetero-oligomers, inhibiting toxicity of native Cry1Ab. On the other hand, the N-terminal domain of Cyt1Aa induces oligomerization of native toxin, and inhibits its toxicity against mosquitoes as well as its hemolytic activity. Regarding insect resistance, we will review the construction of Cry1AMod toxins lacking helix α-1 that are able to form oligomers in absence of binding to cadherin receptor. CryMod toxins kill resistant insects to native Bt toxins affected in cadherin. We will review and discuss recent data that indicated that CryMod toxins overcome other mechanisms of resistance. Overall the mutants reviewed here support that oligomerization is an important step in the mechanism of action of Cry and Cyt toxins.

Insecticidal Cry proteins produced by Bacillus thuringiensis are use worldwide in transgenic crops for efficient pest control. Among the family of Cry toxins, the three domain Cry family is the better characterized regarding their natural evolution leading to a large number of Cry proteins with similar structure, mode of action but different insect specificity. Also, this group is the better characterized regarding the study of their mode of action and the molecular basis of insect specificity. In this review we discuss how Cry toxins have evolved insect specificity in nature and analyse several cases of improvement of Cry toxin action by genetic engineering, some of these examples are currently used in transgenic crops. We believe that the success in the improvement of insecticidal activity by genetic evolution of Cry toxins will depend on the knowledge of the rate-limiting steps of Cry toxicity in different insect pests, the mapping of the specificity binding regions in the Cry toxins, as well as the improvement of mutagenesis strategies and selection procedures.

Bacillus thuringiensis Cry toxins have been widely used in the control of insect pests either as spray products or expressed in transgenic crops. These proteins are pore forming toxins with a complex mechanism of action that involves the sequential interaction with several toxin-receptors. Cry toxins are specific against susceptible larvae and although they are often highly effective, some insect pests are not affected by them or show low susceptibility. In addition, the development of resistance threatens their effectiveness, so strategies to cope with all these problems are necessary. In this review we will discuss and compare the different strategies that have been used to improve insecticidal activity of Cry toxins. The activity of Cry toxins can be enhanced by using additional proteins in the bioassay like serine protease inhibitors, chitinases, Cyt toxins, or a fragment of cadherin receptor containing a toxin-binding site. On the other hand, different modifications performed in the toxin gene such as site directed mutagenesis, introduction of cleavage sites in specific regions of the protein, and deletion of small fragments from the amino-terminal region lead to improved toxicity or overcome resistance, representing interesting alternatives for insect pest control.

Cry11Aa and Cyt1Aa of Bacillus thuringiensis are active against mosquitoes and show synergism. Cyt1Aa functions as a membrane receptor inducing Cry11Aa oligomerization. Here we characterized Cry11Aa helix α-3 mutants impaired in oligomerization and toxicity against Aedes aegypti , indicating that oligomerization of Cry11Aa is important for toxin action. Cyt1Aa did not recover the insecticidal activity of Cry11Aa mutants.

Bacillus thuringiensis subsp. israelensis produces three Cry toxins (Cry4Aa, Cry4Ba and Cry11Aa) that are active against Aedes aegypti larvae. The identification of the rate-limiting binding steps of Cry toxins that are used for insect control in the field, such as those of B. thuringiensis subsp. israelensis, should provide targets for improving insecticides against important insect pests. Previous studies showed that Cry11Aa binds to cadherin receptor fragment CR7–11 (cadherin repeats 7–11) with high affinity. Binding to cadherin has been proposed to facilitate Cry toxin oligomer formation. In the present study, we show that Cry4Ba binds to CR7–11 with 9-fold lower binding affinity compared with Cry11Aa. Oligomerization assays showed that Cry4Ba is capable of forming oligomers when proteolytically activated in vitro in the absence of the CR7–11 fragment in contrast with Cry11Aa that formed oligomers only in the presence of CR7–11. Pore-formation assays in planar lipid bilayers showed that Cry4Ba oligomers were proficient in opening ion channels. Finally, silencing the cadherin gene by dsRNA (double-stranded RNA) showed that silenced larvae were more tolerant to Cry11Aa in contrast with Cry4Ba, which showed similar toxic levels to those of control larvae. These findings show that cadherin binding is not a limiting step for Cry4Ba toxicity to A. aegypti larvae.

BackgroundBacillus thuringiensis Cry toxins are used worldwide in the control of different insect pests important in agriculture or in human health. The Cry proteins are pore-forming toxins that affect the midgut cell of target insects. It was shown that non-toxic Cry1Ab helix α-4 mutants had a dominant negative (DN) phenotype inhibiting the toxicity of wildtype Cry1Ab when used in equimolar or sub-stoichiometric ratios (1∶1, 0.5∶1, mutant∶wt) indicating that oligomer formation is a key step in toxicity of Cry toxins.Methodology/principal findingsThe DN Cry1Ab-D136N/T143D mutant that is able to block toxicity of Cry1Ab toxin, was used to analyze its capacity to block the activity against Manduca sexta larvae of other Cry1 toxins, such as Cry1Aa, Cry1Ac, Cry1Ca, Cry1Da, Cry1Ea and Cry1Fa. Cry1Ab-DN mutant inhibited toxicity of Cry1Aa, Cry1Ac and Cry1Fa. In addition, we isolated mutants in helix α-4 of Cry4Ba and Cry11Aa, and demonstrate that Cry4Ba-E159K and Cry11Aa-V142D are inactive and completely block the toxicity against Aedes aegypti of both wildtype toxins, when used at sub-stoichiometric ratios, confirming a DN phenotype. As controls we analyzed Cry1Ab-R99A or Cry11Aa-E97A mutants that are located in helix α-3 and are affected in toxin oligomerization. These mutants do not show a DN phenotype but were able to block toxicity when used in 10∶1 or 100∶1 ratios (mutant∶wt) probably by competition of binding with toxin receptors.Conclusions/significanceWe show that DN phenotype can be observed among different Cry toxins suggesting that may interact in vivo forming hetero-oligomers. The DN phenotype cannot be observed in mutants affected in oligomerization, suggesting that this step is important to inhibit toxicity of other toxins.

9 p., 1 table, 5 figures and bibliography, The Cyt toxins produced by the bacteria Bacillus thuringiensis show insecticidal activity against some insects, mainly dipteran larvae, being able to kill mosquitoes and black flies. However, they also possess a general cytolytic activity in vitro, showing hemolytic activity in red blood cells. These proteins are composed of two outer layers of α-helix hairpins wrapped around a δ-sheet. With regard to their mode of action, one model proposed that the two outer layers of α-helix hairpins swing away from the δ-sheet, allowing insertion of δ-strands into the membrane forming a pore after toxin oligomerization. The other model suggested a detergent-like mechanism of action of the toxin on the surface of the lipid bilayer. In this work, we cloned the N- and C-terminal domains form Cyt1Aa and analyzed their effects on Cyt1Aa toxin action. The N-terminal domain shows a dominant negative phenotype inhibiting the in vitro hemolytic activity of Cyt1Aa in red blood cells and the in vivo insecticidal activity of Cyt1Aa against Aedes aegypti larvae. In addition, the N-terminal region is able to induce aggregation of the Cyt1Aa toxin in solution. Finally, the C-terminal domain composed mainly of δ-strands is able to bind to the SUV liposomes, suggesting that this region of the toxin is involved in membrane interaction. Overall, our data indicate that the two isolated domains of Cyt1Aa have different roles in toxin action. The N-terminal region is involved in toxin aggregation, while the C-terminal domain is involved in the interaction of the toxin with the lipid membrane., This research was funded in part through National Institutes of Health Grant 1R01 AI066014, Grants DGAPA/UNAM IN218608 and IN210208-N, and CONACyT Grant U48631-Q 478. I.R.d.E. received a José Castillejo postdoctoral grant and a mobility grant for teaching and research staff of Universidad Publica de Navarra-Gobierno de Navarra.

Bacillus thuringiensis Cry toxins are used worldwide as insecticides in agriculture, in forestry, and in the control of disease transmission vectors. In the lepidopteran Manduca sexta, cadherin (Bt-R(1)) and aminopeptidase-N (APN) function as Cry1A toxin receptors. The interaction with Bt-R(1) promotes cleavage of the amino-terminal end, including helix alpha-1 and formation of prepore oligomer that binds to APN, leading to membrane insertion and pore formation. Loops of domain II of Cry1Ab toxin are involved in receptor interaction. Here we show that Cry1Ab mutants located in domain II loop 3 are affected in binding to both receptors and toxicity against Manduca sexta larvae. Interaction with both receptors depends on the oligomeric state of the toxin. Monomers of loop 3 mutants were affected in binding to APN and to a cadherin fragment corresponding to cadherin repeat 12 but not with a fragment comprising cadherin repeats 7-12. In contrast, the oligomers of loop 3 mutants were affected in binding to both Bt-R(1) fragments but not to APN. Toxicity assays showed that either monomeric or oligomeric structures of Cry1Ab loop 3 mutations were severely affected in insecticidal activity. These data suggest that loop 3 is differentially involved in the binding with both receptor molecules, depending on the oligomeric state of the toxin and also that possibly a "ping pong" binding mechanism with both receptors is involved in toxin action.