TASmania: A bacterial Toxin-Antitoxin Systems database

Bacterial Toxin-Antitoxin systems (TAS) are involved in key biological functions including plasmid maintenance, defense against phages, persistence and virulence. They are found in nearly all phyla and classified into 6 different types based on the mode of inactivation of the toxin, with the type II TAS being the best characterized so far. We have herein developed a new in silico discovery pipeline named TASmania, which mines the >41K assemblies of the EnsemblBacteria database for known and uncharacterized protein components of type I to IV TAS loci. Our pipeline annotates the proteins based on a list of curated HMMs, which leads to >2.106 loci candidates, including orphan toxins and antitoxins, and organises the candidates in pseudo- operon structures in order to identify new TAS candidates based on a guilt-by- association strategy. In addition, we classify the two-component TAS with an unsupervised method on top of the pseudo-operon (pop) gene structures, leading to 1567 “popTA” models offering a more robust classification of the TAs families. These results give valuable clues in understanding the toxin/antitoxin modular structures and the TAS phylum specificities. Preliminary in vivo work confirmed six putative new hits in Mycobacterium tuberculosis as promising candidates. The TASmania database is available on the following server https://shiny.bioinformatics.unibe.ch/apps/tasmania/

TASmania: A bacterial Toxin-Antitoxin Systems database.

Bacterial Toxin-Antitoxin systems (TAS) are involved in key biological functions including plasmid maintenance, defense against phages, persistence and virulence. They are found in nearly all phyla and classified into 6 different types based on the mode of inactivation of the toxin, with the type II TAS being the best characterized so far. We have herein developed a new in silico discovery pipeline named TASmania, which mines the >41K assemblies of the EnsemblBacteria database for known and uncharacterized protein components of type I to IV TAS loci. Our pipeline annotates the proteins based on a list of curated HMMs, which leads to >2.106 loci candidates, including orphan toxins and antitoxins, and organises the candidates in pseudo-operon structures in order to identify new TAS candidates based on a guilt-by-association strategy. In addition, we classify the two-component TAS with an unsupervised method on top of the pseudo-operon (pop) gene structures, leading to 1567 "popTA" models offering a more robust classification of the TAs families. These results give valuable clues in understanding the toxin/antitoxin modular structures and the TAS phylum specificities. Preliminary in vivo work confirmed six putative new hits in Mycobacterium tuberculosis as promising candidates. The TASmania database is available on the following server https://shiny.bioinformatics.unibe.ch/apps/tasmania/

Extending the chemical space of CAZymes: how and why?

International audienc

Extending the chemical space of CAZymes

International audienc

Extending the chemical space of CAZymes

International audienc

Biogeochemical controls of the transport and cycling of persistent organic pollutants in the polar oceans

Humanity is currently using more than 200000 synthetic organic compounds in many industrial, agricultural and domestic applications. Many of these chemicals reach the environment and have a harmful effect on ecosystems and humans. Among them, the group of persistent organic pollutants (POPs) comprises several families of compounds that have physical and chemical properties that give them the ability to be distributed and impact globally (semivolatility, high persistence and bioaccumulation capacity due to their hydrophobicity). In the present thesis, the coupling of atmospheric transport and biogeochemical cycles in the Arctic and Southern Ocean has been studied for Hexachlorocyclohexanes (HCHs), Hexachlorobenzene (HCB) and Polychlorinated Biphenyls (PCBs). Three oceanographic cruises were conducted, one in the North Atlantic and the Arctic Ocean (2007) and two in the Southern Ocean surrounding the Antarctic Peninsula (2008 and 2009). During these campaigns, air (gas and particulate), water (dissolved and particulate) and biota (phytoplankton) were sampled simultaneously allowing to report a complete picture of POPs cycling in polar areas. In the case of the Southern Ocean, the largest data set available for PCBs, HCH and HCB has been generated. The atmospheric and seawater concentrations were low, among the lowest reported for the Polar Oceans, and in the case of the Southern Ocean there is a clear historical trend of decreasing concentrations, consistent with reduced emissions in source regions. Long range atmospheric transport was identified as the main POPs input to polar ecosystems agreeing with previous works. However, it has been found that secondary local sources from soil and snow influences strongly the atmospheric concentrations overland in the Antarctic region, and over the adjacent Southern ocean in the case of HCHs. Atmospheric residence times calculated from the measurements were in agreement with the prediction from environmental fate models. The atmospheric residence times were longer for the less hydrophobic PCBs and shorter for the more hydrophobic, consistent with the role of the biological pump sequestering atmospheric PCBs. Once POPs reach the Polar regions the main route of entry of these compounds to surface waters is by atmospheric deposition, mainly by diffusive exchange between the gas and dissolved phase with minor contributions from dry deposition of aerosol bound POPs. Estimated bioconcentration factors revealed that concentration of POPs in phytoplankton were correlated with the chemical hydrophobicity, but some discrepancies with model predictions were observed. The biological and degradative pumps are identified as the two main processes that control the fate and occurrence of POPs in the surface water column, and also are able to modulate the atmospheric transport of POPs to remote areas. POPs such HCHs are prone to be efficiently degraded by bacterial communities in surface waters, depleting the seawater concentrations and increasing the diffusive air-water exchange to the Arctic and Southern Ocean. Conversely, the biological pump decreases the dissolved phase concentrations of the more hydrophobic PCB congeners increasing the air to water fugacity gradients and enhancing the diffusive air-water exchange fluxes. This is the first time that the influence of the biological pump on POP cycling is demonstrated for Oceanic waters. Finally, HCB was close to air-water equilibrium showing that neither the biological and degradative pumps are efficient sequestration processes for the highly persistent and mid-hydrophobic compounds. Overall, the results show clearly that biogeochemical processes occurring in the water column affect the atmospheric deposition and long range transport of POPs to remote regions.The magnitude of these processes may show a clear seasonality and are suitable to be perturbed under the current scenario of climate change.En la actualidad se usan en aplicaciones domésticas más de 200.000 compuestos orgánicos sintéticos. Muchos de estos compuestos químicos que se liberan al medio ambiente son nocivos para el medio ambiente y los humanos. Entre estos compuestos se encuentran los contaminantes orgánicos persistentes (COPs) que comprenden una serie de familias de compuestos que comparten una serie de características físico-químicas que les permiten estar distribuidos globalmente (semivolatilidad, elevada persistencia y capacidad de bioacumulacion por sus características hidrofóbicas). En la presente tesis doctoral se ha estudiado en profundidad el acoplamiento entre el transporte atmosférico y los ciclos biogeoquímicos Hexaclorociclohexanos (HCHs), Hexaclorobenceno (HCB) y Bifenilos Policlorados (PCBs) en los Océanos Polar Ártico y Polar Antártico. Durante esta tesis se han realizado tres campañas oceanográficas, una al Atlántico Norte y al Océano Polar Ártico (2007), y dos en el Océano Polar Antártico y en aguas circundantes a la Peninsula Antártica (2008 y 2009). Durante estas tres campañas oceanográficas se han tomado muestras de aire (gas y particulado), agua (disuelto y particulado) y biota (fitoplankton) de forma simultánea lo que permitió tener una amplio conocimiento de el ciclo de los COPs en zonas polares. En el caso de el Océano Polar Antártico y en aguas circundantes a la Peninsula Antártica se ha generado la mayor cantidad de datos en un mismo trabajo, incluso se han generado datos que hasta ahora no se habían publicado como las concentraciones de fitoplankton. La concentraciones medidas en el la atmósgera y aguas superficiales fueron bajas, siendo en algunos casos las concentraciones más bajas jamás encontradas en el océano polares, en el caso de el Océano Polar Antártico se ha encontrado una significativa tendencia histórica de concentraciones decrecientos lo cual es consistente con la reducción de emisiones de COPs en origen. El transporte atmosférico a larga distancia ha sido identificado como la vía de entrada principal de entrada de los COPs a sistemas polares. Sin embargo, se ha encontrado que hay fuentes secundarias provenientes de el suelo y la nieve con una clara influencia sobre las concentraciones atmosféricas en zonas de el continente Antártico y aguas costeras adyacentes en el caso de los HCHs. Los tiempos de residencia atmosférica calculados están en los mismos rangos con los modelos predictivos. Los tiempos de residencia atmosférica fieron más largos para los compuestos menos hidrofóbicos y más cortos para los más hidrófobicos lo cual es consistente con la bomba biológica. Una vez estos compuestos alcanzan las regions polares la principal ruta de entrada de estos compuestos al agua superficial es por deposición atmosférica, principalmente por intercambio difusivo entre la fase gas y la fase disuelta, se ha comprobado que la contribución de la deposición seca es significativamente menor. Los factores de bioconcentración revelaron que la concentración de COPs en el fitoplankton se correlacionaba con la hidrofobicidad química, pero se encontraron discrepancias con los modelos predictivos. Las bombas biológica y degradative han sido identificadas como los dos procesos principals que controlan el destino y ocurrencia de COPs en la columna de agua superficial e incluso son capaces de modular el transporte atmosférico de COPs a areas remotes. COPs como los HCHs son eficientemente degradados por las comunidades bacterianas de aguas superficiales disminuyendo su concentraciéon aumentando los flujos difusivos de deposición entre la fase gas y la superficie disuelta en el Océano Polar Antártico y en aguas circundantes a la Peninsula Antártica. Por otro lado, la bomba biológica disminuye las concentraciones de el disuelto de los COPs más hidrofóbicos aumentando el gradiente de fugacidades y favoreciendo la deposición por intercambio difusivo aire-agua. La presente tesis es la primera que ha demostrado la influencia de la bomba biológica influye de forma significativa el ciclo de los COPs.El HCB se ha encontrado en equilibrio en ambas zonas de estudio y no se ha demostrado que hubiera influencia de la bomba biológica o de procesos degradativos en aguas superficiales. Como conclusion final se ha demostrado a través de los resultados que los procesos biogeoquímicos en la columna de agua afectan a la deposición atmosférica y el transporte a larga distancia de COPs a regiones remotas. La magnitud de estos procesos muestra una clara estacionalidad que puede ser perturbada en un actual escenario de cambio climático

Enhancing non-canonical amino acid incorporation towards enzyme engineering upgrading Genetic code expansion tool improvement towards biocatalytic reprogramming

International audienceStandard enzyme engineering strategies relies on one or several amino acids permutations among the 20 amino acids (AA). The natural AA repertoire displays only limited chemical functions, which further restrain potentialities of engineering tailored proteins. To circumvent such limitation, systems have been developed over the last decades to incorporate into proteins non-canonical amino acids (ncAAs) with non-naturally encountered chemical functions [1]. The applications of ncAAs use are multiple, including protein labeling [2], protein immobilization, and in depth redesign of enzyme active sites, thus opening avenues for new catalytic opportunities [3,4]. Despite the tremendous potential of ncAAs, their use is still limited because of technical constraints. The main bottleneck consists in the poor incorporation efficiency, which can be dependent or at least related to the ncAA itself, the incorporation position and the target protein. Optimizing the incorporation system is required to overcome these limitations to efficiently produce proteins with ncAA incorporation in a more versatile way and at high production yields.Genetic code expansion is based on the reassignment of a nonsense codon to an ncAA by introducing an orthogonal amino-acyl tRNA synthetase (aaRS)/tRNA pair. In E. coli, the pEVOL system is the historical and most widely used [5]. The pUltra system allows improved incorporation efficiencies in some conditions and can be combined with the pEVOL system for the incorporation of two different ncAAs [6]. While these systems have proven their value, the incorporation efficiency remains highly variable.To go further in improvement and provide to the community a more efficient tool for ncAA incorporation, the pINS system has been developed. We focused on the expression levels of both the tRNA and the aaRS. We have demonstrated that the incorporation efficiency was increased for the three different tested ncAAs, either at the surface or surrounding the catalytic site. In addition, the incorporation position bias observed with standard systems was suppressed. The pINS system allows satisfying incorporation efficiencies with reduced ncAAs concentrations. Finally, the overall production level was increased up to 4-fold compared to pEVOL. The pINS system, making ncAA incorporation more efficient, more reliable and cheaper, should facilitate the use of ncAA in many areas of enzyme engineering

Enhancing non-canonical amino acid incorporation towards enzyme engineering upgrading Genetic code expansion tool improvement towards biocatalytic reprogramming

Standard enzyme engineering strategies relies on one or several amino acids permutations among the 20 amino acids (AA). The natural AA repertoire displays only limited chemical functions, which further restrain potentialities of engineering tailored proteins. To circumvent such limitation, systems have been developed over the last decades to incorporate into proteins non-canonical amino acids (ncAAs) with non-naturally encountered chemical functions [1]. The applications of ncAAs use are multiple, including protein labeling [2], protein immobilization, and in depth redesign of enzyme active sites, thus opening avenues for new catalytic opportunities [3,4]. Despite the tremendous potential of ncAAs, their use is still limited because of technical constraints. The main bottleneck consists in the poor incorporation efficiency, which can be dependent or at least related to the ncAA itself, the incorporation position and the target protein. Optimizing the incorporation system is required to overcome these limitations to efficiently produce proteins with ncAA incorporation in a more versatile way and at high production yields.Genetic code expansion is based on the reassignment of a nonsense codon to an ncAA by introducing an orthogonal amino-acyl tRNA synthetase (aaRS)/tRNA pair. In E. coli, the pEVOL system is the historical and most widely used [5]. The pUltra system allows improved incorporation efficiencies in some conditions and can be combined with the pEVOL system for the incorporation of two different ncAAs [6]. While these systems have proven their value, the incorporation efficiency remains highly variable.To go further in improvement and provide to the community a more efficient tool for ncAA incorporation, the pINS system has been developed. We focused on the expression levels of both the tRNA and the aaRS. We have demonstrated that the incorporation efficiency was increased for the three different tested ncAAs, either at the surface or surrounding the catalytic site. In addition, the incorporation position bias observed with standard systems was suppressed. The pINS system allows satisfying incorporation efficiencies with reduced ncAAs concentrations. Finally, the overall production level was increased up to 4-fold compared to pEVOL. The pINS system, making ncAA incorporation more efficient, more reliable and cheaper, should facilitate the use of ncAA in many areas of enzyme engineering

Substrate recognition and cryo-EM structure of the ribosome-bound TAC toxin of Mycobacterium tuberculosis

International audienceToxins of toxin-antitoxin systems use diverse mechanisms to control bacterial growth. Here, we focus on the deleterious toxin of the atypical tripartite toxin-antitoxin-chaperone (TAC) system of Mycobacterium tuberculosis, whose inhibition requires the concerted action of the antitoxin and its dedicated SecB-like chaperone. We show that the TAC toxin is a bona fide ribonuclease and identify exact cleavage sites in mRNA targets on a transcriptome-wide scale in vivo. mRNA cleavage by the toxin occurs after the second nucleotide of the ribosomal A-site codon during translation, with a strong preference for CCA codons in vivo. Finally, we report the cryo-EM structure of the ribosome-bound TAC toxin in the presence of native M. tuberculosis cspA mRNA, revealing the specific mechanism by which the TAC toxin interacts with the ribosome and the tRNA in the P-site to cleave its mRNA target

Genetic Incorporation of Non-canonical Amino Acids in Anti-HER2 VHH: Expression and Characterization

International audienceNanobodies-or VHH-are small proteins (~120 residues) issued from antibodies with intact recognition for the original target of the antibody. In the present study, we show the possibility of incorporating non-canonical amino acids at precise locations of the sequence via classical genetic techniques (Genetic Code Expansion). We demonstrate that the amount of recombinant protein obtained is compatible with a large production format. We show that this protein can be purified, that its sequence corresponds to the theoretical molecular weight, and that the two non-canonical amino acids are incorporated at the desired locations of the sequence. Finally, we show by surface plasmon resonance (SPR) that the affinity of these VHHs is maintained towards their target, HER2