Unraveling corynebacterial divisome composition by proximity labeling in the living cell

Bacterial cell division is directed by the divisome, a protein complex whose assembly begins with the polymerization of FtsZ at midcell to form a ring (Z-ring) This structure participates in the recruitment of other divisome proteins, that in the case of the model bacilli (Escherichia coli and Bacillus subtilis) have been identified and characterized. However, the order Corynebacteriales (that includes important human pathogens as Mycobacterium tuberculosis and Corynebacterium diphtheriae) lacks recognizable homologues for many of these cell division proteins, and the ones fulfilling these missing functions are yet to be identified. To identify the unknown pieces of the corynebacterial divisome, we developed and optimized a proteomic strategy based on proximity biotinylation in the living cell, using Corynebacterium glutamicum as a model organism. We generated a strain expressing FtsZ fused to an engineered ascorbate peroxidase (APEX2). APEX2 catalyzes the oxidation of phenol biotin in the presence of H2O2 giving rise to a radical that reacts with amino acids of nearby proteins. This allowed us to label the proteomic environment of FtsZ in the living cell, and its purification and identification by Mass Spectrometry. We corroborated that APEX2 is active in the biochemical background of C. glutamicum, and optimized the labelling strategy to guarantee the identification of physiologically relevant FtsZ neighbours. We identified a confident list of 253 FtsZ neighbors, that includes known cell division proteins as well as an important number of non-characterized proteins, which represents putative new divisome components. We focused on hypothetical membrane proteins, that might mediate membrane anchor of the Z-ring, as most of the proteins fulfilling this role in E. coli and B. subtilis are not present in corynebacterial genomes. We generate strains expressing the selected candidates fused to a fluorescent proof to evaluate their subcellular localization and their interaction with FtsZ. The results allowed us to identify new conserved membrane bound components of the corynebacterial divisome. Their precise role in cell division, the molecular details of its interaction with the Z-ring and its regulation by protein phosphorylation are being studied.Agencia Nacinal de Investigación e InnovaciónECOS-Sud France-Uruguay U20B0

Complete Genome Sequence of Bradyrhizobium sp. Strain C-145, a Nitrogen-Fixing Rhizobacterium Used as a Peanut Inoculant in Argentina

We present the complete genome sequence of Bradyrhizobium sp. strain C-145, one of the most widely used nitrogen-fixing rhizobacteria for inoculating peanut crops in Argentina. The genome consists of 9.53 Mbp in a single circular chromosome and was determined using a hybrid long- and short-read assembly approach.Instituto de Microbiología y Zoología Agrícola (IMYZA)Fil: Nievas, Fiorela. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Físico-Químicas y Naturales. Departamento de Biología Molecular. Instituto de Biotecnología Ambiental y Salud; ArgentinaFil: Revale, Santiago. University of Oxford. Wellcome Centre for Human Genetics; Reino UnidoFil: Foresto, Emiliano. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Físico-Químicas y Naturales. Departamento de Biología Molecular. Instituto de Biotecnología Ambiental y Salud; ArgentinaFil: Cossovich, Sacha. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Físico-Químicas y Naturales. Departamento de Biología Molecular. Instituto de Biotecnología Ambiental y Salud; ArgentinaFil: Puente, Mariana Laura. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Microbiología y Zoología Agrícola. Laboratorio de Bacterias Promotoras del Crecimiento Vegetal; ArgentinaFil: Alzari, Pedro. Université de Paris. Institut Pasteur. Unité de Microbiologie Structurale; FranciaFil: Martínez, Mariano. Université de Paris. Institut Pasteur. Unité de Microbiologie Structurale; FranciaFil: Ben-Assaya, Mathilde. Université de Paris. Institut Pasteur. Unité de Microbiologie Structurale; FranciaFil: Mornico, Damien. Institut Pasteur. Département Biologie Computationnelle. Hub de Bioinformatique et Biostatistique; FranciaFil: Santoro, Maricel. Max Planck for Chemical Ecology. Department of Biochemistry; FranciaFil: Martínez-Abarca, Francisco. Estación Experimental del Zaidín. Department of Plant and Soil Microbiology. Structure, Dynamics, and Function of Rhizobacterial Genomes; EspañaFil: Giordano, Walter. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Físico-Químicas y Naturales. Departamento de Biología Molecular. Instituto de Biotecnología Ambiental y Salud (INBIAS-CONICET); ArgentinaFil: Bogino, Pablo. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Físico-Químicas y Naturales. Departamento de Biología Molecular. Instituto de Biotecnología Ambiental y Salud (INBIAS-CONICET); Argentin

Genome sequence of Mesorhizobium mediterraneum strain R31, a nitrogen-fixing rhizobium used as an inoculant for chickpea in Argentina

Here, we report the complete genome sequence of Mesorhizobium mediterraneum R31, a rhizobial strain recommended and used as a commercial inoculant for chickpea in Argentina. The genome consists of 7.25 Mb, distributed into four circular replicons: a chromosome of 6.72 Mbp and three plasmids of 0.29, 0.17, and 0.07 Mbp.Instituto de Microbiología y Zoología Agrícola (IMYZA)Fil: Foresto, Emiliano. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Físico-Químicas y Naturales. Instituto de Biotecnología Ambiental y Salud. Departamento de Biología Molecular; ArgentinaFil: Revale, Santiago. University of Oxford. Wellcome Centre for Human Genetics; Reino UnidoFil: Nievas, Fiorela. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Físico-Químicas y Naturales. Instituto de Biotecnología Ambiental y Salud. Departamento de Biología Molecular; ArgentinaFil: Carezzano, María Evangelina. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Físico-Químicas y Naturales. Instituto de Biotecnología Ambiental y Salud. Departamento de Biología Molecular; ArgentinaFil: Puente, Mariana Laura. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Microbiología y Zoología Agrícola. Laboratorio de Bacterias Promotoras del Crecimiento Vegetal; ArgentinaFil: Alzari, Pedro. Université de Paris. Institut Pasteur. Unité de Microbiologie Structurale; FranciaFil: Martínez, Mariano. Université de Paris. Institut Pasteur. Unité de Microbiologie Structurale; FranciaFil: Ben-Assaya, Mathilde. Université de Paris. Institut Pasteur. Unité de Microbiologie Structurale; FranciaFil: Mornico, Damien. Institut Pasteur. Département Biologie Computationnelle. Hub de Bioinformatique et Biostatistique; FranciaFil: Santoro, Maricel. Max Planck for Chemical Ecology. Department of Biochemistry; FranciaFil: Martínez-Abarca, Francisco. CSIC. Estación Experimental Del Zaidín. Grupo de Ecología Genética de la Rizósfera; EspañaFil: Giordano, Walter. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Físico-Químicas y Naturales. Instituto de Biotecnología Ambiental y Salud (INBIAS-CONICET). Departamento de Biología Molecular; ArgentinaFil: Bogino, Pablo. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas, Físico-Químicas y Naturales. Instituto de Biotecnología Ambiental y Salud (INBIAS-CONICET). Departamento de Biología Molecular; Argentin

Complete genome sequence of Mesorhizobium ciceri Strain R30, a Rhizobium used as a commercial inoculant for Chickpea in Argentina

We report the complete genome sequence of Mesorhizobium ciceri strain R30, a rhizobium strain recommended and used as a commercial inoculant for chickpea in Argentina. The genome consists of almost 7 Mb, distributed into two circular replicons: a chromosome of 6.49 Mb and a plasmid of 0.46 Mb.Fil: Foresto, Emiliano. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Revale, Santiago. University of Oxford; Reino UnidoFil: Primo, Emiliano David. Universidad Nacional de Rio Cuarto. Facultad de Cs.exactas Fisicoquimicas y Naturales. Instituto de Biotecnologia Ambiental y Salud. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Instituto de Biotecnologia Ambiental y Salud.; ArgentinaFil: Nievas, Fiorela Lujan. Universidad Nacional de Rio Cuarto. Facultad de Cs.exactas Fisicoquimicas y Naturales. Instituto de Biotecnologia Ambiental y Salud. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Instituto de Biotecnologia Ambiental y Salud.; ArgentinaFil: Carezzano, Maria Evangelina. Universidad Nacional de Rio Cuarto. Facultad de Cs.exactas Fisicoquimicas y Naturales. Instituto de Biotecnologia Ambiental y Salud. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Instituto de Biotecnologia Ambiental y Salud.; ArgentinaFil: Puente, Mariana Laura. Instituto de Microbiología y Zoología Agrícola; ArgentinaFil: Alzari, Pedro. Institut Pasteur de Paris.; FranciaFil: Martinez, Mariano. Institut Pasteur de Paris.; FranciaFil: Mathilde Ben-Assaya. Institut Pasteur de Paris.; FranciaFil: Mornico, Damien. Institut Pasteur de Paris.; FranciaFil: Santoro, Valeria Maricel. Max Planck For Chemical Ecology,; Alemania. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Departamento de Biología Molecular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Martínez Abarca, Francisco. Estación Experimental del Zaidín; EspañaFil: Giordano, Walter Fabian. Universidad Nacional de Rio Cuarto. Facultad de Cs.exactas Fisicoquimicas y Naturales. Instituto de Biotecnologia Ambiental y Salud. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Instituto de Biotecnologia Ambiental y Salud.; ArgentinaFil: Bogino, Pablo Cesar. Universidad Nacional de Rio Cuarto. Facultad de Cs.exactas Fisicoquimicas y Naturales. Instituto de Biotecnologia Ambiental y Salud. - Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - Cordoba. Instituto de Biotecnologia Ambiental y Salud.; Argentin

Nuevos componentes del divisoma de Corynebacteriales identificados por una estrategia proteómica de marcado por proximidad en las células vivas

La división celular bacteriana es un proceso dirigido por el divisoma, complejo macromolecular cuyo ensamblaje comienza con la polimerización de la proteína FtsZ en el sitio de división. FtsZ participa en el reclutamiento de otras proteínas del divisoma, que para Escherichia coli y Bacillus subtilis han sido bien caracterizadas. Sin embargo, el suborden Corynebacterineae (incluye importantes patógenos humanos) carece de homólogos reconocibles para muchas de estas proteínas del divisoma. Este trabajo se centra en descifrar la arquitectura molecular del divisoma en este grupo de bacterias. Desarrollamos una estrategia proteómica basada en la biotinilación por proximidad para estudiar el divisoma de Corynebacterium glutamicum. Generamos una cepa que expresa FtsZ fusionada a una ascorbato peroxidasa ingenierizada (APEX2). APEX2 cataliza la oxidación de fenol biotina en presencia de H2O2 dando lugar a un radical que reacciona con aminoácidos de proteínas cercanas. Esto permitió marcar el entorno proteómico de FtsZ en la célula viva para su purificación e identificación por Espectrometría de Masa. Hemos identificado 253 vecinos confiables de FtsZ, que incluye proteínas de división celular conocidas y un gran número de proteínas no caracterizadas. Nos centramos en proteínas de membrana hipotéticas, que podrían mediar el anclaje del anillo Z a la membrana. Generamos cepas que expresan los candidatos seleccionados fusionados a una sonda fluorescente para evaluar su localización subcelular y su interacción con FtsZ. Los resultados permitieron identificar nuevos miembros conservados del divisoma de corinebacterias. Actualmente estudiamos su papel en la división celular, los detalles moleculares de su interacción con el anillo Z y su regulación mediante fosforilación de proteínas.Agencia Nacinal de Investigación e InnovaciónECOS-Sud France-Uruguay U20B0

A novel nuclease is the executing part of a bacterial plasmid defense system

Cells are continuously facing the risk of taking up foreign DNA that can compromise genomic and cellular integrity. Therefore, bacteria are in a constant arms race with mobile genetic elements such as phages, transposons and plasmids. They have developed several active strategies against invading DNA molecules that can be seen as a bacterial ‘innate immune system’. Anti-phage systems are usually organized in ‘defense islands’ and can consist of restriction-modification (R-M) systems, CRISPR-Cas, and abortive infection (Abi) systems. Despite recent advances in the field, much less is known about plasmid defense systems. We have recently identified the MksBEFG system in Corynebacterium glutamicum as a novel plasmid defense system which comprises homologues of the condensin system MukFEB. Here, we investigated the molecular arrangement of the MksBEFG complex. Importantly, we identified MksG as a novel nuclease that degrades plasmid DNA and is, thus, the executing part of the system. The crystal structure of MksG revealed a dimeric assembly through its DUF2220 C-terminal domains. This domain is homologous to the TOPRIM domain of the topoisomerase II family of enzymes and contains the corresponding divalent ion binding site that is essential for DNA cleavage in topoisomerases, explaining the in vitro nuclease activity of MksG. We further show that the MksBEF subunits exhibit an ATPase cycle similar to MukBEF in vitro and we reason that this reaction cycle, in combination with the nuclease activity provided by MksG, allows for processive degradation of invading plasmids. Super-resolution localization microscopy revealed that the Mks system is spatially regulated via to the polar scaffold protein DivIVA. Introduction of plasmids increases the diffusion rate and alters the localization of MksG, indicating an activation of the system in vivo

Legionella para-effectors target chromatin and promote bacterial replication

Legionella pneumophila replicates intracellularly by secreting effectors via a type IV secretion system. One of these effectors is a eukaryotic methyltransferase (RomA) that methylates K14 of histone H3 (H3K14me3) to counteract host immune responses. However, it is not known how L. pneumophila infection catalyses H3K14 methylation as this residue is usually acetylated. Here we show that L. pneumophila secretes a eukaryotic-like histone deacetylase (LphD) that specifically targets H3K14ac and works in synergy with RomA. Both effectors target host chromatin and bind the HBO1 histone acetyltransferase complex that acetylates H3K14. Full activity of RomA is dependent on the presence of LphD as H3K14 methylation levels are significantly decreased in a Δ lphD mutant. The dependency of these two chromatin-modifying effectors on each other is further substantiated by mutational and virulence assays revealing that the presence of only one of these two effectors impairs intracellular replication, while a double knockout (Δ lphDΔromA ) can restore intracellular replication. Uniquely, we present evidence for “para-effectors”, an effector pair, that actively and coordinately modify host histones to hijack the host response. The identification of epigenetic marks modulated by pathogens opens new vistas for the development of innovative therapeutic strategies to counteract bacterial infection and strengthening host defences

Central role and structure of the membrane pseudokinase YukC in the antibacterial <i>Bacillus subtilis</i> Type VIIb Secretion System

Posté dans BioRxiv le 9 mai 2020We previously linked TSHZ3 haploinsufficiency to autism spectrum disorder (ASD) and showed that embryonic or postnatal Tshz3 deletion in mice results in behavioral traits relevant to the two core domains of ASD, namely social interaction deficits and repetitive behaviors. Here, we provide evidence that cortical projection neurons (CPNs) and striatal cholinergic interneurons (SCINs) are two main and complementary players in the TSHZ3-linked ASD syndrome. We show that in the cerebral cortex, TSHZ3 is expressed in CPNs and in a proportion of GABA interneurons, while not in cholinergic interneurons or glial cells. TSHZ3-expressing cells, which are predominantly SCINs in the striatum, represent a low proportion of neurons in the ascending cholinergic projection system. We then characterized two new conditional knockout (cKO) models generated by crossing Tshz3 flox/flox with Emx1-Cre ( Emx1-cKO ) or Chat-Cre ( Chat-cKO ) mice to decipher the respective role of CPNs and SCINs. Emx1-cKO mice show altered excitatory synaptic transmission onto CPNs and plasticity at corticostriatal synapses, with neither cortical neuron loss nor impaired layer distribution. These animals present social interaction deficits but no repetitive patterns of behavior. Chat-cKO mice exhibit no loss of SCINs but changes in the electrophysiological properties of these interneurons, associated with repetitive patterns of behavior without social interaction deficits. Therefore, dysfunction in either CPNs or SCINs segregates with a distinct ASD behavioral trait. These findings provide novel insights onto the implication of the corticostriatal circuitry in ASD by revealing an unexpected neuronal dichotomy in the biological background of the two core behavioral domains of this disorder

The Antibacterial Type VII Secretion System of Bacillus subtilis: Structure and Interactions of the Pseudokinase YukC/EssB

International audienceType VIIb secretion systems (T7SSb) were recently proposed to mediate different aspects of Firmicutes physiology, including bacterial pathogenicity and competition. However, their architecture and mechanism of action remain largely obscure. Here, we present a detailed analysis of the T7SSb-mediated bacterial competition in Bacillus subtilis, using the effector YxiD as a model for the LXG secreted toxins. By systematically investigating protein-protein interactions, we reveal that the membrane subunit YukC contacts all T7SSb components, including the WXG100 substrate YukE and the LXG effector YxiD. YukC's crystal structure shows unique features, suggesting an intrinsic flexibility that is required for T7SSb antibacterial activity. Overall, our results shed light on the role and molecular organization of the T7SSb and demonstrate the potential of B. subtilis as a model system for extensive structure-function studies of these secretion machineries. IMPORTANCE Type VII secretion systems mediate protein extrusion from Gram-positive bacteria and are classified as T7SSa and T7SSb in Actinobacteria and in Firmicutes, respectively. Despite the genetic divergence of T7SSa and T7SSb, the high degree of structural similarity of their WXG100 substrates suggests similar secretion mechanisms. Recent advances revealed the structures of several T7SSa cytoplasmic membrane complexes, but the molecular mechanism of secretion and the T7SSb architecture remain obscure. Here, we provide hints on the organization of T7SSb in B. subtilis and a highresolution structure of its central pseudokinase subunit, opening new perspectives for the understanding of the T7SSb secretion mechanism by using B. subtilis as an amenable bacterial model

FtsEX-independent control of RipA-mediated cell separation in Corynebacteriales

International audienceThe bacterial cell wall is a multi-layered mesh, whose major component is peptidoglycan (PG), a sugar polymer cross-linked by short peptide stems. During cell division, a careful balance of PG synthesis and degradation, precisely coordinated both in time and space, is necessary to prevent uncontrolled destruction of the cell wall. In Corynebacteriales, the D,L endopeptidase RipA has emerged as a major PG hydrolase for cell separation, and RipA defaults have major implications for virulence of the human pathogens Mycobacterium tuberculosis and Corynebacterium diphtheriae . However, the precise mechanisms by which RipA mediates cell separation remain elusive. Here we report phylogenetic, biochemical, and structural analysis of the Corynebacterium glutamicum homologue of RipA, Cg1735. The crystal structures of full-length Cg1735 in two different crystal forms revealed the C-terminal NlpC/P60 catalytic domain obtruded by its N-terminal conserved coiled-coil domain, which locks the enzyme in an autoinhibited state. We show that this autoinhibition is relieved by the extracellular core domain of the transmembrane septal protein Cg1604. The crystal structure of Cg1604 revealed a (β/α) protein with an overall topology similar to that of receiver domains from response regulator proteins. The atomic model of the Cg1735–Cg1604 complex, based on bioinformatical and mutational analysis, indicates that a conserved, distal-membrane helical insertion in Cg1604 is responsible for Cg1735 activation. The reported data provide important insights into how intracellular cell division signal(s), yet to be identified, control PG hydrolysis during RipA-mediated cell separation in Corynebacteriales