72 research outputs found
Proteomic analysis to understand Cr(VI) homeostasis in Streptomyces sp. MC1
Chromium is a heavy metal widely used in a variety of industrial processes (leather tanning, steel production, metal corrosion inhibition). Hexavalent chromium is carcinogenic and presents higher toxicity than trivalent form since Cr(VI) is more water-soluble and mobile than Cr(III). Industrial effluents containing Cr(VI) are released into water courses, mostly without proper treatment, resulting in anthropogenic contamination. Over the last years, bacteria-mediated removal or stabilization of heavy metal into no or less toxic forms has become in an effective biotechnological process. In this sense, several physiological studies on Streptomyces sp. MC1, an actinobacteria isolated from a polluted soil in the province of Tucumán (Argentina), demonstrated be able to grow in presence of Cr(VI) and remove the metal both in liquid medium and contaminated soils. However, the molecular mechanisms involved are unknown in this actinobacteria. MS-based proteomics have become a powerful tool to understand the mechanisms that underlie physiological processes. In the present work, we use MS-based, label-free and quantitative proteomic analyses in order to identify enzymes involves in oxidative stress response caused by the presence of Cr(VI) in our actinobacteria strain MC1. Sampling points for proteomics analyses were established according to the growth of Streptomyces sp. MC1 in minimal medium (MM) amended with Cr(VI) at 50 mg L-1 and MM without the metal (control condition). Cells were harvested after 18 and 24 h of incubation in control condition and MM with Cr(VI) respectively. These sampling points allowed obtaining comparable and metabolically active cells (exponential phase of growth). Cr(VI) removal was 10% at the time that cells were harvested (24 h). A total of 1981 different proteins were detected in the proteome. It represents approximately 22% of the predicted protein sequences for this strain. 518 of these proteins passed our significance parameters which 186 of them were up-regulated in the condition supplemented with Cr(VI). Analysis with the software BlastKOALA showed that up-regulated proteins were distributed in metabolic pathways that result essential for a correct cellular operation. Overall, the proteins were related to carbon and energy metabolism, genetic information processing, oxidative stress response and membrane transports. Interestingly, enzymes from pentose phosphate pathway increasing significantly their abundance in presence of chromium. About, 10 different oxidoreductases enzymes were up-regulated in presence of the metal. Regarding oxidative stress response, key enzymes like superoxide dismutase, catalase, mycothiol synthase, and mycothiol amidase were identified with an increment in their abundance. The proteome analysis performed in Streptomyces sp. MC1 allowed us to identify the proteins involves in the homeostasis of Cr(VI). These results serve as basement to study and improve the heavy metal removal by actinobacteria.Fil: Sineli, Pedro Eugenio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Aparicio, Juan Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaFil: Pernodet, Jean Luc. Institut de Biologie Intégrative de la Cellule; FranciaFil: Polti, Marta Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Planta Piloto de Procesos Industriales Microbiológicos; ArgentinaLV Annual SAIB Meeting and XIV PABMB CongressSaltaArgentinaSociedad Argentina de Investigaciones Bioquímica
An Iterative Nonribosomal Peptide Synthetase Assembles the Pyrrole-Amide Antibiotic Congocidine in Streptomyces ambofaciens
SummaryCongocidine (netropsin) is a pyrrole-amide (oligopyrrole, oligopeptide) antibiotic produced by Streptomyces ambofaciens. We have identified, in the right terminal region of the S. ambofaciens chromosome, the gene cluster that directs congocidine biosynthesis. Heterologous expression of the cluster and in-frame deletions of 8 of the 22 genes confirm the involvement of this cluster in congocidine biosynthesis. Nine genes can be assigned specific functions in regulation, resistance, or congocidine assembly. In contrast, the biosynthetic origin of the precursors cannot be easily inferred from in silico analyses. Congocidine is assembled by a nonribosomal peptide synthetase (NRPS) constituted of a free-standing module and several single-domain proteins encoded by four genes. The iterative use of its unique adenylation domain, the utilization of guanidinoacetyl-CoA as a substrate by a condensation domain, and the control of 4-aminopyrrole-2-carboxylate polymerization constitute the most original features of this NRPS
Cyclodipeptide synthases, a family of class-I aminoacyl-tRNA synthetase-like enzymes involved in non-ribosomal peptide synthesis
Cyclodipeptide synthases (CDPSs) belong to a newly defined family of enzymes that use aminoacyl-tRNAs (aa-tRNAs) as substrates to synthesize the two peptide bonds of various cyclodipeptides, which are the precursors of many natural products with noteworthy biological activities. Here, we describe the crystal structure of AlbC, a CDPS from Streptomyces noursei. The AlbC structure consists of a monomer containing a Rossmann-fold domain. Strikingly, it is highly similar to the catalytic domain of class-I aminoacyl-tRNA synthetases (aaRSs), especially class-Ic TyrRSs and TrpRSs. AlbC contains a deep pocket, highly conserved among CDPSs. Site-directed mutagenesis studies indicate that this pocket accommodates the aminoacyl moiety of the aa-tRNA substrate in a way similar to that used by TyrRSs to recognize their tyrosine substrates. These studies also suggest that the tRNA moiety of the aa-tRNA interacts with AlbC via at least one patch of basic residues, which is conserved among CDPSs but not present in class-Ic aaRSs. AlbC catalyses its two-substrate reaction via a ping-pong mechanism with a covalent intermediate in which l-Phe is shown to be transferred from Phe-tRNAPhe to an active serine. These findings provide insight into the molecular bases of the interactions between CDPSs and their aa-tRNAs substrates, and the catalytic mechanism used by CDPSs to achieve the non-ribosomal synthesis of cyclodipeptides
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Minimum Information about a Biosynthetic Gene cluster
A wide variety of enzymatic pathways that produce specialized metabolites in bacteria, fungi and plants are known to be encoded in biosynthetic gene clusters. Information about these clusters, pathways and metabolites is currently dispersed throughout the literature, making it difficult to exploit. To facilitate consistent and systematic deposition and retrieval of data on biosynthetic gene clusters, we propose the Minimum Information about a Biosynthetic Gene cluster (MIBiG) data standard.Chemistry and Chemical Biolog
Etude de la résistance aux macrolides chez Streptomyces ambofaciens et Streptomyces lividans
Les antibiotiques macrolides inhibent la synthèse protéique en se liant aux ribosomes. Streptomyces ambofaciens produit la spiramycine et possède plusieurs mécanismes de résistance à la spiramycine et aux macrolides. J'ai caractérisé, le déterminant de résistance srmC et montré qu'il était constitué des trois gènes srmC1, srmC2 et srmC3. srmC1 et srmC2 codent les sous-unités d'un transporteur de la famille ABC (ATP Binding Cassette) et confèrent la résistance à certains macrolides, dont la spiramycine, mais aussi à la daunorubicine. SrmC1/C2 sont co-transcrits et leur expression est réprimée par SrmC3, un répresseur transcriptionnel de la famille TetR. Les gènes srmC ne sont pas localisés parmi les gènes, de biosynthèse de la spiramycine et ne sont pas indispensables chez S. ambofaciens. En étudiant chez Streptomyces lividans le mécanisme d'induction de l'expression de srmC1/C2, j'ai mis en évidence l'existence dans cette souche d'un nouveau mécanisme de résistance aux macrolides et à d'autres antibiotiques, inductible par un macrolide, la rosaramicine. Des orthologues de srmC1/C2, désignés sclA et sclB, codant les sous-unités d'un transporteur ABC, sont impliqués dans cette résistance. Ils confèrent une résistance multi-drogue (macrolides, daunorubicine, bromure d'éthidium). Leur expression est réprimée par SclR (répresseur transcriptionnel de la famille TetR.). L'étude d'un mutant SclA SclR suggère qu'un ou plusieurs autres déterminants participent également à la résistance multi-drogue inductible par la rosaramicine. Parmi les gènes de résistance aux macrolides de S. ambofaciens, certains ont des orthologues chez S. lividans, un non-producteur de macrolide, d'autres non. Ceci suggère que certains déterminants de résistance de S. ambofaciens sont liés à la biosynthèse de spiramycine et jouent un rôle crucial dans la protection de la souche. D'autres, dont srmC, protégeraient la souche contre des produits toxiques présents dans l'environnement.Macrolide antibiotics inhibit protein synthesis by binding to ribosomes. Streptomyces ambofaciens produces spiramycin and possesses several resistance mechanisms to spiramycine and other macrolides. The srmC determinant was studied in the work reported here. It is comprised of three genes: srmC1, srmC2 and srmC3. srmC1/srmC2 encode the two sub-units of an ABC (ATP Binding Cassette) transporter. They confer resistance to several macrolides, including spiramycin, and to daunorubicin. The two genes srmC1/srmC2 are co-transcribed and their expression is repressed by SrmC3, a repressor of the TetR family. The srmC genes are not localized in the spiramycin biosynthetic gene cluster and are dispensable in S. ambofaciens. During the study of the induction of srmCl/C2 expression in S. lividans, a new mechanism of resistance to macrolides and other antibiotics, inducible by the macrolide rosaramicin was discovered in this strain. Genes sclA and sclB, orthologous to srmC1/C2, encoding an ABC transporter are involved in this resistance. sclA and sclB confer multidrug resistance (resistance to macrolides daunorubicin and ethidium bromide). Their expression is controlled by SclR (a TetR-like repressor). The study of a SclA SclR mutant strain suggested that other resistance determinant(s) might be involved in the rosaramicin-inducible resistance. Among macrolide resistance genes from S. ambofaciens, some have homologues in S. lividans, others do not. This suggests that some S. ambofaciens resistant determinants could be directly linked to spiramycin biosynthesis and important for self-protection against spiramycin. Others, including srmC might protect the strain against other toxic compounds encountered in the environment.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF
Caractérisation de voies de biosynthèse d'antibiotiques de la famille des pyrrolamides
Les pyrrolamides constituent une famille de produits naturels dotés de diverses activités biologiques et synthétisés par des actinobactéries. La congocidine et la distamycine, les molécules les plus connues de cette famille, sont capables de se lier à l'ADN de façon non covalente selon une certaine spécificité de séquence (succession de 4 paires de base A/T). Récemment, les gènes et la voie de biosynthèse de la congocidine ont été identifiés et caractérisés chez S. ambofaciens. Ceci a révélé un mécanisme original impliquant notamment de nouvelles enzymes et de nouvelles voies pour la biosynthèse des trois précurseurs nécessaires à l assemblage de la congocidine. Nous avons entrepris d étudier la régulation de la biosynthèse de la congocidine chez S. ambofaciens et d isoler et de caractériser les groupes de gènes de biosynthèse de deux autres pyrrolamides, la distamycine et les pyrronamycines (produites respectivement par S distallicus et un streptomyces non caractérisé). L'objectif de cette étude est, dans un premier temps, d améliorer notre compréhension des mécanismes impliqués lors de la biosynthèse de ces molécules (comme le mécanisme d incorporation des pyrroles) et, par la suite, de manipuler les gènes identifiés pour synthétiser de nouvelles molécules pyrrolamides hybrides.Pyrrolamides constitute a family of natural products with various biological activities, synthesized by actinobacteria. Congocidine (also called netropsin) and distamycin are the best characterized pyrrolamides, largely studied due to their ability to bind into the minor groove of the DNA double helix in a sequence specific manner (succession of four A/T bases). Recently, the congocidine biosynthetic pathway has been characterized in Streptomyces ambofaciens. We showed that an iterative Non Ribosomal Peptide Synthetase with an unusual architecture assembles congocidine, using precursors with undocumented biosynthetic pathways. With the aim of developing a combinatorial biosynthesis approach for the development of new pyrrolamides, we undertook the study of the regulation of congocidine biosynthesis in S. ambofaciens and the isolation of the distamycin and pyrronamycins biosynthetic gene clusters. Characterization of these clusters will result in a more detailed understanding of pyrrolamide biosynthesis (e.g. mechanism of pyrrole polymerization), and provide new tools (enzymes) and building blocks (precursors) necessary for combinatorial biosynthesis.PARIS11-SCD-Bib. électronique (914719901) / SudocSudocFranceF
Etude de la biosynthèse de l'antibiotique spiramycine par Streptomyces ambofaciens
Streptomyces ambofaciens synthétise l'antibiotique macrolide la spiramycine. La région contenant les gènes de biosynthèse de la spiramycine a été séquencé chez S. ambofaciens. L'analyse de cette séquence d'environ 100 kb révèle la présence de 50 gènes, dont 41 sont probablement impliqués dans la biosynthèse de spiramycine, dans sa régulation ou dans la résistance à la spiramycine. Afin d'étudier le rôle de ces gènes dans la biosynthèse de spiramycine, nous avons construit des souches dans lesquelles certains gènes ont été inactivés par délétion en phase. Pour cela, nous avons développé des cassettes excisables facilement par recombinaison spécifique de site. Deux gènes régulateurs, srmR et srmS, ont été identifiés parmi les gènes de biosynthèse de la spiramycine. L'inactivation de chacun de ces deux gènes abolit la production de la spiramycine tandis que la surexpression de chacun d'eux augmente d'un facteur trois le niveau de production de la spiramycine. L'analyse d'expression par RT-PCR de tous les gènes de biosynthèse de la spiramycine, chez la souche sauvage, dans les mutants srmR et srmS et dans le double mutant srmR, srmS, a été effectuée. Ces résultats indiquent que SrmR est nécessaire à l'expression de srmS ; SrmS contrôlant quant à lui l'expression de la plupart des gènes de biosynthése de la spiramycine. La spiramycine est produite sous forme d'un mélange de spiramycine I, II et III. La spiramycine I est la forme la plus active. Nous avons identifié et inactivé le gène, orf6*, codant l'acétyltransférase responsable de la production de la spiramycine II et III. Le niveau de production de spiramycine I a été augmenté en surexprimant srmR dans ce mutant.Streptomyces ambofaciens synthesizes the 16-membered macrolide antibiotic spiramycin. The biosynthetic gene cluster for spiramycin has been characterized in S. ambofaciens. Sequence analysis of a region of more than 100 kb spanning the entire cluster revealed the presence of 50 genes, 41 of them being most probably involved in spiramycin biosynthesis, its regulation or resistance to the antibiotic. In order to study the role of the gene products in spiramycin biosynthesis, we constructed strains in which some genes were inactivated by in-frame deletion. For this purpose, we developed cassettes that can be easily excised by site-specific recombination. Two regulatory genes, srmR and srmS, were identified in the biosynthetic gene cluster. The disruption of each of these two regulatory genes eliminated spiramycin production while the over-expression of each of them increased three fold the level of spiramycin production. Expression analysis by RT-PCR for all the genes of the cluster in the wild type strain, in srmR and srmS deletion mutants and in the srmR, srmS double deletion mutant was performed. These results, together with complementation experiments, indicated that SrmR is required for srmS expression, SrmS being a pathway-specific activator that controls most of the spiramycin biosynthetic genes. Spiramycin is normally produced as a mixture of spiramycin I, II and III. Spiramycin I is the most active form of the antibiotic. We identified and inactivated the gene encoding the acetyltransferase responsible for the production of spiramycin II and III. The level of production of this mutant strain was further increased by over-expression of the regulatory gene srmR.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF
Caractérisation de voies de biosynthèse d'antibiotiques de la famille des pyrrolamides
La congocidine (nétropsine) est un antibiotique de la famille des pyrrolamides produit par Streptomyces ambofaciens. Nous avons identifié, dans la région terminale droite du chromosome de S. ambofaciens, le groupe de gènes qui dirige la biosynthèse de congocidine. L'expression du groupe de gènes dans un hôte hétérologue et la délétion de huit des 22 gènes confirme l'implication de ce groupe de gènes dans la biosynthèse de la congocidine. Il est possible d'attribuer à neuf de ces gènes une fonction spécifique dans la régulation, la résistance ou l'assemblage de la congocidine. La congocidine est assemblée par une synthétase de peptides non ribosomiques (NRPS) présentant plusieurs aspects originaux. Elle est constituée par un module libre et plusieurs domaines isolés codés par quatre gènes et utilise de manière itérative un domaine d'adénylation. L'étude du gène cgc1 suggère que Cgc1 serait un régulateur orphelin apparenté à ceux des systèmes à deux composants et activant la transcription des gènes cgc. La recherche d'autres groupes de gènes dirigeant la synthèse de pyrrolamides a permis d'identifier chez Streptomyces neptrosis la majorité des gènes de biosynthèse de la nétropsine, molécule similaire à la congocidine, et chez Streptomyces distallicus la majorité des gènes de biosynthèse de la distamycine. Les résultats préliminaires obtenus montrent que des gènes homologues aux gènes cgc, organisés de manière similaire, dirigent la biosynthèse de ces deux pyrrolamides.Congocidine (netropsin) is a pyrrole-amide antibiotic produced by Streptomyces ambofaciens. We have identified, in the right terminal region of the chromosome of S. ambofaciens, the gene cluster that directs congocidine biosynthesis. Heterologous expression of the cluster and in-frame deletion of eight of the 22 genes confirmed the involvement of this cluster in congocidine biosynthesis. Nine genes can be assigned functions in regulation, resistance or congocidine assembly. Congocidine is assembled by a non-ribosomal peptide synthetase (NRPS) exhibiting some original features. This NRPS is constituted of a freestanding module and several single domain proteins encoded by four genes and it uses iteratively a single adenylation domain. The study of cgc1 suggests that Cgc1 could be an orphan response regulator related to those of two component systems and activating the transcription of the cgc genes. Other gene clusters directing the biosynthesis of pyrrole-amide antibiotics have been identified. Most of the gene cluster directing the biosynthesis of neptrosin (a compound identical to congocidine) was identified in Streptomyces neptrosis and most of the gene cluster involved in distamycin biosynthesis was identified in Streptomyces distallicus. The preliminary results obtained indicated that homologues of the cgc genes, with a comparable genetic organization, direct the biosynthesis of these two pyrrole-amide antibiotics.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF
Résistance ribosomique aux macrolides et leur effet sur la fidélité de traduction
Les macrolides forment un groupe homogène d'agents antibactériens produits par des Streptomyces et d'autres actinomycètes. Ils inhibent la synthèse protéique des bactéries par liaison à la sous-unité ribosomique 50S, ce qui empêche son assemblage ou son fonctionnement. La première partie de ce manuscrit porte sur l'effet des macrolides sur la fidélité de traduction. Nous avons utilisé comme système rapporteur la luciférase de Vibrio harveyi avec un codon stop introduit au début du gène luxB pour mesurer in vivo la suppression de ce codon stop. L'érythromycine augmente la trans-lecture du codon stop UAG et diminue donc la fidélité de traduction, en accord avec l'hypothèse selon laquelle les macrolides agissent en début d'élongation. Cet effet a été confirmé par l'étude du taux global d'erreur en utilisant l'électrophorèse 2-D des protéines. La deuxième partie porte sur la résistance naturelle aux macrolides chez les mycobactéries du complexe M. tuberculosis (CMT), résistance généralement attribuée à la structure spécifique de la paroi des mycobactéries. Nous avons identifié un gène codant une méthyltransférase de type Erm, modifiant les ribosomes et conférant la résistance aux macrolides. Ce gène, appelé ermMT, est conservé chez tous les membres du CMT mais est affecté par une délétion dans certaines souches vaccinales de M. bovis BCG comme BCG Pasteur. Cette dernière souche est sensible aux macrolides, alors que les autres membres du CMT sont résistants. L'expression de ermMT dans des mycobactéries sensibles aux macrolides confère la résistance. La comparaison des niveaux de résistance et de l'affinité pour l'érythromycine des ribosomes de souches exprimant ermMT ou d'autres gènes erm indique que ermMT confère une résistance de type I aux macrolides, lincosamides et streptogramines, correspondant à la mono-méthylation de A2058 dans l'ARN 23S. Nos résultats montrent que ermMT joue un rôle majeur dans la résistance naturelle aux macrolides chez les mycobactéries du CMT.Macrolide antibiotics constitute a homogenous group of antibacterial agents produced by Streptomyces or related Actinobacteria. They inhibit protein synthesis in bacteria by binding to the 50S ribosomal subunit, preventing its assembly or inhibiting its function. In the first part of this thesis the effect of macrolides on translation accuracy was studied. We have used the reporter system based on Vibrio harveyi luciferase with a stop codon inserted in the proximal part of the luxB gene for the in vivo measurement of the nonsense codon readthrough. Erythromycin stimulated the leadthrough of the UAG stop codon and thus the decrease of the translation accuracy. This is in agreement with the hypothesis that macrolides influence the early stages of elongation process. The misreading effect of macrolides was confirmed by the study of global error frequencies using the 2-D gel electrophoresis of proteins. The second part deals with the intrinsic macrolide resistance of the Mycobacterium tuberculosis complex (MTC), generally attributed to the low permeability of the mycobacterial cell wall. However we have shown that a gene, whose product confers macrolide resistance by ribosome modification, was present in all members of the MTC. It was named ermMT (erm.37). Part of the ermMT is deleted in some vaccinal strains of Mycobacterium bovis BCG, such as the Pasteur strain. The Pasteur strain was susceptible to macrolides, whereas MTC species were resistant to them. The expression of ermMT in the macrolide-sensitive Mycobacterium strains conferred macrolide resistance. Comparison of the resistance patterns and ribosomal affinity for erythromycin of Mycobacterium host strains expressing ermMT or other erm genes indicates that ermMT confers a type I resistance to macrolides, lincosamides and streptogramins, coiresponding to the mono-methylation of A2058 in 23S rRNA. Our results indicate that ermMT plays a major role in the intrinsic macrolide resistance of members of the MTC.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF
A set of modular and integrative vectors for synthetic biology in Streptomyces
International audienceWith the development of synthetic biology in the field of (actinobacteria) specialized metabolism, new tools are needed for the design or refactoring of biosynthetic gene clusters. If libraries of synthetic parts (such as promoters or ribosome binding sites) and DNA cloning methods have been developed, to our knowledge, not many vectors designed for the flexible cloning of biosynthetic gene clusters have been constructed.We report here the construction of a set of 12 standardized and modular vectors designed to afford the construction or the refactoring of biosynthetic gene clusters in Streptomyces species, using a large panel of cloning methods. Three different resistance cassettes and four orthogonal integration systems are proposed. In addition, FRT sites were incorporated to allow the recycling of antibiotic markers and to limit the risks of unwanted homologous recombination in Streptomyces strains, when several vectors are used. The functionality and proper integration of the vectors in three commonly used Streptomyces strains, as well as the functionality of the Flp-catalysed excision were all confirmed.To illustrate some possible uses of our vectors, we refactored the albonoursin gene cluster from Streptomyces noursei using the Biocrick assembly method. We also used the seamless Ligase Chain Reaction cloning method to assemble a transcription unit in one of the vectors and genetically complement a mutant strain.IMPORTANCE One of the strategies employed today to obtain new bioactive molecules with potential applications for human health (for example antimicrobial or anticancer agents) is synthetic biology. Synthetic biology is used to biosynthesize new unnatural specialized metabolites, or to force the expression of otherwise silent natural biosynthetic gene clusters. To assist the development of synthetic biology in the field of specialized metabolism, we constructed and are offering to the community a set of vectors that were intended to facilitate DNA assembly and integration in actinobacteria chromosome. These vectors are compatible with various DNA cloning and assembling methods. They are standardized and modular, allowing the easy exchange of a module by another one of the same nature. Although designed for the assembly or the refactoring of specialized metabolite gene clusters, they have a broader potential utility, for protein production or genetic complementation, for example
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