Disrupting the Acyl Carrier Protein/SpoT Interaction In Vivo: Identification of ACP Residues Involved in the Interaction and Consequence on Growth

In bacteria, Acyl Carrier Protein (ACP) is the central cofactor for fatty acid biosynthesis. It carries the acyl chain in elongation and must therefore interact successively with all the enzymes of this pathway. Yet, ACP also interacts with proteins of diverse unrelated function. Among them, the interaction with SpoT has been proposed to be involved in regulating ppGpp levels in the cell in response to fatty acid synthesis inhibition. In order to better understand this mechanism, we screened for ACP mutants unable to interact with SpoT in vivo by bacterial two-hybrid, but still functional for fatty acid synthesis. The position of the selected mutations indicated that the helix II of ACP is responsible for the interaction with SpoT. This suggested a mechanism of recognition similar to one used for the enzymes of fatty acid synthesis. Consistently, the interactions tested by bacterial two-hybrid of ACP with fatty acid synthesis enzymes were also affected by the mutations that prevented the interaction with SpoT. Yet, interestingly, the corresponding mutant strains were viable, and the phenotypes of one mutant suggested a defect in growth regulation

La thioestérase YbgC, l'Acyl Carrier Protein et le métabolisme des phospholipides chez Escherichia coli

AIX-MARSEILLE2-BU Sci.Luminy (130552106) / SudocSudocFranceF

Rôle de l'interaction entre l'AcyL Carrier protein et SpoT (un lien génétique et physique entre la réponse stringente et le métabolisme des lipides)

Lors d une carence nutritionnelle, les bactéries synthétisent du (p)ppGpp dont l accumulation déclenche la réponse stringente, caractérisée par un arrêt de la croissance et une modulation de l expression génétique. E .coli possède deux protéines impliquées dans le métabolisme du (p)ppGpp, RelA et SpoT. Alors que RelA répond à une carence en acides aminés par un mécanisme bien défini, SpoT répond à une carence en acides gras ou en carbone par un mécanisme toujours inconnu. Au cours de ma thèse, nous avons caractérisé une interaction physique entre SpoT et l Acyl Carrier Protein (ACP), co-facteur central de la synthèse des acides gras. La perte de cette interaction empêche l accumulation de (p)ppGpp lors d une carence en acides gras, suggérant que l interaction ACP/SpoT est impliquée dans la réponse au stress SpoT-dépendante. Ainsi, nous avons proposé un modèle dans lequel une modification d ACP signale une carence en acides gras à SpoT, entraînant un changement conformationnel de SpoT et la synthèse de (p)ppGpp. Afin d étudier l importance de cette interaction dans la physiologie bactérienne, nous avons construit des souches dans lesquelles l interaction entre SpoT et ACP est abolie en introduisant des mutations ponctuelles dans le gène spoT. Ces souches présentent des phénotypes de croissance ainsi que des altérations de l enveloppe cellulaire. De plus, dans ces souches la réponse à la carence en acides gras et l interaction ACP/SpoT sont altérées dans un contexte relA mais pas dans un contexte relA+, suggérant un rôle de RelA dans l interaction ACP/SpoT. L étude de la conservation de l interaction ACP/SpoT parmi différentes espèces bactériennes nous a permis de montrer que cette interaction n est conservée que dans une minorité de bactéries contenant des enzymes de type SpoT. Enfin, nous avons caractérisé un réseau d interactions centré autour de SpoT, liant des protéines impliquées dans la traduction et dans le métabolisme des lipides. L ensemble de ces résultats suggèrent que l interaction ACP/SpoT est importante pour la physiologie bactérienne, notamment en conditions de carence.Bacteria respond to various nutritional stresses by producing (p)ppGpp. The accumulation of this nucleotide triggers the stringent response, which is characterized by growth arrest and the modulation of gene expression. E. coli contains two enzymes involved in (p)ppGpp metabolism, RelA and SpoT. RelA responds to amino acid starvation by a well known mechanism, whereas SpoT produces (p)ppGpp during fatty acid or carbon starvation but the mechanism for this response is unknown. During my thesis, we characterized a physical interaction between SpoT and Acyl Carrier Protein (ACP), a central co-factor in fatty acid synthesis. The loss of this interaction prevents (p)ppGpp accumulation in response to fatty acid starvation, supporting the idea that the ACP/SpoT interaction is involved in SpoT-dependent stress response. This led us to propose a model in which an ACP modification signals fatty acid starvation to SpoT, triggering a conformational switch in SpoT and so leading to (p)ppGpp synthesis. Then, in order to study the importance of the ACP/SpoT interaction in bacterial physiology, we have engineered E. coli strains in which this interaction is abolished by introducing specific point mutations in spoT gene. These strains present growth phenotypes and envelope defects. Furthermore, the response to fatty acids starvation and the interaction between ACP and SpoT mutated proteins in these strains are affected in a relA context but not in a relA+ context, suggesting a role for RelA in the ACP/SpoT interaction. Studying the conservation of the ACP/SpoT interaction between bacterial species, we show that this interaction is only present in bacteria containing SpoT enzymes. Lastly, we characterized an interaction network centred on SpoT, linking translation and lipid metabolism proteins. All these data suggest that the ACP/SpoT interaction is important for bacterial physiology, even more during starvation condition.AIX-MARSEILLE2-BU Sci.Luminy (130552106) / SudocSudocFranceF

Linking glucose metabolism to the stringent response through the PTS

International audienc

The bacterial two-hybrid system based on adenylate cyclase reconstitution in Escherichia coli.

International audienceThe bacterial two-hybrid system based on the reconstitution of adenylate cyclase in Escherichia coli (BACTH) was described 14years ago (Karimova, Pidoux, Ullmann, and Ladant, 1998, PNAS, 95:5752). For microbiologists, it is a practical and powerful alternative to the use of the widely spread yeast two-hybrid technology for testing protein-protein interactions. In this review, we aim at giving the reader clear and most importantly simple instructions that should break any reticence to try the technique. Yet, we also add recommendations in the use of the system, related to its specificities. Finally, we expose the advantages and disadvantages of the technique, and review its diverse applications in the literature, which should help in deciding if it is the appropriate method to choose for the case at hand

Bacterial interactomes: from interactions to networks.

International audienceIn order to ensure their function(s) in the cell, proteins are organized in machineries, underlaid by a complex network of interactions. Identifying protein interactions is thus crucial to our understanding of cell functioning. Technical advances in molecular biology and genomic technology now allow for the systematic study of the interactions occurring in a given organism. This review first presents the techniques readily available to microbiologists for studying protein-protein interactions in bacteria, as well as their usability for high-throughput studies. Two types of techniques need to be considered: (1) the isolation of protein complexes from the organism of interest by affinity purification, and subsequent identification of the complex partners by mass spectrometry and (2) two-hybrid techniques, in general based on the production of two recombinant proteins whose interaction has to be tested in a reporter cell. Next, we summarize the bacterial interactomes already published. Finally, the strengths and pitfalls of the techniques are discussed, together with the potential prospect of interactome studies in bacteria

Prévention du no-reflow (étude concernant l'utilisation du guard wire au cours de l'angioplastie en phase aigue d'infarctus du myocarde. Faisabilité et sécurité)

STRASBOURG-Medecine (674822101) / SudocPARIS-BIUM (751062103) / SudocSudocFranceF

Bacteria Possessing Two RelA/SpoT-Like Proteins Have Evolved a Specific Stringent Response Involving the Acyl Carrier Protein-SpoT Interaction ▿

Bacteria respond to nutritional stress by producing (p)ppGpp, which triggers a stringent response resulting in growth arrest and expression of resistance genes. In Escherichia coli, RelA produces (p)ppGpp upon amino acid starvation by detecting stalled ribosomes. The SpoT enzyme responds to various other types of starvation by unknown mechanisms. We previously described an interaction between SpoT and the central cofactor of lipid synthesis, acyl carrier protein (ACP), which is involved in detecting starvation signals in lipid metabolism and triggering SpoT-dependent (p)ppGpp accumulation. However, most bacteria possess a unique protein homologous to RelA/SpoT (Rsh) that is able to synthesize and degrade (p)ppGpp and is therefore more closely related to SpoT function. In this study, we asked if the ACP-SpoT interaction is specific for bacteria containing two RelA and SpoT enzymes or if it is a general feature that is conserved in Rsh enzymes. By testing various combinations of SpoT, RelA, and Rsh enzymes and ACPs of E. coli, Pseudomonas aeruginosa, Bacillus subtilis and Streptococcus pneumoniae, we found that the interaction between (p)ppGpp synthases and ACP seemed to be restricted to SpoT proteins of bacteria containing the two RelA and SpoT proteins and to ACP proteins encoded by genes located in fatty acid synthesis operons. When Rsh enzymes from B. subtilis and S. pneumoniae are produced in E. coli, the behavior of these enzymes is different from the behavior of both RelA and SpoT proteins with respect to (p)ppGpp synthesis. This suggests that bacteria have evolved several different modes of (p)ppGpp regulation in order to respond to nutrient starvation

Degradation of Exogenous Fatty Acids in Escherichia coli

International audienceMany bacteria possess all the machineries required to grow on fatty acids (FA) as a uniquesource of carbon and energy. FA degradation proceeds through the  -oxidation cycle that producesacetyl-CoA and reduced NADH and FADH cofactors. In addition to all the enzymes required for -oxidation, FA degradation also depends on sophisticated systems for its genetic regulation andfor FA transport. The fact that these machineries are conserved in bacteria suggests a crucial rolein environmental conditions, especially for enterobacteria. Bacteria also possess specific enzymesrequired for the degradation of FAs from their environment, again showing the importance ofthis metabolism for bacterial adaptation. In this review, we mainly describe FA degradation inthe Escherichia coli model, and along the way, we highlight and discuss important aspects of thismetabolism that are still unclear. We do not detail exhaustively the diversity of the machineries foundin other bacteria, but we mention them if they bring additional information or enlightenment onspecific aspects