Dynamic subcellular localization of a respiratory complex controls bacterial respiration

International audienceRespiration, an essential process for most organisms, has to optimally respond to changes in the metabolic demand or the environmental conditions. The branched character of their respiratory chains allows bacteria to do so by providing a great metabolic and regulatory flexibility. Here, we show that the native localization of the nitrate reductase, a major respiratory complex under anaerobiosis in Escherichia coli, is submitted to tight spatiotemporal regulation in response to metabolic conditions via a mechanism using the transmembrane proton gradient as a cue for polar localization. These dynamics are critical for controlling the activity of nitrate reductase, as the formation of polar assemblies potentiates the electron flux through the complex. Thus, dynamic subcellular localization emerges as a critical factor in the control of respiration in bacteria

History of Maturation of Prokaryotic Molybdoenzymes—A Personal View

In prokaryotes, the role of Mo/W enzymes in physiology and bioenergetics is widely recognized. It is worth noting that the most diverse family of Mo/W enzymes is exclusive to prokaryotes, with the probable existence of several of them from the earliest forms of life on Earth. The structural organization of these enzymes, which often include additional redox centers, is as diverse as ever, as is their cellular localization. The most notable observation is the involvement of dedicated chaperones assisting with the assembly and acquisition of the metal centers, including Mo/W-bisPGD, one of the largest organic cofactors in nature. This review seeks to provide a new understanding and a unified model of Mo/W enzyme maturation

Distribution and dynamics of OXPHOS complexes in the bacterial cytoplasmic membrane

International audienceOxidative phosphorylation (OXPHOS) is an essential process for most living organisms mostly sustained by protein complexes embedded in the cell membrane. In order to thrive, cells need to quickly respond to changes in the metabolic demand or in their environment. An overview of the strategies that can be employed by bacterial cells to adjust the OXPHOS outcome is provided. Regulation at the level of gene expression can only provide a means to adjust the OXPHOS outcome to long-term trends in the environment. In addition, the actual view is that bioenergetic membranes are highly compartmentalized structures. This review discusses what is known about the spatial organization of OXPHOS complexes and the timescales at which they occur. As exemplified with the commensal gut bacterium Escherichia coli, three levels of spatial organization are at play: supercomplexes, membrane microdomains and polar assemblies. This review provides a particular focus on whether dynamic spatial organization can fine-tune the OXPHOS through the definition of specialized functional membrane microdomains. Putative mechanisms responsible for spatio-temporal regulation of the OXPHOS complexes are discussed. This article is part of a Special Issue entitled Organization and dynamics of bioenergetic systems in bacteria, edited by Conrad Mullineaux. (C) 2015 Elsevier B.V. All rights reserved

Study of the interaction of respiratory complexes with their membrane coenzymes (the case of the Escherichia coli Nitrate reductase A)

Au cours de ma thèse, je me suis intéressée à l'interaction du complexe Nitrate Reductase A (NarGHI) avec les quinones et les lipides de la membrane chez E. coli. Nous avons identifié que les intermédiaires ménasmiquinones interagissent avec une liaison hydrogène avec l'histidine 66 du site Qd. Par ailleurs, nous avons mis en évidence par la fixation spécifique d'une molécule de cardiolipine est indispensable au fonctionnement du complexe NarGHI en permettant la fixation du quinol. Enfin, nous avons démontré l'existence d'une liaison fonctionnelle entre la voie de biosynthèse des hèmes et les complexes respiratoires via la protéine HemG, qui couple la réduction des quinones avec l'oxydation du protoporphyrinogène IX. Ces éléments prouvent qu'une voie catalytique peut contribuer à la synthèse ATP. L'ensemble de ces résultats indique une étroite interconnexion physique et fonctionnelle entre tous les éléments qui composent la membrane cytoplasmique d'E. coliIn this thesis, I study the interaction between the nitrate reductase A comlex (NarGHI) with the quinines and lipids of the E. coli cytoplasmic membrane. We demonstrate that His66 present at the Qd site is directly hydrogen bonded to both menasemiquinone and ubisemiquinone species. In addition, we show that functionning of the enzyme complex is controlled by cardiolipin binding in a specific cavity allowing quinol binding at the nearby QD site. Finally, we relealed that heme biosynthesis is a quinone-depended metabolic reaction during anaerobic growth of E. coli, in wich the HemG protein will direct electron transfer issued from oxidation of a heme biosynthetic intermediate towards quinone molecules via interaction between quinones, lipids and membrane- associated complexes that couple respiration and anabolic pathways to ATP generation in specialized domains of E. coli membraneAIX-MARSEILLE2-Bib.electronique (130559901) / SudocSudocFranceF

EBEC 2020: The conference that should have taken place!

International audienc

The Prokaryotic Mo/W-bisPGD Enzymes Family

International audienceOver the past two decades, prominent importance of molybdenum/tungsten-containing enzymes in prokaryotes has been put forward by studies originating from different fields providing a unique combination of knowledge. Proteomic or bioinformatic studies underpinned that the list of molybdenum/tungsten-containing enzymes is far from being complete with, to date, more than 50 different enzymes involved in the biogeochemical nitrogen, carbon and sulfur cycles. In particular, the vast majority of prokaryotic molybdenum-/tungsten-containing enzymes belong to the so-called DMSO reductase family. Despite its extraordinary diversity, this family is characterized by the presence of a Mo/W-bis(pyranopterin guanosine dinucleotide) cofactor at the active site with a yet uncovered reactivity. This chapter highlights the substrate specificity of their catalytic site, the modular variation of their structural organization and their contribution to the bioenergetics of prokaryotes

Supramolecular Organization in Prokaryotic Respiratory Systems

International audienceProkaryotes are characterized by an extreme flexibility of their respiratory systems allowing them to cope with various extreme environments. To date, supramolecular organization of respiratory systems appears as a conserved evolutionary feature as supercomplexes have been isolated in bacteria, archaea, and eukaryotes. Most of the yet identified supercomplexes in prokaryotes are involved in aerobic respiration and share similarities with those reported in mitochondria. Supercomplexes likely reflect a snapshot of the cellular respiration in a given cell population. While the exact nature of the determinants for supramolecular organization in prokaryotes is not understood, lipids, proteins, and subcellular localization can be seen as key players. Owing to the well-reported supramolecular organization of the mitochondrial respiratory chain in eukaryotes, several hypotheses have been formulated to explain the consequences of such arrangement and can be tested in the context of prokaryotes. Considering the inherent metabolic flexibility of a number of prokaryotes, cellular distribution and composition of the supramolecular assemblies should be studied in regards to environmental signals. This would pave the way to new concepts in cellular respiration

Synthesis of the NarP response regulator of nitrate respiration in Escherichia coli is regulated at multiple levels by Hfq and small RNAs

ABSTRACT Two-component systems (TCS) and small RNAs (sRNA) are widespread regulators that participate in the response and the adaptation of bacteria to their environments. They mostly act at the transcriptional and post-transcriptional levels, respectively, and can be found integrated in regulatory circuits, where TCSs control sRNAs transcription and/or sRNAs post-transcriptionally regulate TCSs synthesis. In response to nitrate and nitrite, the paralogous NarQ-NarP and NarX-NarL TCSs regulate the expression of genes involved in anaerobic respiration of these alternative electron acceptors. In addition to the previously reported repression of NarP synthesis by the SdsN 137 sRNA, we show here that RprA, another Hfq-dependent sRNA, also negatively controls narP . Interestingly, the repression of narP by RprA actually relies on two independent controls. The first is via the direct pairing of the central region of RprA to the narP translation initiation region and presumably occurs at the translation initiation level. In contrast, the second control requires only the very 5’ end of the narP mRNA, which is targeted, most likely indirectly, by the full-length or the shorter, processed, form of RprA. In addition, our results raise the possibility of a direct role of Hfq in narP control, further illustrating the diversity of post-transcriptional regulation mechanisms in the synthesis of TCSs

Synthesis of the NarP response regulator of nitrate respiration in Escherichia coli is regulated at multiple levels by Hfq and small RNAs

International audienceAbstract Two-component systems (TCS) and small RNAs (sRNA) are widespread regulators that participate in the response and the adaptation of bacteria to their environments. TCSs and sRNAs mostly act at the transcriptional and post-transcriptional levels, respectively, and can be found integrated in regulatory circuits, where TCSs control sRNAs transcription and/or sRNAs post-transcriptionally regulate TCSs synthesis. In response to nitrate and nitrite, the paralogous NarQ-NarP and NarX-NarL TCSs regulate the expression of genes involved in anaerobic respiration of these alternative electron acceptors to oxygen. In addition to the previously reported repression of NarP synthesis by the SdsN137 sRNA, we show here that RprA, another Hfq-dependent sRNA, also negatively controls narP. Interestingly, the repression of narP by RprA actually relies on two independent mechanisms of control. The first is via the direct pairing of the central region of RprA to the narP translation initiation region and presumably occurs at the translation initiation level. In contrast, the second requires only the very 5′ end of the narP mRNA, which is targeted, most likely indirectly, by the full-length or the shorter, processed, form of RprA. In addition, our results raise the possibility of a direct role of Hfq in narP control, further illustrating the diversity of post-transcriptional regulation mechanisms in the synthesis of TCSs

Membrane-Associated Maturation of the Heterotetrameric Nitrate Reductase of Thermus thermophilus

The nar operon, coding for the respiratory nitrate reductase of Thermus thermophilus (NRT), encodes a di-heme b-type (NarJ) and a di-heme c-type (NarC) cytochrome. The role of both cytochromes and that of a putative chaperone (NarJ) in the synthesis and maturation of NRT was studied. Mutants of T. thermophilus lacking either NarI or NarC synthesized a soluble form of NarG, suggesting that a putative NarCI complex constitutes the attachment site for the enzyme. Interestingly, the NarG protein synthesized by both mutants was inactive in nitrate reduction and misfolded, showing that membrane attachment was required for enzyme maturation. Consistent with its putative role as a specific chaperone, inactive and misfolded NarG was synthesized by narJ mutants, but in contrast to its Escherichia coli homologue, NarJ was also required for the attachment of the thermophilic enzyme to the membrane. A bacterial two-hybrid system was used to demonstrate the putative interactions between the NRT proteins suggested by the analysis of the mutants. Strong interactions were detected between NarC and NarI and between NarG and NarJ. Weaker interaction signals were detected between NarI, but not NarC, and both NarG and NarH. These results lead us to conclude that the NRT is a heterotetrameric (NarC/NarI/NarG/NarH) enzyme, and we propose a model for its synthesis and maturation that is distinct from that of E. coli. In the synthesis of NRT, a NarCI membrane complex and a soluble NarGJH complex are synthesized in a first step. In a second step, both complexes interact at the cytoplasmic face of the membrane, where the enzyme is subsequently activated with the concomitant conformational change and release of the NarJ chaperone from the mature enzyme