To produce their energy in the absence of dioxygen (anaerobiosis), bacteria employ anaerobic respiration which depends on quinones. In anaerobiosis, the function of ubiquinone (UQ) is unknown. Its biosynthesis is not characterized but is thought to implicate hydroxylases of a new type. By studying the model bacterium Escherichia coli, we wish to elucidate the biosynthesis of UQ in anaerobiosis, to characterize the associated proteins and to understand the physiological functions of UQ.
E. coli, like many microorganisms, is a facultative anaerobe: it can grow under anaerobic (absence of dioxygen) or aerobic conditions in order to adapt to the varying oxygen tensions met in different environments. Ubiquinone (UQ) is known to contribute to aerobic metabolism but is also produced in anaerobic conditions by unidentified enzymes, some of which are different from those used in aerobiosis. The objectives of the project are to: <br />1) Identify the genes involved in anaerobic UQ biosynthesis and characterize the proteins. <br />2) Characterize biochemically and structurally proteins specifically involved in aerobic UQ biosynthesis (UbiJ, K) and demonstrate the existence of a multiprotein complex.<br />3) Understand the remodeling between aerobic and anaerobic UQ biosynthesis. <br />4) Compare the effect of mutations in (an)aerobiosis-specific ubi genes for E. coli grown in laboratory cultures.<br /><br />Our results may reveal a new class of enzymes which can perform hydroxylation reactions in the absence of dioxygen. The characterization of a multiprotein complex regrouping most proteins involved in the aerobic biosynthesis of UQ will shed light on the molecular organization of a complex biosynthetic pathway that produces an important cellular metabolite. The characterization of the dynamic and of the physiological importance of UQ biosynthesis in anaerobic conditions will reveal an important aspect of microaerobic/anaerobic metabolism of widespread facultative aerobic proteo-bacteria.
To provide a comprehensive description of aerobic and anaerobic UQ biosynthesis in E. coli and to assess the physiological involvement of UQ in diverse cellular functions in laboratory cultures, we combine multi-disciplinary approaches: (i) microbial genetics and physiology, (ii) molecular biology and cellular imaging, (iii) protein chemistry structural biology and organic synthesis for UQ intermediates not commercially available.
Protein biochemistry techniques (overexpression and purification, SEC-MALLS, X-ray crystallography, site-directed mutagenesis, fluorescence of intrinsic residues) will be used to characterize the UbiJ and UbiK proteins, their interaction with other Ubi proteins and their capacity to interact with lipids.
We will study the multiprotein Ubi complex by native electrophoresis techniques (BN-PAGE) coupled to immunodetection, by microscopy of Ubi proteins fused to reporter fluorescent proteins (GFP, m-Cherry) and by tandem affinity purification (SPA-tag) followed by proteomic analysis.
The remodeling of UQ biosynthesis between aerobic and anaerobic conditions will be evaluated by quantitative reverse transcription PCR (qRT-PCR), by immunodetection of particular proteins and by fluorescence microscopy.
The identification of genes specifically implicated in anaerobic UQ biosynthesis will be carried out by a genetic approach, by a comparative genomic approach or by a biochemical approach involving the purification of the putative anaerobic Ubi complex and its characterization by proteomics. The newly-identified proteins will be characterized biochemically by partner 3.
The biochemical characterization of UbiJ and UbiK demonstrated that these proteins form a heterotrimer (UbiK2-UbiJ1) and that UbiJ is able to bind palmitoleic acid, an endogenous lipid of E. coli. These results were published in June 2017 together with the description that the new gene ubiK is implicated in aerobic UQ biosynthesis in E. coli.
We showed that several Ubi proteins co-migrate at 1 MDa on BN-PAGE gels. Fluorescence microscopy allowed to detect only some Ubi proteins but those were shown to co-localize inside the cell. These results agree with the existence of multiprotein Ubi complex implicated in aerobic UQ biosynthesis and we are actively trying to purify this complex and establish its composition.
Via a genetic approach, we have identified two new genes of unknown function which are essential for UQ biosynthesis in anaerobic conditions. We suspect that these proteins may catalyze the hydroxylation reaction of the anaerobic UQ biosynthesis pathway and we are currently trying to purify these proteins and establish their function in activity assays.
Regarding the physiological function of UQ in anaerobic conditions, we refuted the proposal by Sauer and Sevin that UQ participates to the stability of E. coli membranes and its resistance to osmotic stress (Nat Chem Biol, 2014). Indeed, unlike these authors, we did not observe any salt sensitivity of E. coli mutants deprived of UQ assessed either in aerobic or anaerobic conditions. We will now concentrate on studying other potential functions of UQ under anaerobiosis like its participation to microaerobic respiration.
We think that the hydroxylases from the anaerobic UQ biosynthetic pathway may belong to a new class of enzymes capable of catalyzing hydroxylation reactions in the absence of dioxygen. The discovery and characterization of such enzymes, probably widespread among anaerobic microorganisms, may open new leads for the development of hydroxylation catalysts that respect the principles of green chemistry.
Via our characterization of the dynamic of UQ biosynthesis in anaerobiosis, its cellular organization and physiological importance, we will contribute to obtain a better understanding of the anaerobic-microaerobic metabolism of a large class of proteobacteria, some of which are virulent. Our previous studies demonstrate the importance of UQ for the virulence of Salmonella. Thus UQ may also represent a virulence factor in anaerobic conditions. If true, our identification of genes –not conserved in humans- implicated in UQ biosynthesis in anaerobic conditions may open new perspectives to develop novel antibacterial molecules.
Loiseau, L., Fyfe, C., Aussel, L., Hajj Chehade, M., Hernandez, S.B., Faivre, B., et al. (2017). The UbiK protein is an accessory factor necessary for bacterial ubiquinone (UQ) biosynthesis and forms a complex with the UQ biogenesis factor UbiJ. J Biol Chem. doi: 10.1074/jbc.M117.789164.
Pelosi, L., Ducluzeau, A.L., Loiseau, L., Barras, F., Schneider, D., Junier, I., et al. (2016). Evolution of Ubiquinone Biosynthesis: Multiple Proteobacterial Enzymes with Various Regioselectivities To Catalyze Three Contiguous Aromatic Hydroxylation Reactions. mSystems 1(4), e00091-00016. doi: 10.1128/mSystems.00091-16.
Respiration is a fundamental process of life because it generates an electrochemical gradient across membranes that is used for energy production and active transport. Quinones are central to respiration because they function as obligatory electron and proton shuttles in respiratory chains. In the model bacterium Escherichia coli, the quinone pool is composed of ubiquinone (UQ) which participates mostly to aerobic respiration and two naphtoquinones involved mainly in anaerobic respiration. Quinones are biosynthesized lipid molecules that localize in membranes. In addition to respiration, quinones are implicated in important functions like formation of disulfide bonds in proteins, transcriptional regulation, antioxidant action and membrane stabilization.
The biosynthesis of UQ is a complex process that requires at least 11 different Ubi proteins, among which two were identified by our consortium (JBC in 2013 and J Bacteriol. in 2014). E. coli synthesizes UQ when grown in aerobic and anaerobic conditions but has developed specific strategies to do so. Indeed, our preliminary data establish that E. coli possesses several Ubi proteins, conserved among eubacteria, that function only in either conditions. The biosynthesis and physiological functions of UQ in anaerobic and microaerobic conditions have been understudied despite the fact that variable and low oxygen tensions are frequently encountered by E. coli in several ecological niches. Our projects aims at providing a comprehensive description of aerobic and anaerobic UQ biosynthesis in E. coli and at assessing the physiological involvement of UQ in diverse cellular functions in laboratory cultures and for colonization of different hosts. To reach this goal, the three partners will combine multi-disciplinary approaches: (i) microbial genetics and physiology, (ii) molecular biology and cellular imaging, (iii) protein chemistry structural biology and organic synthesis for UQ intermediates not commercially available.
We will focus our biochemical and structural studies on the Ubi proteins that we have identified recently in order to elucidate their function notably with regard to a Ubi-complex which regroups several Ubi proteins. In addition, we propose several independent approaches to identify UQ anaerobic hydroxylases which may be considered as the prototypes of a new class of enzymes that perform hydroxylation reactions independently from oxygen. Such enzymes are likely widespread among obligate anaerobes, but they have not been identified so far since very few of these microorganisms are genetically tractable. Therefore, the possibility to use E. coli to identify anaerobic hydroxylases represents a unique opportunity to uncover the details of a reaction which is of major interest for chemists. In order to complete our comprehension of the roles of UQ in anaerobic-microaerobic conditions, we propose in vivo experiments that will compare the phenotypes of E. coli mutants affected for UQ biosynthesis in either aerobic or anaerobic conditions or in both. Overall, our results will elucidate why E. coli produces UQ in the presence and absence of dioxygen and how it does so.
Monsieur Fabien Pierrel (Laboratoire Techniques de l'Ingénierie Médicale et de la Complexité - Informatique, Mathématiques et Applications, Grenoble)
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
INSTITUT PASTEUR Département de Microbiologie STRESS ADAPTATION AND METABOLISM- INSTITUT PASTEUR
TIMC-IMAG Laboratoire Techniques de l'Ingénierie Médicale et de la Complexité - Informatique, Mathématiques et Applications, Grenoble
CNRS DR12 - LCB Centre National de la Recherche Scientifique - Délégation Provence et Corse - Laboratoire de Chimie Bactérienne
LCPB- CNRS UMR8229- College de France Laboratoire de Chimie des Processus Biologiques
Help of the ANR 541,254 euros
Beginning and duration of the scientific project: October 2015 - 48 Months