UMR8261 Expression Génétique Microbienne

CNRS / Université Paris Diderot Paris 7

Directeur : Harald Putzer, Directeur-adjoint : Ciarán Condon

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Resp : Miklos de Zamaroczy

Liliana Mora


Tel:    +33 1 58 41 51 54

CNRS - FRE 3630 (UPR 9073)
Institut de Biologie Physico-Chimique
(pièces 103-104)

13, rue Pierre et Marie Curie
75005 Paris, France


   Our research relies on functional and structural approaches to study the inhibition of protein synthesis by antimicrobial cytotoxins (colicins). Colicin-producing Escherichia coli strains kill neighbouring competing bacteria by releasing cytotoxins into the surroundings. The transfer of RNase- or DNase-type colicins, synthesized during times of environmental stress, from the extracellular environment to their cytoplasmic targets necessitates a cascade of molecular interactions between the colicin domains and inner- and outer-membrane components.

   Our group aims to decipher how nuclease type colicins are taken up by the target cells and how this leads to cell killing. We are interested in dissecting several macromolecular associations that are necessary for the delivery of the toxic catalytic colicin domain to the cytoplasm. We have shown that the import process of these toxic enzymes requires their endoproteolytic processing and the subsequent translocation of the catalytic domain across the inner membrane of the target cells. Our goals are to elucidate how nuclease colicins parasitize different membrane systems (e.g., protease FtsH, signal peptidase LepB), particularly during the late import steps. Our investigations also extend to the killing activities exerted by these colicins and to the mechanisms that protect the colicin-producing cells from both the endo- and exogenic presence of their own toxins. Our experimental approaches rely on a multidisciplinary set of experimental techniques that lead to the structural and functional characterization of hijacked membrane partners that are associated with different domains of nuclease type colicins.

   Colicin D is a toxic RNase of transfer RNAs that parasitizes several membrane components in order to enter the cell. The N-terminal domain is required for import and the C-terminal domain carries the toxic enzyme (de Zamaroczy et al. (2002) Biochimie). Several interactions between colicin D and the energy transducer protein TonB are essential for its import. This process is inhibited by specific tonB mutations, but it is restored by suppressor mutations introduced into the colicin D structural gene.(Mora et al (2005) J Bact).

   It was demonstrated that there is a requirement for an endoproteolytic cleavage of two different RNase colicins (D and E3) for their import into the cytoplasm of target cells. In particular, we showed that the inner membrane ATPase-protease FtsH is essential for colicin processing (PF: processed form) and for the translocation of the toxic domains across the cytoplasmic membrane. (Chauleau et al. (2011) J Biol Chem; de Zamaroczy et al. (2012) Biochem Soc).

   Genetic evidence revealed that the inner-membrane signal-peptidase LepB is a specific requirement for colicin D import, but that its peptidase activity per se is not required for colicin D processing. We reported a model suggesting that the direct interaction between LepB and the central domain of colicin D specifically promotes conformational changes in the tRNase domain necessary for its access to FtsH (de Zamaroczy et al. (2001) Mol Cell ; Chauleau et al. (2011) J Biol Chem).

Immunity complex of the transfer-RNase colicin D

   We solved the crystal structure of colicin D tRNase domain in complex with its cognate immunity protein (ImmD). Together with functional studies, we showed that the active site pocket of colicin D is blocked by ImmD (Graille et al. (2004) EMBO J). In addition to this major interaction, we also have shown a second interaction between ImmD and the central domain (CD) of colicin D. Thus, ImmD is “sandwiched” between these two domains of the full-length colicin D. This structure ensures that the colicin D - ImmD complex is naturally secreted in a nontoxic state (Mora et al. (2008) J Biol Chem).

   Currently, our goals are to get insights into:

• Functions of LepB and FtsH in colicin biology

• The tRNase colicin D, how it recognizes its tRNA-Arg substrate, its catalytic mechanism

• Endoproteolytic cleavage of DNase-type colicins and other bacteriocins during import

   These studies also will be useful in the general context of protein transport into bacteria. The current crisis in antibiotic resistance observed with E. coli obtained from human clinical isolates provides an important incentive for researchers to discover novel antibiotics. The mechanistic analysis of colicin penetration into target cells will be of a major interest in developing medical protocols in humans to exploit the killing action of colicins against pathogenic E. coli strains.



Main Publications

Mora, L., Moncoq, K., England, P., Oberto, J. & de Zamaroczy, M. (2015) The stable interaction between signal-peptidase LepB of Escherichia coli and nuclease bacteriocins promotes toxin entry into the cytoplasm.J Biol Chem. 290, 30783-30796; doi: 10.1074/jbc

Roque, S., Cerciat, M., Gaugué, I., Mora, L., Floch, A., de Zamaroczy, M., Heurgué-Hamard, V. & Kervestin, S. (2015) Interaction between the poly(A)-binding protein Pab1 and the eukaryotic release factor eRF3 regulates translation termination but not mRNA decay in Saccharomyces cerevisiae. RNA 21, 124-134.

Mora, L. & de Zamaroczy, M. (2014) In vivo Processing of DNase colicins E2 and E7 is required for their import into the cytoplasm of target cells. PLoS One. 2014 May 19;9(5):e96549. doi: 10.1371/journal.pone.0096549. eCollection 2014

de Zamaroczy, M. & Mora, L. (2012) Hijacking of cellular functions for processing and delivery of colicins E3 and D into the cytoplasm. Biochem. Soc. T. 40, 1486-1491

Chauleau, M., Mora, L., Serba, J. & de Zamaroczy, M. (2011) FtsH-dependent processing of the RNase colicins D and E3 means that only the cytotoxic domains are imported into the cytoplasm. J. Biol Chem. 286, 29397-29407.

Liger, D., Mora, L., Lazar, N., Figaro, S., Henri, J., Scrima, N., Buckingham R. H., van Tilbeurgh, H., Heurgué-Hamard, V. & Graille, M (2011) Mechanism of activation of methyltransferases involved in translation by the Trm112 "hub" protein.  Nucleic Acids Res. 39, 6249-6259.

de Zamaroczy, M., and Chauleau, M. (2011). Colicin killing: foiled cell defense and hijacked cell functions. In Procaryotic antimicrobial peptides: from genes to applications, D. Drider and S. Rebuffat, eds. Chapter 14, (Springer Science Media, Springer New York, London), pp. 255-288.

Diago-Navarro, E., Mora, L., Buckingham, R.H., Díaz-Orejas, R. & Lemonnier, M. (2009) Novel Escherichia coli RF1 mutants with decreased translation termination activity and increased sensitivity to the cytotoxic effect of the bacterial toxins Kid and RelE. Mol. Microbiol. 71, 66-78.

Mora, L., Klepsch, M., Buckingham, R.H., Heurgué-Hamard, V., Kervestin, S. & de Zamaroczy, M. (2008) Dual roles of the central domain of colicin D tRNase in TonB-mediated import and in immunity. J Biol Chem. 283, 4993-5003.

Mora, L., Heurgue-Hamard, V., de Zamaroczy, M., Kervestin, S. and Buckingham, R. H. (2007) Methylation of bacterial release factors RF1 and RF2 is required for normal translation termination in vivo. J. Biol. Chem. 282, 35638-35645.

Vestergaard, B., Sanyal, S., Roessle, M., Mora, L., Buckingham, R. H., Kastrup, J. S., Gajhede, M., Svergun, D. I. and Ehrenberg, M. (2005) The SAXS solution structure of RF1 differs from its crystal structure and is similar to its ribosome-bound cryo-EM structure. Molecular Cell, 20, 929-38.

Graille, M., Heurgué-Hamard, V., Champ, S., Mora, L., Scrima, N., Ulryck, N., van Tilbeurgh, H. & Buckingham, R.H. (2005) Molecular basis for bacterial class I release factor methylation by PrmC. Mol. Cell, 20, 917-927.

Heurgue-Hamard, V., Champ, S., Mora, L., Merkulova-Rainon, T., Kisselev, L. L., and Buckingham, R. H. (2005).The glutamine residue of the conserved GGQ motif in Saccharomyces cerevisiae release factor eRF1 is methylated by the product of the YDR140w gene. J. Biol. Chem. 280, 2439-2445.

Mora, L., Diaz, N., Buckingham, R. H., and de Zamaroczy, M. (2005) Import of the transfer RNase colicin D requires site-specific interaction with the energy-transducing protein TonB. J. Bacteriol. 187, 2693-2697.

Graille, M., Mora, L., Buckingham, R.H., Van Tilbeurgh, H. & de Zamaroczy, M. (2004) Structural inhibition of the colicin D tRNase by the tRNA-mimicking immunity protein. EMBO J. 23, 1474-82.

Mora, L., Heurgué-Hamard, V., Champ, S., Ehrenberg, M., Kisselev, L. & Buckingham, R.H. (2003) The essential role of the invariant GGQ motif in the function and the stability in vivoof bacterial release factors RF1 and RF2. Mol. Microbiol. 47, 267-275.

Mora, L., Zavialov, A.V., Ehrenberg, M. & Buckingham, R.H. (2003) Stop codon recognition and interactions with peptide release factor RF3 of truncated and chimeric RF1 and RF2 from Escherichia coli. Mol. Microbiol. 50, 1467-1476.

Menez, J., Buckingham, R.H., de Zamaroczy, M. & Karmazyn-Campelli, C. (2002) Peptidyl-tRNA hydrolase in Bacillus subtilis, encoded by spoVC, is essential to vegetative growth, whereas the homologous enzyme in Saccharomyces cerevisiae is dispensible. Mol. Microbiol. 147, 1581-1589.

Zavialov, A.V., Mora, L., Buckingham, R.H. & Ehrenberg, M. (2002) Release of peptide promoted by the GGQ-motif of class 1 release factors regulates the GTPase activity of RF3. Molecular Cell, 10, 789-798.

de Zamaroczy, M. & Buckingham, R.H. (2002) Importation of nuclease colicins into E. coli cells: endonucleolytic cleavage and its prevention by the immunity protein. Biochimie, 84, 423-432.

de Zamaroczy, M., Mora, L., Lecuyer, A., Geli, V. & Buckingham, R.H. (2001) Cleavage of colicin D is necessary for cell killing and requires the inner membrane peptidase LepB. Molecular Cell, 8, 159-168.

de Zamaroczy, M., Mora, L., Lecuyer, A., Geli, V. & Buckingham, R.H. (2001) Cleavage of colicin D is necessary for cell killing and requires the inner membrane peptidase LepB. Molecular Cell. 8, 159-168.

Dinçbas-Renqvist, V., Engström, Å., Mora, L., Heurgué-Hamard, V., Buckingham, R.H. & Ehrenberg, M. (2000) A post-translational modification in the GGQ motif of RF2 from Escherichia coli stimulates termination of translation. EMBO J. 19, 6900-6907.



Karine MONCOQ CNRS UMR 7099, IBPC, Paris
Structure du complexe LepB / colicine D.

Patrick ENGLAND Institut Pasteur, PFBMI, Paris
Etudes biophysiques de LepB en interaction avec la colicine D pendant l’import.




















Former Lab Members


2000 - 2001 : Aurélie Lecuyer, 9 mois, Master 2, Université Paris 6 (UPMC)

2002 : Nancy Diaz, 6 mois, Licence (L3), Université Montpellier II

2004 : Agnès Hebert, 5 mois, Licence (L3), Université Paris 7 (UDD)

1998 - 2004 : Stéphanie Champ, AI / CNRS

2005 : Mirjam Klepsch, 5 mois, Master 2, ERASMUS, Université de Bayreuth, Allemagne

2008 : Kevin Fidelin, 2 mois, stage de BTS, ENCPB, Paris

2005 - 2008 : Nathalie Scrima, TCN / CNRS

2009 : Richard Buckingham, DR1 émérite CNRS (retraite)

2010 : Justyna Serba, 6 mois, Master 2, ERASMUS, Université de Poznan, Pologne

2011 : Hanna Kasprzak, 6 mois, Master 2, ERASMUS, Université de Poznan, Pologne

2009 - 2011 : Mathieu Chauleau, Doctorat d’Université Paris 11 (ED GGC)

1996 - 2012 : Jean-Hervé Alix, ancien MCHC, Université Paris 7 (UDD) (retraite)

2013 : Marta Cerqueira Mendes, 6 mois, Master 2, ERASMUS, Technical University Lisbon, Portugal

2013 : Mélanie Lemor, 2 mois Master 1, Université Blaise Pascal, Clermont-Ferrand

2001 - 2013 : Valérie Heurgué-Hamard, CR1 CNRS.




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