UMR8261 Expression Génétique Microbienne

CNRS / Université Paris Diderot Paris 7

Directeur : Harald Putzer, Directeur-adjoint : Ciarán Condon

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TRANSCRIPTIONAL AND POST-TRANSCRIPTIONAL CONTROLS OF GENE EXPRESSION

Resp : Harald Putzer

The ability to regulate gene expression is a fundamental property of all life forms from the simplest virus to the most complex mamammal. To understand how  the overall developement of an organism is programmed, it is crucial to understand the different mechanisms which control the expression of individual genes. Our group concentrates on mechanisms controlling mRNA levels. There are two major ways RNA levels are controlled, either by a control of their synthesis (transcription initiation and elongation) or their degradation (by mRNA nucleases). The different projects use either E. coli or B. subtilis as model organisms and can be divided into two major subjects.

 

Thme 1 : Biosynthesis of the translational apparatus and the role of mRNA stability/processing in gene expression

 

Resp : Harald Putzer

 

Theme 2 : The use of amino sugars by E. coli and B. subtilis.

 

Resp : Jackie Plumbridge

 

 

Biosynthesis of the translational apparatus and the role of mRNA stability/processing in gene expression

Resp : Harald Putzer

   The structural genes and the proteins they encode are often conserved between different bacterial species (e.g., Gram positive and Gram negative bacteria). In contrast, the mechanisms that control the expression of these genes can be surprisingly different. This is clearly seen when one compares the two model organisms Escherichia coli (Gram-) and Bacillus subtilis (Gram+), which are separated by more than 2 billion years of evolution. Thus, it is important to study more than one organism in great detail in order to better understand the different strategies used to modulate gene expression in prokaryotes.

   We are interested in co- and post-transcriptional mechanisms that control the synthesis of components of the translational apparatus in B. subtilis, and we are particularly interested in mechanisms that involve transcriptional anti-termination and mRNA processing/degradation.

   Recently, we have extended our studies to the characterization of migrating B. subtilis communities (“swarming”), which is an example of social behavior that allows us to analyze gene expression in single cells.

Control of expression of proteins of the translational apparatus

   The biosynthesis of the translational apparatus (ribosomes, aminoacy-tRNA synthetases, translation factors, etc.) consumes more than half of the cellular energy and thus requires efficient control. In B. subtilis, and Gram-positive bacteria in general, our knowledge of ribosomal protein expression is still limited, notably concerning the strategies used to co-ordinate their synthesis with that of ribosomal RNAs. We have elucidated for the first time, on a molecular level, a mechanism that controls the expression of a ribosomal protein in a Gram-positive organism (the infC-rpmI-rplT operon in B. subtilis). Interestingly, B. subtilis and E. coli use the same effector molecule, ribosomal protein L20, and molecular mimicry. However, they do so in very different contexts: Bacillus uses transcriptional anti-termination while E. coli regulates expression by translational auto-repression. Although limited to only a few examples, this illustrates that molecular mimicry is a very powerful concept that can be integrated into very diverse control mechanisms.

   Expression of aminoacyl-tRNA synthetases in B. subtilis also is controlled by another mechanism that is not observed in E. coli: tRNA-dependent transcriptional anti-termination. Our studies on these diverse regulatory mechanisms also have revealed the importance of strategic mRNA cleavages that can directly influence gene expression. This has led us to identify and characterize the ribonucleases that are implicated in these cleavages.

The role of mRNA degradation/maturation in gene expression

   Controlling the degradation of mRNA can be a very efficient means for regulating gene expression. It is a powerful tool that permits the rapid adaptation to nutritional and environmental changes, and it allows the cell to efficiently control the stoichiometry of proteins encoded by an operon.

   RNase E is the major ribonuclease initiating mRNA degradation in E. coli but B. subtilis has no orthologue of this key enzyme. While studying the fate of various mRNAs, we identified two novel ribonucleases named RNases J1/J2 and RNase Y. Surprisingly, we found that ribonucleases J and Y have an endonucleolytic cleavage specificity that is similar to that of E. coli RNase E, despite a total absence of sequence similarity between these three enzymes. This enzymatic activity has thus been invented independently at least three times, illustrating an impressive case of convergent evolution. It is likely that the three RNases E, J and Y represent the key enzymes in eubacterial RNA metabolism. Indeed, all eubacterial species have at least one of these three enzymes and many actually have all three

Figure 1 :

 

Figure 1 : Distribution of RNases E/G (green), J (blue) and/ or Y (red) in prokaryotic organisms (as a percentage of the total number of organisms within a phylum). RNases present in the majority of a phylum are shown on the right. All Archaea contain an RNase J-like activity that can be partitioned into two major subdivisions that correspond to orthologs of the eukaryotic cleavage and polyadenylation specificity factor (CPSF73) and bacterial RNase J.

   RNase J1 is a dual activity ribonuclease that shows both endo- and 5’-3’ exonucleolytic cleavages. We have resolved the 3D structure of this ribonuclease and shown that both activities are catalyzed within a single active site

Figure 2 :

The structure also highlighted the presence of a mononucleotide binding pocket that explains why the 5’-3’ exonucleolytic activity requires a monophosphate or hydroxyl moiety on the 5’ end of the RNA, which is the product generally created by an endonucleolytic cleavage.

   Transcriptomic and proteomic analyses of B. subtilis RNase J and Y mutants have confirmed the global importance of these enzymes in RNA metabolism.

   We have observed that a depletion of RNase Y increases bulk mRNA stability more than 2-fold, and we favor the hypothesis that the major mRNA decay initiating pathway is endonucleolytic rather than exonucleolytic

Figure 3 :

This suggests that the degradation/maturation of mRNA is more similar between Gram-positive and Gram-negative bacteria than previously thought. In this model, the 5’ exonucleolytic activity of RNase J serves primarily to degrade RNA fragments produced by endonucleolytic cleavage and that is otherwise protected from 3’ exonucleolytic attack by a secondary structure at the 3’ end (e.g., a transcription terminator).

   We now want to analyze the role of RNases J and Y in the regulation of specific genes in B. subtilis, and we want to identify novel strategies of regulating gene expression based on the mRNA stability. The study of their respective functions and how their expression is controlled will help to better understand mRNA metabolism in Bacilli.

The migrating Bacillus subtilis community: a study of the spatio-temporal expression in a single cell

   Social behavior of bacterial communities assures survival and dissemination in diverse habitats. Migration “in mass” (swarming) on a nutrient surface precedes classic biofilm formation, and it can be considered as a mechanism for territorial expansion. Dendritic migration of B. subtilis can occur in the form of a cellular monolayer for several hours. This phenomenon can be exploited for the quantitative analysis of spatio-temporal gene expression in single cells. It allows us to analyze the localization of mRNA and RNases implicated in their metabolism. This experimental system widens our approach to study gene expression and reflects bacterial behavior as it is observed in nature.

 

 

The use of amino sugars by E. coli and B. subtilis.

Resp : Jackie Plumbridge

   Bacteria are capable of using a wide variety of substances as sources of carbon and energy. Amino sugars, as well as supplying carbon, are also a good source of nitrogen and moreover amino sugars are essential components of all cells. In bacteria amino sugars form the backbone of the peptidoglycan of the cell wall and in Gram negative bacteria they are part of the outer membrane lipopolysaccharride. Our primary interest is the regulation of the use of amino sugars, especially N-acetylglucosamine (GlcNAc) and glucosamine (GlcN), and their physiological roles in E. coli.

Comparison of the regulation by the homologous transcription factors, Mlc and NagC in Escherichia coli.

   In both E. coli and B. subtilis the amino sugars are taken up by the phosphotransferase system (PTS), a complex phosphorylation cascade, which results in the simultaneous transport and phosphorylation of the sugar. In E. coli, the genes for use of GlcNAc are controlled by the NagC repressor. NagC also controls several related operons e.g. for use of chitobiose, the dimer of GlcNAc (Fig. 1).

Fig.1

An orthologue of NagC in E. coli is Mlc, which controls the genes for uptake of glucose by the PTS. These two transcription factors are 70% similar and this homology includes the helix-turn-helix (HTH) DNA-binding motifs of the two proteins. Likewise, the operator sequences they recognise on DNA are also very similar. We are studying how NagC and Mlc discriminate between each others targets and have shown that the HTH is not the primary specificity determinant for DNA binding but instead operator specificity is defined by an adjacent sequence, described as a "linker", joining the DNA binding domain to the oligomerisation/effector binding domain of the protein.

   Despite strong overall homology and similar DNA binding sites, the signals which prevent DNA binding are very different for the two proteins. The inducing signal for most transcriptional regulators is a small molecule related to the metabolism of the operon controlled. For NagC this is GlcNAc-6P, the product of the PTS transport of GlcNAc (Fig. 2).

Fig.2

However inactivation of Mlc binding to DNA requires its sequestration by membranes which contain the glucose transporter when it is actively transporting glucose (Fig. 2). We have identified amino acids important for these two signalling mechanisms.

Comparison of the use of amino sugars by E. coli and B. subtilis.

   Comparison of the use of amino sugars by E. coli and B. subtilis.
Unlike E. coli and most other Bacilli, B. subtilis grows better with GlcN than GlcNAc as carbon source. We have shown this is due to the presence of a unique operon (gamAP) encoding duplicate genes for the transport and metabolism of GlcN (Fig. 3). Homologous transcription factors of the GntR family (NagR and GamR) are responsible for repression of these two regulons. Although derived from a common ancestor both their DNA binding characteristics and inducer recognition properties are strongly diverged.

Physiology of amino sugar utilisation: catabolism, anabolism and recycling.

   Since amino sugars are essential components of the bacterial cell wall, all bacteria must balance the expression and activity of the catabolic amino sugar-degrading enzymes with that of the essential biosynthetic enzyme, GlcN6P synthase (GlmS) (Fig.1). A key enzyme is the nagB encoded GlcN6P deaminase. In E. coli this enzyme is allosteric, whereas in B. subtlis there are two NagB isozymes (NagB and GamA (Fig. 3) and neither is allosteric.

Fig.3

The conservation of NagB allostery in certain bacteria like E. coli and in mammals including man, suggests it has a regulatory importance. The peptidoglycan (PG) of the cell wall is undergoing continuous degradation and resynthesis and its amino sugar components, at least in E. coli, are subject to a dedicated recycling procedure. Both NagA and, as we have shown, NagE of the GlcNAc degradation pathway, are important components of the recycling pathway (Fig. 1). The role of NagB allostery in E. coli is an intriguing question which we are addressing by multiple techniques, including genetics, biochemistry, biophysics and metabolomics

 

 

Collaborations

 

Theme 1

Béatrice Golinelli - Collège de France, Paris

Francis-André Wollman - IBPC, Paris

Philippe Bessieres - INRA, Jouy-en-Josas

Isabelle Martin-Verstraete - Institut Pasteur, Paris

Simone Seror - Institut de Génétique et Microbiologie, Orsay

Erhard Bremer – University Göttingen, Germany

Adrian Daerr - Université Paris 7

Rut Carballido-Lopez - Micalis, Jouy-en-Josas

Arnaud Chastanet - Micalis, Jouy-en-Josas

Theme 2

Ian Blomfield - University of Kent, Canterbury, UK

Bernard Badet - Institut de Chimie des substances natural (ICSN), Gif-sur-Yvette

Lionello Bossi and Nara Figueroa-Bossi - Centre de Génétique Moléculaire (CGM) Gif-sur-Yvette

Mario Calcagno - National Autonomous University of Mexico (UNAM), Mexico

Hannes Link - Institute of Molecular Systems Biology, ETH, Zurich, Switzerland

Jacques Oberto - Institut de Génétique et Microbiologie (IGM), Université Paris XI, Orsay

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Main publications


Theme 1

Laalami S., Zig L. and H. Putzer (2014). Initiation of mRNA decay in bacteria. Cell Mol Life Sci, DOI 10.1007/s00018-013-1472-4.

Jamalli A., Hébert A., Zig L. and H. Putzer (2014). Control of expression of the ribonucleases J1 and J2 in B. subtilis (2013). J. Bacteriol., 196, 318-324.

Schroeter R., Hoffmann T., Voigt B., Meyer H., Bleisteiner M., Muntel J., Jürgen B., Albrecht D., Becher D., Lalk M., Evers S., Bongaerts J.,lMaurer K., Putzer H., Hecker M., Schweder T. and E. Bremer (2013). Stress responses of the industrial workhorse Bacillus licheniformis to osmotic challenges. PloS ONE 8(11) : e80956.

Gaugué I., Oberto J., Putzer H. and J. Plumbridge (2013). The Use of Amino Sugars by Bacillus subtilis: Presence of a Unique Operon for the Catabolism of Glucosamine. PLoS ONE 8(5) : e63025.

Laalami S., Bessières P., Rocca A., Zig L., Nicolas P. and H. Putzer (2013). Bacillus subtilis RNase Y activity in vivo analysed by tiling microarrays. PLoS ONE 8(1) : e54062. 

Taverniti V., Forti F., Ghisotti D. and H. Putzer (2011). Mycobacterium smegmatis RNase J is a 5’-3’ exo-/endoribonuclease and both RNase J and RNase E are involved in ribosomal RNA maturation. Mol. Microbiol., 82, 1260-1276.

Laalami S. and H. Putzer (2011). mRNA degradation and maturation in prokaryotes : the global players. Biomolecular Concepts,2, 491-506.

Bruscella P., Shahbabian K., Laalami S. and H. Putzer (2011). RNase Y is responsible for uncoupling the expression of translation factor IF3 from that of the ribosomal proteins L35 and L20 in Bacillus subtilis. Mol. Microbiol.,81, 1526-1541

Dorléans A., Li de la Sierra Gallay I., Piton J., Zig L., Winieski L., Putzer H. and C. Condon (2011). Molecular basis for the recognition and cleavage of RNA by the bifunctional 5'-3' exo/endoribonuclease RNase J. Structure, 19, 1252-1261

Hamze K., Autret S., Hinc K., Julkowska D., Laalami S., Briandet R., Renault M., Absalon C., Holland I.B., Putzer H. & S.J. Séror (2011). Single cell in situ analysis in a B. subtilis swarming community identifies three subpopulations differentially expressing hag (flagellin), including specialized swarmers. Microbiology, 157, 2456-2469.

Brill J., Hoffmann T., Putzer H. and E. Bremer (2011). T-box-mediated control of the anabolic proline biosynthetic genes of Bacillus subtilis. Microbiology, 157, 977-987.

Mathy N, Hébert A., Mervelet P., Bénard L., Dorléans A., Li de la Sierra-Gallay I., Noirot P., Putzer H. and C. Condon (2010). Bacillus subtilis ribonucleases J1 and J2 form a complex with altered enzyme behaviour. Mol. Microbiol., 75, 489-498.

Shahbabian K., Jamalli A., Zig L. and H. Putzer (2009). RNase Y, a novel endoribonuclease, initiates riboswitch turnover in B. subtilis. EMBO J., 28, 3523-3533.

Mäder U., Zig L., Kretschmer J., Homuth G. and H. Putzer (2008). mRNA processing by RNases J1 and J2 affects Bacillus subtilis gene expression on a global scale. Mol. Microbiol., 70, 183-196.

Li de la Sierra-Gallay I., Zig L., Jamalli A. and H. Putzer (2008). Structural insights into the dual activity of RNase J. Nat. Struct. Mol. Biol., 15, 206-212.

Choonee N., Even S., Zig L. and H. Putzer (2007). Ribosomal protein L20 controls expression of the Bacillus subtilis infC operon via a transcription attenuation mechanism. Nucleic Acids Res., 35, 1578-1588.

Even S., Pellegrini O., Zig L., Labas V., Vinh J., Brechemier-Baey D. and H. Putzer (2005). Ribonucleases J1 and J2 : two novel endoribonucleases in B. subtilis with functional homology to E. coli RNase E. Nucleic Acids Res., 33, 2141-2152.

Even S., Brito R., Condon C. and H. Putzer (2003). Aminoacyl-tRNA synthetase gene regulation in bacteria. Rec. Res. Dev. Biol., Vol. 1, p. 201-218.

Pellegrini O., Nezzar J., Marchfelder A., Putzer H. and C. Condon (2003). Endonucleolytic processing of CCA-less tRNA precursors by RNase Z in Bacillus subtilis. EMBO J., 22, 4534-4543.

Condon C. and H. Putzer (2002). The phylogenetic distribution of bacterial ribonucleases. Nucleic Acids Res., 30, 5339-5346.

Putzer H., Condon C., Brechemier-Baey D., Brito R. and M. Grunberg-Manago (2002). Transfer RNA mediated antitermination in vitro. Nucleic Acids Res., 30, 3026-3033.

Condon C., Rourera J. Brechemier-Baey D. and H. Putzer (2002). Ribonuclease has few, if any, mRNA substrates in Bacillus subtilis. J. Bacteriol., 184, 2845-2849.

Putzer H. and S. Laalami (2002). Regulation of the expression of aminoacyl-tRNA synthetases and translational factors, p. 388-415. In: J. Lapointe and L. Brakier-Gingras (eds.), Translational Mechanisms. Landes Bioscience, Georgetown, Tx.

Condon C., Brechemier-Baey D., Beltchev B., Grunberg-Manago M. and H. Putzer (2001). Identification of the gene encoding the 5S ribosomal RNA maturase in Bacillus subtilis: mature 5S rRNA is dispensable for ribosome function. RNA, 7, 1-12.

Main publications 2009-2015


Theme 2

Bréchemier-Baey D, Pennetier C, Plumbridge J. (2015) Dual Inducer recognition by an Mlc homologue. Microbiology 161: 1694-1706

Plumbridge J. (2015) Regulation of the use of amino sugars by E. coli and B. subtilis: same genes different control. J. Mol. Microbiol. Biotechnol. 50th PTS Anniversary Symposium volume 25: 154-167.

Bréchemier-Baey D, Domínguez-Ramírez L, Oberto J, Plumbridge J. (2015) Operator recognition by the ROK transcription factor family members, NagC and Mlc.  Nucleic Acids Res. 43: 361-372  

Plumbridge J, Bossi L, Oberto J, Wade JT, Figueroa-Bossi N. (2014) Interplay of transcriptional and small RNA-dependent control mechanisms regulates chitosugar uptake in Escherichia coli and Salmonella. Mol. Microbiol. 92(4): 648-658

Gaugué I, Oberto J, Plumbridge J. (2014) Regulation of amino sugar utilisation in Bacillus subtilis by the GntR family regulators, NagR and GamR. Mol. Microbiol. 92: 100-115

Gaugué I, Oberto J, Putzer H, Plumbridge J. (2013) The use of amino sugars by Bacillus subtilis: Presence of a unique operon for the catabolism of glucosamine PLOS One 8: e63025

Cournac  A,  Plumbridge J. (2013) DNA looping in prokaryotes:experimental and theoretical approaches. 2013 J. Bacteriol.  195: 1109-1119

Bréchemier-Baey D, Domínguez-Ramírez L, Plumbridge J. (2012) The linker sequence, joining the DNA binding domain of the homologous transcription factors Mlc and NagC, to the rest of the protein determines the specificity of their target recognition in Escherichia coli. Sep;85(5):1007-1.

McVicker G, Sun L, Sohanpal BK, Gashi K, Williamson RA, Plumbridge J, Blomfield IC. (2011) SlyA protein activates fimB gene expression and type 1 fimbriation in Escherichia coli K-12.  J Biol Chem. Sep 16;286(37):32026-35.

Pennetier C, Oberto J, Plumbridge J. (2010) An Antisense Transcript from within the  ptsG Promoter Region in Escherichia coli. J Mol Microbiol Biotechnol. July 29;18(4):230-240.

Alvarez-Añorve LI, Bustos-Jaimes I, Calcagno ML, Plumbridge J. (2009) Allosteric regulation of glucosamine-6-phosphate deaminase (NagB) and growth of Escherichia coli on glucosamine. J Bacteriol. 191: 6401-7. Epub 2009 Aug 21.

Plumbridge J. (2009) An alternative route for recycling of N-acetylglucosamine from peptidoglycan involves the N-acetylglucosamine phosphotransferase system in Escherichia coli. J Bacteriol. 2009 191: 5641-7. Epub 2009 Jul 17.

El Qaidi S, Allemand F, Oberto J, Plumbridge J (2009) Repression of galP, the galactose transporter in Escherichia coli, requires the specific regulator of N-acetylglucosamine metabolism. Mol. Microbiol. 71(1):146-57

El Qaidi S, Plumbridge J. (2008) Switching control of expression of ptsG from the Mlc regulon to the NagC regulon. J. Bacteriol. 190 4677-4686 (Erratum in  J. Bacteriol. (2008) 190 5733.)

Pennetier C, Domínguez-Ramírez L, Plumbridge J. (2008) Different regions of Mlc and NagC, homologous transcriptional repressors controlling expression of the glucose and N-acetylglucosamine phosphotransferase systems in Escherichia coli, are required for inducer signal recognition." Mol. Microbiol. 67 364-377

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Members

 

Harald Putzer CNRS DR1
Soumaya Laalami CNRS CR1
Jackie Plumbridge DREM
Saravuth Ngo TCN CNRS
Liliana Mora IE1HC
Lina Hamouche Post-doc
Marina Cavaiuolo Post-doc

 

Former members

Anna Liponska Ph. D. student,12/2012-03/2016

Lina Hamouche Ph. D. student, 02.2013/03.2016

Marcin Sowa Erasmus student 10.2015/02.2016

Josef Deutscher, DREM, 09.2013/02.2016

Josette Rouvière-Yaniv, DREM CNRS, -08/2015

Sylvain Roques, IE, 03/2013-02/2015

Léna Zig, AI CNRS, 03/2004-09/2014

Anna Rocca, CR1 CNRS, 01/2011-12/2013

Valerio Taverniti, Ph. D. student (Université de Milan), 01/2010 – 01/2011

Patrice Bruscella, Post-doc, 01/2008 – 05/2010

Karen Shahbabian, Ph. D. student (Université Paris VII), 03/2007 – 05/2010

Mme A. Jamalli, Ph. D. student (Université Paris VII), 10/2006 – 09/2010

Agnès Hebert, M2 student (Université Paris VII), 01/2006 – 06/2006

Hélène Launay, technician, 03/2005 – 12/2005

Nasslie Choonee, M2 and Ph. D. student(Université Paris VII), 10/2004 – 09/2007

Carlos Costa, Erasmus student, 03/2004 – 07/2004

Sergine Even, Post-doc, 10/2002 – 10/2004

Dominique Brechemier-Baey, IE CNRS, 1999 - 2004

Renata Brito, Erasmus student, 03/2002 – 07/2002 et 03/2003 -07/2003

Sylvana Pavlovic, M1 student(Université Paris VII), 07/2001 – 08/2001

Jeanette Brill, Ph. D. student (Université de Marburg, Allemagne), 09/2000 – 12/2000

Sonia Benhamida, M2 student(Université Paris XI), 01/1999 – 06/1999

Dong Luo, Ph. D. student (Université Paris XI), 1994 - 1998

Josette Leautey, AI CNRS, 1991 - 1998

Nathalie Gendron, Ph. D. student (Université Paris VII), 1990 - 1993

 

 

Photos

 

2004

Sergine, Nasslee, Carlos...

Hélène, Agnès, Nasslee...

 

 

2009

Patrice, Karen, Valerio...

 

2011

 


 

 

 

 

 

 

 

 

 

 

 

 

 


 

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