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1. The essential role of mRNA degradation in understanding and engineering E. coli metabolism

Author

Laurence Girbal, Charlotte Roux, Delphine Ropers, Muriel Cocaign-Bousquet, Thibault A. Etienne, Agamemnon J. Carpousis, Eliane Hajnsdorf, Toulouse Biotechnology Institute (TBI), Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Institut de biologie physico-chimique (IBPC (FR_550)), Centre National de la Recherche Scientifique (CNRS), Modeling, simulation, measurement, and control of bacterial regulatory networks (IBIS), Laboratoire Adaptation et pathogénie des micro-organismes [Grenoble] (LAPM), Centre National de la Recherche Scientifique (CNRS)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-Université Joseph Fourier - Grenoble 1 (UJF)-Inria Grenoble - Rhône-Alpes, Institut National de Recherche en Informatique et en Automatique (Inria)-Institut National de Recherche en Informatique et en Automatique (Inria)-Institut Jean Roget, Laboratoire de microbiologie et génétique moléculaires (LMGM), Centre de Biologie Intégrative (CBI), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), This work was supported by the French National Research Agency [ANR-18-CE43-0010]., ANR-18-CE43-0010,RIB-ECO,Ingénierie du cycle de vie des ARN pour une approche économique de l'énergétique microbienne: application à la bioconversion des sources de carbone dérivées de la biomasse(2018), CNRS UMR8261, Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-Université Joseph Fourier - Grenoble 1 (UJF)-Centre National de la Recherche Scientifique (CNRS)-Inria Grenoble - Rhône-Alpes, Analyse, ingénierie et contrôle des micro-organismes (MICROCOSME), Inria Grenoble - Rhône-Alpes, Institut National de Recherche en Informatique et en Automatique (Inria)-Institut National de Recherche en Informatique et en Automatique (Inria)-Université Grenoble Alpes (UGA), Laboratoire de microbiologie et génétique moléculaires - UMR5100 (LMGM), Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), and Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Systems biology, RNA Stability, [SDV]Life Sciences [q-bio], Bioengineering, Biology, Applied Microbiology and Biotechnology, Metabolic engineering, 03 medical and health sciences, Transcription (biology), Gene expression, Transcriptional regulation, Protein biosynthesis, Escherichia coli, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, 2. Zero hunger, 0303 health sciences, Messenger RNA, Heterologous protein production, 030302 biochemistry & molecular biology, Translation (biology), Cell biology, Microbe engineering, Metabolism, Metabolic Engineering, mRNA degradation, Metabolic Networks and Pathways, Biotechnology
Abstract

International audience; Metabolic engineering strategies are crucial for the development of bacterial cell factories with improved performance. Until now, optimal metabolic networks have been designed based on systems biology approaches integrating large-scale data on the steady-state concentrations of mRNA, protein and metabolites, sometimes with dynamic data on fluxes, but rarely with any information on mRNA degradation. In this review, we compile growing evidence that mRNA degradation is a key regulatory level in E. coli that metabolic engineering strategies should take into account. We first discuss how mRNA degradation interacts with transcription and translation, two other gene expression processes, to balance transcription regulation and remove poorly translated mRNAs. The many reciprocal interactions between mRNA degradation and metabolism are also highlighted: metabolic activity can be controlled by changes in mRNA degradation and in return, the activity of the mRNA degradation machinery is controlled by metabolic factors. The mathematical models of the crosstalk between mRNA degradation dynamics and other cellular processes are presented and discussed with a view towards novel mRNA degradation-based metabolic engineering strategies. We show finally that mRNAdegradation-based strategies have already successfully been applied to improve heterologous protein synthesis. Overall, this review underlines how important mRNA degradation is in regulating E. coli metabolism and identifies mRNA degradation as a key target for innovative metabolic engineering strategies in biotechnology.

Published
2021

2. Genomewide Stabilization of mRNA during a 'Feast-to-Famine' Growth Transition in Escherichia coli

Author

Laurence Girbal, Delphine Ropers, Manon Morin, Brice Enjalbert, and Muriel Cocaign-Bousquet

Subjects
Molecular Biology and Physiology, Transcription, Genetic, carbon starvation, RNA Stability, lcsh:QR1-502, Down-Regulation, posttranscriptional regulation, Biology, metabolic transition, Microbiology, lcsh:Microbiology, Transcriptome, 03 medical and health sciences, Bacterial Proteins, Transcription (biology), Stress, Physiological, Gene expression, Transcriptional regulation, Escherichia coli, mRNA stability, RNA, Messenger, Molecular Biology, Gene, 030304 developmental biology, 2. Zero hunger, Regulation of gene expression, 0303 health sciences, Messenger RNA, 030306 microbiology, Escherichia coli Proteins, Gene Expression Profiling, MRNA stabilization, transcriptomic, Gene Expression Regulation, Bacterial, Adaptation, Physiological, QR1-502, Carbon, Cell biology, Up-Regulation, Glucose, Genome, Bacterial, Research Article
Abstract

The ability to rapidly respond to changing nutrients is crucial for E. coli to survive in many environments, including the gut. Reorganization of gene expression is the first step used by bacteria to adjust their metabolism accordingly. It involves fine-tuning of both transcription (transcriptional regulation) and mRNA stability (posttranscriptional regulation). While the forms of transcriptional regulation have been extensively studied, the role of mRNA stability during a metabolic switch is poorly understood. Investigating E. coli genomewide transcriptome and mRNA stability during metabolic transitions representative of the carbon source fluctuations in many environments, we have documented the role of mRNA stability in the response to nutrient changes. mRNAs are globally stabilized during carbon depletion. For a few genes, this leads directly to expression upregulation. As these genes are regulators of stress responses and metabolism, our work sheds new light on the likely importance of posttranscriptional regulations in response to environmental stress., Bacteria have to continuously adjust to nutrient fluctuations from favorable to less-favorable conditions and in response to carbon starvation. The glucose-acetate transition followed by carbon starvation is representative of such carbon fluctuations observed in Escherichia coli in many environments. Regulation of gene expression through fine-tuning of mRNA pools constitutes one of the regulation levels required for such a metabolic adaptation. It results from both mRNA transcription and degradation controls. However, the contribution of transcript stability regulation in gene expression is poorly characterized. Using combined transcriptome and mRNA decay analyses, we investigated (i) how transcript stability changes in E. coli during the glucose-acetate-starvation transition and (ii) if these changes contribute to gene expression changes. Our work highlights that transcript stability increases with carbon depletion. Most of the stabilization occurs at the glucose-acetate transition when glucose is exhausted, and then stabilized mRNAs remain stable during acetate consumption and carbon starvation. Meanwhile, expression of most genes is downregulated and we observed three times less gene expression upregulation. Using control analysis theory on 375 genes, we show that most of gene expression regulation is driven by changes in transcription. Although mRNA stabilization is not the controlling phenomenon, it contributes to the emphasis or attenuation of transcriptional regulation. Moreover, upregulation of 18 genes (33% of our studied upregulated set) is governed mainly by transcript stabilization. Because these genes are associated with responses to nutrient changes and stress, this underscores a potentially important role of posttranscriptional regulation in bacterial responses to nutrient starvation. IMPORTANCE The ability to rapidly respond to changing nutrients is crucial for E. coli to survive in many environments, including the gut. Reorganization of gene expression is the first step used by bacteria to adjust their metabolism accordingly. It involves fine-tuning of both transcription (transcriptional regulation) and mRNA stability (posttranscriptional regulation). While the forms of transcriptional regulation have been extensively studied, the role of mRNA stability during a metabolic switch is poorly understood. Investigating E. coli genomewide transcriptome and mRNA stability during metabolic transitions representative of the carbon source fluctuations in many environments, we have documented the role of mRNA stability in the response to nutrient changes. mRNAs are globally stabilized during carbon depletion. For a few genes, this leads directly to expression upregulation. As these genes are regulators of stress responses and metabolism, our work sheds new light on the likely importance of posttranscriptional regulations in response to environmental stress.

Published
2020

3. The stability of an mRNA is influenced by its concentration: a potential physical mechanism to regulate gene expression

Author

Claire Moulis, Anne-Laure Finoux, Muriel Cocaign-Bousquet, Laurence Girbal, Sébastien Nouaille, Sophie Mondeil, Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), Centre de génétique moléculaire (CGM), Centre National de la Recherche Scientifique (CNRS), Institut National de la recherche Agronomique, Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), and Nouaille, Sebastien

Subjects
0301 basic medicine, RNA Stability, [SDV]Life Sciences [q-bio], 030106 microbiology, Biotechnologies, Biology, 03 medical and health sciences, Species Specificity, Transcription (biology), Gene expression, phylogénie, Genetics, Escherichia coli, RNA, Messenger, expression arn m, Gene, bactérie, Regulation of gene expression, Messenger RNA, Base Sequence, Lactococcus lactis, Gene regulation, Chromatin and Epigenetics, MRNA stabilization, Gene Expression Regulation, Bacterial, biology.organism_classification, Cell biology, RNA, Bacterial, 030104 developmental biology, facteur de transcription, Genome, Bacterial
Abstract

International audience; Changing mRNA stability is a major post-transcriptional way of controlling gene expression, particularly in newly encountered conditions. As the concentration of mRNA is the result of an equilibrium between transcription and degradation, it is generally assumed that at constant transcription, any change in mRNA concentration is the consequence of mRNA stabilization or destabilization. However, the literature reports many cases of opposite variations in mRNA concentration and stability in bacteria. Here, we analyzed the causal link between the concentration and stability of mRNA in two phylogenetically distant bacteria Escherichia coli and Lactococcus lactis. Using reporter mRNAs, we showed that modifying the stability of an mRNA had unpredictable effects , either higher or lower, on its concentration, whereas increasing its concentration systematically reduced stability. This inverse relationship between the concentration and stability of mRNA was generalized to native genes at the genome scale in both bacteria. Higher mRNA turnover in the case of higher concentrations appears to be a simple physical mechanism to regulate gene expression in the bacterial kingdom. The consequences for bacterial adaptation of this control of the stability of an mRNA by its concentration are discussed.

Published
2017

4. Multiplexing polysome profiling experiments to study translation in Escherichia coli

Author

Sébastien Nouaille, Marie-Pierre Duviau, Laurence Girbal, Muriel Cocaign-Bousquet, Huong Le Nguyen, Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), INRA (french national institute for agricultural research), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), and Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)

Subjects
[SDV.BIO]Life Sciences [q-bio]/Biotechnology, Gene Expression, Biochemistry, Ribosome, Multiplexing, Database and Informatics Methods, Gene expression, Protein biosynthesis, 3' Untranslated Regions, 0303 health sciences, Multidisciplinary, Organic Compounds, Reverse Transcriptase Polymerase Chain Reaction, Chemistry, Messenger RNA, Escherichia coli Proteins, Monosaccharides, 030302 biochemistry & molecular biology, Translation (biology), Cell biology, Nucleic acids, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, Physical Sciences, Telecommunications, Engineering and Technology, Medicine, RNA extraction, Cellular Structures and Organelles, Sequence Analysis, Research Article, Bioinformatics, Science, Carbohydrates, Biotechnologies, Research and Analysis Methods, 03 medical and health sciences, Extraction techniques, Sequence Motif Analysis, Polysome, Genetics, Escherichia coli, RNA, Messenger, 030304 developmental biology, Biology and life sciences, Three prime untranslated region, Organic Chemistry, Chemical Compounds, Cell Biology, Arabinose, Polyribosomes, Protein Biosynthesis, RNA, Protein Translation, Ribosomes
Abstract

International audience; Polysome profiling is a widely used method to monitor the translation status of mRNAs. Although it is theoretically a simple technique, it is labor intensive. Repetitive polysome frac-tionation rapidly generates a large number of samples to be handled in the downstream processes of protein elimination, RNA extraction and quantification. Here, we propose a multiplex polysome profiling experiment in which distinct cellular extracts are pooled before loading on the sucrose gradient for fractionation. We used the multiplexing method to study translation in E. coli. Multiplexing polysome profiling experiments provided similar mRNA translation status to that obtained with the non-multiplex method with comparable distribution of mRNA copies between the polysome profiling fractions, similar ribosome occupancy and ribosome density. The multiplexing method was used for parallel characterization of gene translational responses to changing mRNA levels. When the mRNA level of two native genes, cysZ and lacZ was increased by transcription induction, their global translational response was similar, with a higher ribosome load leading to increased ribosome occupancy and ribosome densities. However the pattern and the magnitude of the translational response were gene specific. By reducing the number of polysome profiling experiments, the multiplexing method saved time and effort and reduced cost and technical bias. This method would be useful to study the translational effect of mRNA sequence-dependent parameters that often require testing multiple samples and conditions in parallel.

Published
2019

5. Detachment of the RNA degradosome from the inner membrane of Escherichia coli results in a global slowdown of mRNA degradation, proteolysis of RNase E and increased turnover of ribosome-free transcripts

Author

Marie Bouvier, Lydia Hadjeras, Agamemnon J. Carpousis, Muriel Cocaign-Bousquet, Quentin Morin‐Ogier, Isabelle Canal, Leonora Poljak, Laurence Girbal, Laboratoire de microbiologie et génétique moléculaires (LMGM), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS), Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), French National Research Agency (ANR) [ANR-13-BSV6-0005], Centre de Biologie Intégrative (CBI), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), ANR-13-BSV6-0005,memRNase,Localisation membranaire de la RNase E : rôle dans la maturation, la surveillance et la dégradation des ARNm(2013), Laboratoire de microbiologie et génétique moléculaires - UMR5100 (LMGM), Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), and Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)

Subjects
RNA Stability, RNase P, Biology, Microbiology, Ribosome, 03 medical and health sciences, Multienzyme Complexes, Endoribonucleases, Escherichia coli, Inner membrane, Nucleoid, RNA, Messenger, Molecular Biology, membrane, expression arn m, Research Articles, 030304 developmental biology, Polyribonucleotide Nucleotidyltransferase, 0303 health sciences, Messenger RNA, 030306 microbiology, Escherichia coli Proteins, RNA, MRNA stabilization, Gene Expression Regulation, Bacterial, Cell biology, RNA, Bacterial, [SDV.MP]Life Sciences [q-bio]/Microbiology and Parasitology, ribosome, Proteolysis, RNA-seq, Ribosomes, RNA Helicases, Research Article
Abstract

Summary The reason for RNase E attachment to the inner membrane is largely unknown. To understand the cell biology of RNA degradation, we have characterized a strain expressing RNase E lacking the membrane attachment site (cytoplasmic RNase E). Genome‐wide data show a global slowdown in mRNA degradation. There is no correlation between mRNA stabilization and the function or cellular location of encoded proteins. The activity of cRNase E is comparable to the wild‐type enzyme in vitro, but the mutant protein is unstable in vivo. Autoregulation of cRNase E synthesis compensates for protein instability. cRNase E associates with other proteins to assemble a cytoplasmic RNA degradosome. CsrB/C sRNAs, whose stability is regulated by membrane‐associated CsrD, are stabilized. Membrane attachment of RNase E is thus necessary for CsrB/C turnover. In contrast to mRNA stability, ribosome‐free transcripts are sensitive to inactivation by cRNase E. Our results show that effects on RNA degradation are not due to the differences in the activity or level of cRNase E, or failure to assemble the RNA degradosome. We propose that membrane attachment is necessary for RNase E stability, functional interactions with membrane‐associated regulatory factors and protection of ribosome‐free transcripts from premature interactions with RNase E in the nucleoid., Attachment of RNase E to the inner membrane of E. coli is important for multiple processes including efficient degradation of mRNA, interactions with membrane‐associated regulatory factors involved in the posttranscriptional control of gene expression, and the protection of nascent transcripts in the nucleoid from degradation by RNase E. Detachment of RNase E from the inner membrane results in its proteolysis, which could be important for downregulating RNase E activity under stress conditions.

Published
2019

6. PNPase is involved in the coordination of mRNA degradation and expression in stationary phase cells of Escherichia coli

Author

Muriel Cocaign-Bousquet, Cecília M. Arraiano, Laurence Girbal, Clémentine Dressaire, Sandrine Laguerre, Vânia Pobre, Instituto de Tecnologia Química e Biológica António Xavier (ITQB), Molecular, Structural and Cellular Microbiology (MOSTMICRO), Universidade Nova de Lisboa (NOVA), Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), Collaborative INTERREG Interbio project (2011-2012), Fundacao para a Ciencia e Tecnologia (FCT-Portugal) - FEDER funds through COMPETE2020 - Programa Operacional Competitividadee Internacionalizacao (POCI) [LISBOA-01-0145-FEDER-007660 UID/CBQ/04612/2013], European Union's Horizon 2020 research and innovation program [635536], FCT Post-Doctoral Fellowships [SFRH/BPD/65528/2009, SFRH/BPD/87188/2012], [PTDC/BIA-MIC/1399/2014], Universidade Nova de Lisboa = NOVA University Lisbon (NOVA), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), European Project: 635536,H2020,H2020-LEIT-BIO-2014-1,EmPowerPutida(2015), Girbal, Laurence, and Arraiano, Cecilia Maria

Subjects
0301 basic medicine, RNA, Untranslated, [SDV.BIO]Life Sciences [q-bio]/Biotechnology, lcsh:QH426-470, RNase P, messenger rna, lcsh:Biotechnology, RNA Stability, 030106 microbiology, Mutant, RNase R, Purine nucleoside phosphorylase, Biotechnologies, Biology, RNA decay, PNPase, E. coli, transcriptome, gene expression regulation, 03 medical and health sciences, Transcription (biology), lcsh:TP248.13-248.65, Genetics, RNA, Messenger, analyse du transcriptome, Gene expression regulation, Messenger RNA, MRNA stabilization, Non-coding RNA, Cell biology, lcsh:Genetics, 030104 developmental biology, Exoribonucleases, Mutation, escherichia coli, arn messager, Transcriptome, Genome, Bacterial, Half-Life, Research Article, Biotechnology
Abstract

Background Exoribonucleases are crucial for RNA degradation in Escherichia coli but the roles of RNase R and PNPase and their potential overlap in stationary phase are not well characterized. Here, we used a genome-wide approach to determine how RNase R and PNPase affect the mRNA half-lives in the stationary phase. The genome-wide mRNA half-lives were determined by a dynamic analysis of transcriptomes after transcription arrest. We have combined the analysis of mRNA half-lives with the steady-state concentrations (transcriptome) to provide an integrated overview of the in vivo activity of these exoribonucleases at the genome-scale. Results The values of mRNA half-lives demonstrated that the mRNAs are very stable in the stationary phase and that the deletion of RNase R or PNPase caused only a limited mRNA stabilization. Intriguingly the absence of PNPase provoked also the destabilization of many mRNAs. These changes in mRNA half-lives in the PNPase deletion strain were associated with a massive reorganization of mRNA levels and also variation in several ncRNA concentrations. Finally, the in vivo activity of the degradation machinery was found frequently saturated by mRNAs in the PNPase mutant unlike in the RNase R mutant, suggesting that the degradation activity is limited by the deletion of PNPase but not by the deletion of RNase R. Conclusions This work had identified PNPase as a central player associated with mRNA degradation in stationary phase. Electronic supplementary material The online version of this article (10.1186/s12864-018-5259-8) contains supplementary material, which is available to authorized users.

Published
2018

7. Roles of the F-domain in [FeFe] hydrogenase

Author

Isabelle Meynial-Salles, Charles Gauquelin, Isabelle André, Carole Baffert, David Guieysse, Emilien Etienne, Laurence Girbal, Emma Kamionka, Bruno Guigliarelli, Christophe Léger, Vincent Fourmond, Philippe Soucaille, Pierre Richaud, Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Bioénergétique et Ingénierie des Protéines (BIP ), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS), Biologie végétale et microbiologie environnementale - UMR7265 (BVME), Institut de Biosciences et Biotechnologies d'Aix-Marseille (ex-IBEB) (BIAM), Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Environnement, Bioénergie, Microalgues et Plantes (EBMP), Institut de biologie structurale et microbiologie (IBSM), Université de la Méditerranée - Aix-Marseille 2-Université Paul Cézanne - Aix-Marseille 3-Université de Provence - Aix-Marseille 1-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), National French EPR network (RENARD) [IR3443], EU [1944-32670], Provence Alpes Cote d'Azur (PACA) [DEB 09-621], ANR-12-BS08-0014,ECCHYMOSE,Etudes d'hydrogénases à Fer par électrochimie: mécanisme et optimisation pour la photoproduction d'hydrogène(2012), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Bioénergie et Microalgues (EBM), Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés ( LISBP ), Institut National de la Recherche Agronomique ( INRA ) -Institut National des Sciences Appliquées - Toulouse ( INSA Toulouse ), Institut National des Sciences Appliquées ( INSA ) -Institut National des Sciences Appliquées ( INSA ) -Centre National de la Recherche Scientifique ( CNRS ), Bioénergétique et Ingénierie des Protéines ( BIP ), Aix Marseille Université ( AMU ) -Centre National de la Recherche Scientifique ( CNRS ), Bioénergie et Microalgues ( EBM ), Institut de Biosciences et Biotechnologies d'Aix-Marseille (ex-IBEB) ( BIAM ), Centre National de la Recherche Scientifique ( CNRS ) -Aix Marseille Université ( AMU ) -Direction de Recherche Fondamentale (CEA) ( DRF (CEA) ), Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Centre National de la Recherche Scientifique ( CNRS ) -Aix Marseille Université ( AMU ) -Direction de Recherche Fondamentale (CEA) ( DRF (CEA) ), Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ) -Commissariat à l'énergie atomique et aux énergies alternatives ( CEA ), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, Hydrogenase, Clostridium acetobutylicum, Stereochemistry, [SDV]Life Sciences [q-bio], Mutant, Mutation, Missense, Biophysics, Electron transfer pathway, 010402 general chemistry, Photochemistry, 01 natural sciences, Biochemistry, [ CHIM ] Chemical Sciences, H-cluster, 03 medical and health sciences, Electron transfer, [Fe-Fe] hydrogenase, Bacterial Proteins, Protein Domains, Hyda, Ferredoxin, ComputingMilieux_MISCELLANEOUS, chemistry.chemical_classification, biology, accessory domains, Cell Biology, biology.organism_classification, ferredoxin, Enzyme assay, 0104 chemical sciences, 030104 developmental biology, Enzyme, Amino Acid Substitution, chemistry, biology.protein
Abstract

International audience; The role of accessory Fe-S clusters of the F-domain in the catalytic activity of M3-type [FeFe] hydrogenase and the contribution of each of the two Fe-S surface clusters in the intermolecular electron transfer from ferredoxin are both poorly understood. We designed, constructed, produced and spectroscopically, electrochemically and biochemically characterized three mutants of Clostridium acetobutylicum CaHydA hydrogenase with modified Fe-S clusters: two site-directed mutants, HydA_C100A and HydA_C48A missing the FS4C and the FS2 surface Fe-S clusters, respectively, and a HydA_Delta DA mutant that completely lacks the F-domain. Analysis of the mutant enzyme activities clearly demonstrated the importance of accessory clusters in retaining full enzyme activity at potentials around and higher than the equilibrium 2H(+)/H-2 potential but not at the lowest potentials, where all enzymes have a similar turnover rate. Moreover, our results, combined with molecular modelling approaches, indicated that the FS2 cluster is the main gate for electron transfer from reduced ferredoxin.

Published
2018

8. Large-Scale Measurement of mRNA Degradation in Escherichia coli: To Delay or Not to Delay

Author

Muriel Cocaign-Bousquet, Agamemnon J. Carpousis, Ignacio González, Annick Moisan, Marie Bouvier, Sébastien Nouaille, Nathalie Villa-Vialaneix, Christine Gaspin, Sandrine Laguerre, Laurence Girbal, Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), Unité de Mathématiques et Informatique Appliquées de Toulouse (MIAT INRA), Institut National de la Recherche Agronomique (INRA), Laboratoire de microbiologie et génétique moléculaires (LMGM), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de microbiologie et génétique moléculaires - UMR5100 (LMGM), Centre de Biologie Intégrative (CBI), Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), and Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS)

Subjects
0301 basic medicine, RNA half-life, Chemistry, [SDV]Life Sciences [q-bio], Linear model, Bacterial growth, medicine.disease_cause, Correlation, Genome-wide measurement, 03 medical and health sciences, 030104 developmental biology, Exponential decay, Linear regression, MRNA degradation, Biophysics, medicine, RNA decay modeling, Delay in the degradation kinetics, Scale measurement, Escherichia coli
Abstract

International audience; In this study, we compared different computational methods used for genome-wide determination of mRNA half-lives in Escherichia coli with a special focus on the impact on considering a delay before the onset of mRNA decay after transcription arrest. A wide variety of datasets were analyzed coming from different technical methods for mRNA quantification (microarrays, RNA-seq, and RT-qPCR) and different bacterial growth conditions. The exponential decay of mRNA levels was fitted using both linear and exponential models and with or without a delay. We showed that for all the models, independently of mRNA quantification methods and growth conditions, ignoring the delay resulted in only a modest overestimation of the half-life. For approximately 80% of the mRNAs, differences in mRNA half-life values were less than 34 s. The correlation between half-lives estimated with and without a delay was extremely high. However, the slope of the linear regression between the half-lives with and without a delay tended to decrease with the delay. For the few mRNAs for which taking into account the delay influenced the estimated half-life, the impact was dependent on the model and the growth condition. The smallest impact was obtained for the linear model.

Published
2018

9. The Csr system regulates genome-wide mRNA stability and transcription and thus gene expression in Escherichia coli

Author

Muriel Cocaign-Bousquet, Thomas Esquerré, Agamemnon J. Carpousis, Laurence Girbal, Catherine Turlan, Marie Bouvier, Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), Universite de Toulouse, Region Midi-Pyrenees, ANR-13-BSV6-0005,memRNase,Localisation membranaire de la RNase E : rôle dans la maturation, la surveillance et la dégradation des ARNm(2013), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), and Girbal, Laurence

Subjects
0301 basic medicine, Transcription, Genetic, [SDV]Life Sciences [q-bio], RNA Stability, 030106 microbiology, Repressor, Gene Expression, RNA-binding protein, Biology, Article, 03 medical and health sciences, Transcription (biology), Gene expression, Transcriptional regulation, Escherichia coli, RNA, Messenger, Gene, Genetics, Regulation of gene expression, Multidisciplinary, Escherichia coli Proteins, Gene Expression Profiling, Membrane Proteins, RNA-Binding Proteins, Gene Expression Regulation, Bacterial, Carbon, Gene expression profiling, Repressor Proteins, RNA, Bacterial, Genome, Bacterial
Abstract

Bacterial adaptation requires large-scale regulation of gene expression. We have performed a genome-wide analysis of the Csr system, which regulates many important cellular functions. The Csr system is involved in post-transcriptional regulation, but a role in transcriptional regulation has also been suggested. Two proteins, an RNA-binding protein CsrA and an atypical signaling protein CsrD, participate in the Csr system. Genome-wide transcript stabilities and levels were compared in wildtype E. coli (MG1655) and isogenic mutant strains deficient in CsrA or CsrD activity demonstrating for the first time that CsrA and CsrD are global negative and positive regulators of transcription, respectively. The role of CsrA in transcription regulation may be indirect due to the 4.6-fold increase in csrD mRNA concentration in the CsrA deficient strain. Transcriptional action of CsrA and CsrD on a few genes was validated by transcriptional fusions. In addition to an effect on transcription, CsrA stabilizes thousands of mRNAs. This is the first demonstration that CsrA is a global positive regulator of mRNA stability. For one hundred genes, we predict that direct control of mRNA stability by CsrA might contribute to metabolic adaptation by regulating expression of genes involved in carbon metabolism and transport independently of transcriptional regulation.

Published
2016

10. Dynamic Analysis of the Lactococcus lactis Transcriptome in Cheeses Made from Milk Concentrated by Ultrafiltration Reveals Multiple Strategies of Adaptation to Stresses

Author

Marina Cretenet, Sergine Even, Sophie Jeanson, Michel Piot, Yves Le Loir, Laurence Girbal, Vincent Ulvé, Pascal Loubière, Sébastien Nouaille, Valérie Laroute, Muriel Cocaign-Bousquet, Sylvie Lortal, Science et Technologie du Lait et de l'Oeuf (STLO), Institut National de la Recherche Agronomique (INRA)-AGROCAMPUS OUEST, Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT), Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro)-Institut national d'enseignement supérieur pour l'agriculture, l'alimentation et l'environnement (Institut Agro), Institut National des Sciences Appliquées (INSA), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), and Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA)

Subjects
Time Factors, Lysis, Ultrafiltration, fromage, Applied Microbiology and Biotechnology, ULTRAFILTRATION, Transcriptome, 03 medical and health sciences, chemistry.chemical_compound, Stress, Physiological, Gene expression, Animals, LACTOCOCCUS LACTIS, Amino Acids, 030304 developmental biology, bactérie, [SDV.EE]Life Sciences [q-bio]/Ecology, environment, 2. Zero hunger, milk, 0303 health sciences, Microbial Viability, CHEESE, Ecology, biology, 030306 microbiology, Gene Expression Profiling, Hydrolysis, gène, Lactococcus lactis, Proteins, Gene Expression Regulation, Bacterial, bacterium, biology.organism_classification, Streptococcaceae, lait, chemistry, Biochemistry, CCPA, Food Microbiology, transcriptome, Bacteria, Food Science, Biotechnology
Abstract

Lactococcus lactis is used extensively for the production of various cheeses. At every stage of cheese fabrication, L. lactis has to face several stress-generating conditions that result from its own modification of the environment as well as externally imposed conditions. We present here the first in situ global gene expression profile of L. lactis in cheeses made from milk concentrated by ultrafiltration (UF-cheeses), a key economical cheese model. The transcriptomic response of L. lactis was analyzed directly in a cheese matrix, starting from as early as 2 h and continuing for 7 days. The growth of L. lactis stopped after 24 h, but metabolic activity was maintained for 7 days. Conservation of its viability relied on an efficient proteolytic activity measured by an increasing, quantified number of free amino acids in the absence of cell lysis. Extensive downregulation of genes under CodY repression was found at day 7. L. lactis developed multiple strategies of adaptation to stressful modifications of the cheese matrix. In particular, expression of genes involved in acidic- and oxidative-stress responses was induced. L. lactis underwent unexpected carbon limitation characterized by an upregulation of genes involved in carbon starvation, principally due to the release of the CcpA control. We report for the first time that in spite of only moderately stressful conditions, lactococci phage is repressed under UF-cheese conditions.

Published
2011

11. Optimized over-expression of [FeFe] hydrogenases with high specific activity in Clostridium acetobutylicum

Author

Thomas Happe, Philippe Soucaille, Alexey Silakov, Laurence Girbal, Gregory von Abendroth, Christian Croux, and Sven T. Stripp

Subjects
0303 health sciences, Hydrogenase, Clostridium acetobutylicum, biology, Renewable Energy, Sustainability and the Environment, Chemistry, Energy Engineering and Power Technology, Chlamydomonas reinhardtii, 010402 general chemistry, Condensed Matter Physics, biology.organism_classification, 01 natural sciences, Redox, 0104 chemical sciences, law.invention, 03 medical and health sciences, Fuel Technology, Biochemistry, law, Yield (chemistry), Recombinant DNA, Homologous recombination, Gene, 030304 developmental biology
Abstract

It was previously shown that Clostridium acetobutylicum is capable to over-express various [FeFe] hydrogenases although the protein yield was low. In this study we report on doubling the yield of the clostridial hydrogenase by replacing the native gene hydA1Ca with a recombinant one via homologous recombination. The purified protein HydA1Ca shows an unexpected high specific activity (up to 2257 μmol H2 min−1 mg−1) for hydrogen evolution. Furthermore, the highly active green algal hydrogenase HydA1Cr from Chlamydomonas reinhardtii was heterologously expressed in C. acetobutylicum, and purified with increased yield (1 mg protein per liter of cells) and high activity (625 μmol H2 min−1 mg−1). EPR studies demonstrate intact H-clusters for homologously and heterologously expressed [FeFe] hydrogenases in the CO-inhibited oxidized redox state, and prove the high efficiency of the C. acetobutylicum expression system.

Published
2008

12. Escherichia coli under Ionic Silver Stress: An Integrative Approach to Explore Transcriptional, Physiological and Biochemical Responses

Author

Frédéric Jamme, Isabelle Fourquaux, Claire Saulou-Bérion, Laurence Girbal, Muriel Cocaign-Bousquet, Muriel Mercier-Bonin, Ignacio González, Jean-Nicolas Audinot, Brice Enjalbert, Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), Materials Research and Technology (MRT) Department, Luxembourg Institute of Science and Technology (LIST), Centre de Microscopie Électronique Appliquée à la Biologie (CMEAB), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Hôpital de Rangueil, CHU Toulouse [Toulouse]-CHU Toulouse [Toulouse], Synchrotron SOLEIL, ANR-07-BLAN-0196,BIOPLEASURE,Plasma - surface engineering for biofilm prevention(2007), Génie et Microbiologie des Procédés Alimentaires (GMPA), Institut National de la Recherche Agronomique (INRA)-AgroParisTech, Hôpital de Rangueil, CHU Toulouse [Toulouse]-CHU Toulouse [Toulouse]-Toulouse Réseau Imagerie-Genotoul ( TRI-Genotoul), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Synchrotron SOLEIL (SSOLEIL), Centre National de la Recherche Scientifique (CNRS), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Toulouse Réseau Imagerie-Genotoul ( TRI-Genotoul), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), AgroParisTech-Institut National de la Recherche Agronomique (INRA), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), and Girbal, Laurence

Subjects
Silver, Transcription, Genetic, [PHYS.PHYS.PHYS-BIO-PH]Physics [physics]/Physics [physics]/Biological Physics [physics.bio-ph], lcsh:Medicine, Biology, medicine.disease_cause, Cell membrane, 03 medical and health sciences, chemistry.chemical_compound, Ribosomal protein, Stress, Physiological, E. Coli, Gene expression, medicine, Extracellular, lcsh:Science, Escherichia coli, 030304 developmental biology, Regulation of gene expression, 0303 health sciences, Multidisciplinary, Escherichia coli K12, Ag nanoparticles, 030306 microbiology, Escherichia coli Proteins, lcsh:R, Gene Expression Regulation, Bacterial, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, Silver nitrate, medicine.anatomical_structure, chemistry, Biochemistry, Silver Nitrate, lcsh:Q, escherichia coli, Intracellular, [PHYS.PHYS.PHYS-DATA-AN]Physics [physics]/Physics [physics]/Data Analysis, Statistics and Probability [physics.data-an], Research Article
Abstract

International audience; For a better understanding of the systemic effect of sub-lethal micromolar concentrations of ionic silver on Escherichia coli, we performed a multi-level characterization of cells under Ag +-mediated stress using an integrative biology approach combining physiological, biochemical and transcriptomic data. Physiological parameters, namely bacterial growth and survival after Ag + exposure, were first quantified and related to the accumulation of intracel-lular silver, probed for the first time by nano secondary ion mass spectroscopy at sub-micrometer lateral resolution. Modifications in E. coli biochemical composition were evaluated under Ag +-mediated stress by in situ synchrotron Fourier-transform infrared micro-spectroscopy and a comprehensive transcriptome response was also determined. Using multivariate statistics, correlations between the physiological parameters, the extracellular concentration of AgNO 3 and the intracellular silver content, gene expression profiles and micro-spectroscopic data were investigated. We identified Ag +-dependent regulation of gene expression required for growth (e.g. transporter genes, transcriptional regulators, ribo-somal proteins), for ionic silver transport and detoxification (e.g. copA, cueO, mgtA, nhaR) and for coping with various types of stress (dnaK, pspA, metA,R, oxidoreductase genes). The silver-induced shortening of the acyl chain of fatty acids, mostly encountered in cell membrane, was highlighted by microspectroscopy and correlated with the down-regulated expression of genes involved in fatty acid transport (fadL) and synthesis/modification of lipid A (lpxA and arnA). The increase in the disordered secondary structure of proteins in the presence of Ag + was assessed through the conformational shift shown for amides I and II, and further correlated with the up-regulated expression of peptidase (hfq) and chaperone (dnaJ), and regulation of transpeptidase expression (ycfS and ycbB). Interestingly, as these transpeptidases act on the structural integrity of the cell wall, regulation of their expression may explain the morphological damage reported under Ag+-mediated stress. This result clearly demonstrates that the cell membrane is a key target of ionic silver.

Published
2015

13. A Genome-Scale Integration and Analysis of Lactococcus lactis Translation Data

Author

Flora Picard, Laurence Girbal, Muriel Cocaign-Bousquet, Vassily Hatzimanikatis, Julien Racle, Ecole Polytechnique Fédérale de Lausanne (EPFL), Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), and Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS)

Subjects
control analysis, [SDV]Life Sciences [q-bio], MESSENGER-RNA, PROTEIN-SYNTHESIS, ESCHERICHIA-COLI, SACCHAROMYCES-CEREVISIAE, GENE-EXPRESSION, WIDE ANALYSIS, IN-VIVO, RIBOSOME, POLYSOME, EFFICIENCY, ribosome translation, Ribosome, [SDV.IDA]Life Sciences [q-bio]/Food engineering, Protein biosynthesis, Biology (General), Databases, Protein, Genetics, 0303 health sciences, Ecology, biology, 030302 biochemistry & molecular biology, systems biology, Lactococcus lactis, Computational Theory and Mathematics, Modeling and Simulation, functional genomics, Functional genomics, Research Article, QH301-705.5, Computational biology, 03 medical and health sciences, Cellular and Molecular Neuroscience, Eukaryotic translation, Bacterial Proteins, Polysome, [SPI.GPROC]Engineering Sciences [physics]/Chemical and Process Engineering, Molecular Biology, Gene, Ecology, Evolution, Behavior and Systematics, 030304 developmental biology, protein networks, post-transcriptional gene expression, Computational Biology, Ribosomal RNA, biology.organism_classification, bacterial physiology, Polyribosomes, Protein Biosynthesis, Genome, Bacterial
Abstract

Protein synthesis is a template polymerization process composed by three main steps: initiation, elongation, and termination. During translation, ribosomes are engaged into polysomes whose size is used for the quantitative characterization of translatome. However, simultaneous transcription and translation in the bacterial cytosol complicates the analysis of translatome data. We established a procedure for robust estimation of the ribosomal density in hundreds of genes from Lactococcus lactis polysome size measurements. We used a mechanistic model of translation to integrate the information about the ribosomal density and for the first time we estimated the protein synthesis rate for each gene and identified the rate limiting steps. Contrary to conventional considerations, we find significant number of genes to be elongation limited. This number increases during stress conditions compared to optimal growth and proteins synthesized at maximum rate are predominantly elongation limited. Consistent with bacterial physiology, we found proteins with similar rate and control characteristics belonging to the same functional categories. Under stress conditions, we found that synthesis rate of regulatory proteins is becoming comparable to proteins favored under optimal growth. These findings suggest that the coupling of metabolic states and protein synthesis is more important than previously thought., Author Summary Post-transcriptional regulation is important for the understanding of gene expression control. Our work is a genome-scale analysis of the translation steps of protein synthesis from transcripts. We have developed a mathematical model to integrate and analyze experimental ribosome density of hundreds of transcripts of Lactococcus lactis, providing robust estimation of polysome sizes. Using a mechanistic approach we have modeled for the first time in bacteria the protein synthesis rate for each gene and determined by control analysis the limiting rate between initiation, elongation and termination. Highly expressed proteins belonged to the group of the proteins with high synthesis rate and were controlled by elongation. Unexpectedly, a significant number of genes under elongation limitation were found although initiation was generally believed to be limiting. In addition, we showed that translation rate and control were in agreement with cellular requirements in cells growing in optimal environment but also in cells under nutritional limitation. This work provided a better understanding of translational regulation in bacteria and demonstrated how protein synthesis control was closely related to cellular metabolic states.

Published
2013

14. Synchrotron FTIR microspectroscopy of Escherichia coli at single-cell scale under silver-induced stress conditions

Author

Claire Saulou, Muriel Mercier-Bonin, Isabelle Fourquaux, Paul Dumas, Claude Maranges, Frédéric Jamme, Muriel Cocaign-Bousquet, Laurence Girbal, Génie et Microbiologie des Procédés Alimentaires (GMPA), Institut National de la Recherche Agronomique (INRA)-AgroParisTech, Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Université de Toulouse (UT), Unité de recherche sur les Biopolymères, Interactions Assemblages (BIA), Institut National de la Recherche Agronomique (INRA), Synchrotron SOLEIL (SSOLEIL), Centre National de la Recherche Scientifique (CNRS), Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT), Centre de Microscopie Électronique Appliquée à la Biologie (CMEAB), Toulouse Réseau Imagerie-Genotoul ( TRI-Genotoul), Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), The authors gratefully acknowledge the 'BioPleasure' project of the Research National Agency (ANR-07-BLAN-0196-01) for funding., The research described in this paper was performed at the French national synchrotron facility SOLEIL (Gif-sur- Yvette, France), using the SMIS beamline (proposal no 20090556)., ANR-07-BLAN-0196,BIOPLEASURE,Plasma - surface engineering for biofilm prevention(2007), AgroParisTech-Institut National de la Recherche Agronomique (INRA), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), Université Fédérale Toulouse Midi-Pyrénées, Institut National des Sciences Appliquées (INSA), Hôpital de Rangueil, CHU Toulouse [Toulouse]-CHU Toulouse [Toulouse]-Toulouse Réseau Imagerie-Genotoul ( TRI-Genotoul), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Hôpital de Rangueil, and CHU Toulouse [Toulouse]-CHU Toulouse [Toulouse]

Subjects
In situ, Analytical chemistry, medicine.disease_cause, Biochemistry, Analytical Chemistry, 03 medical and health sciences, Stress, Physiological, Spectroscopy, Fourier Transform Infrared, medicine, Escherichia coli, Fourier transform infrared spectroscopy, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biochemistry [q-bio.BM], Protein secondary structure, 030304 developmental biology, 0303 health sciences, biology, Ionic silver, 030306 microbiology, Chemistry, Synchrotron FTIR-ATR microspectroscopy, Single-cell scale, [CHIM.MATE]Chemical Sciences/Material chemistry, biology.organism_classification, [SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology, [SDV.BBM.BC]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biomolecules [q-bio.BM], Antibacterial effect, Transmission electron microscopy, Attenuated total reflection, Ultrastructure, Biophysics, Silver Nitrate, Single-Cell Analysis, [SPI.SIGNAL]Engineering Sciences [physics]/Signal and Image processing, Synchrotrons, Bacteria
Abstract

International audience; The present work was focused on elucidating biochemical changes in the model bacterium Escherichia coli exposed to ionic silver mediated stress, at a single-cell scale. In order to achieve this, in situ synchrotron Fourier-transform infrared (sFTIR) microspectroscopy was performed, for the first time, on individual cells by attenuated total reflectance (ATR) combined with the use of zinc-selenide hemisphere for high spatial resolution. In a first part, the potential of the method was evaluated on bacteria subjected to a lethal 100 μM AgNO3 concentration for 2 h compared to untreated 100 % viable cells. Differences in cell composition were assessed for the C–H stretching and protein spectral regions, indicating that the inhibitory action was targeted against both fatty acids and proteins. Transmission electron microscopy (TEM) confirmed morphological damages of the cell ultrastructure. The relevance of ATR-sFTIR microspectroscopy for highlighting the heterogeneity in Ag+-mediated effects within a given bacterial population was also pointed out. In a second part, cells were exposed to sub-lethal Ag+ concentrations (

Published
2013

15. Dual role of transcription and transcript stability in the regulation of gene expression in Escherichia coli cells cultured on glucose at different growth rates

Author

Thomas Esquerré, Muriel Cocaign-Bousquet, Laurence Girbal, Catherine Turlan, Sandrine Laguerre, Agamemnon J. Carpousis, Laboratoire de microbiologie et génétique moléculaires (LMGM), Centre de Biologie Intégrative (CBI), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), Universite de Toulouse, Region Midi-Pyrenees, Université Toulouse III - Paul Sabatier (UPS), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), Girbal, Laurence, Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de microbiologie et génétique moléculaires - UMR5100 (LMGM), Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), and Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)

Subjects
expression génique, Transcription, Genetic, Operon, RNA Stability, [SDV]Life Sciences [q-bio], Biology, 03 medical and health sciences, Transcription (biology), Gene expression, Escherichia coli, Genetics, Transcriptional regulation, RNA, Messenger, microbiologie, Molecular Biology, Gene, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, Regulation of gene expression, 0303 health sciences, Messenger RNA, Gene knockdown, 030306 microbiology, Gene Expression Regulation, Bacterial, Molecular biology, Glucose
Abstract

Microorganisms extensively reorganize gene expression to adjust growth rate to changes in growth conditions. At the genomic scale, we measured the contribution of both transcription and transcript stability to regulating messenger RNA (mRNA) concentration in Escherichia coli. Transcriptional control was the dominant regulatory process. Between growth rates of 0.10 and 0.63 h−1, there was a generic increase in the bulk mRNA transcription. However, many transcripts became less stable and the median mRNA half-life decreased from 4.2 to 2.8 min. This is the first evidence that mRNA turnover is slower at extremely low-growth rates. The destabilization of many, but not all, transcripts at high-growth rate correlated with transcriptional upregulation of genes encoding the mRNA degradation machinery. We identified five classes of growth-rate regulation ranging from mainly transcriptional to mainly degradational. In general, differential stability within polycistronic messages encoded by operons does not appear to be affected by growth rate. We show here that the substantial reorganization of gene expression involving downregulation of tricarboxylic acid cycle genes and acetyl-CoA synthetase at high-growth rates is controlled mainly by transcript stability. Overall, our results demonstrate that the control of transcript stability has an important role in fine-tuning mRNA concentration during changes in growth rate.

Published
2013

16. Role of mRNA stability during bacterial adaptation

Author

Pascal Loubière, Isabelle Queinnec, Muriel Cocaign-Bousquet, Emma Redon, Flora Picard, Laurence Girbal, Clémentine Dressaire, Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), Équipe Méthodes et Algorithmes en Commande (LAAS-MAC), Laboratoire d'analyse et d'architecture des systèmes (LAAS), Université Toulouse - Jean Jaurès (UT2J)-Université Toulouse 1 Capitole (UT1), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Toulouse - Jean Jaurès (UT2J)-Université Toulouse 1 Capitole (UT1), Université Fédérale Toulouse Midi-Pyrénées, Institut National de la Recherche Agronomique Integrative Biology Program (agroBI), Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse 1 Capitole (UT1)-Université Toulouse - Jean Jaurès (UT2J)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse 1 Capitole (UT1)-Université Toulouse - Jean Jaurès (UT2J), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Université Toulouse Capitole (UT Capitole), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université Toulouse - Jean Jaurès (UT2J), Université de Toulouse (UT)-Université Toulouse III - Paul Sabatier (UT3), Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Institut National Polytechnique (Toulouse) (Toulouse INP), Université de Toulouse (UT)-Université Toulouse Capitole (UT Capitole), and Université de Toulouse (UT)

Subjects
Codon Adaptation Index, Science, [SDV]Life Sciences [q-bio], RNA Stability, Biostatistics, Microbiology, Bacterial genetics, Transcriptomes, 03 medical and health sciences, Transcription (biology), Genome Analysis Tools, Microbial Physiology, Gene expression, Genome-Wide Association Studies, Bacterial Physiology, Gene, Biology, 030304 developmental biology, 2. Zero hunger, Regulation of gene expression, 0303 health sciences, Messenger RNA, Multidisciplinary, biology, 030306 microbiology, Systems Biology, Lactococcus lactis, Statistics, Bacteriology, Gene Expression Regulation, Bacterial, Genomics, biology.organism_classification, Molecular biology, Cell biology, Medicine, Genome Expression Analysis, Mathematics, Research Article
Abstract

International audience; Bacterial adaptation involves extensive cellular reorganization. In particular, growth rate adjustments are associated with substantial modifications of gene expression and mRNA abundance. In this work we aimed to assess the role of mRNA degradation during such variations. A genome-wide transcriptomic-based method was used to determine mRNA half-lives. The model bacterium Lactococcus lactis was used and different growth rates were studied in continuous cultures under isoleucine-limitation and in batch cultures during the adaptation to the isoleucine starvation. During continuous isoleucine-limited growth, the mRNAs of different genes had different half-lives. The stability of most of the transcripts was not constant, and increased as the growth rate decreased. This half-life diversity was analyzed to investigate determinants of mRNA stability. The concentration, length, codon adaptation index and secondary structures of mRNAs were found to contribute to the determination of mRNA stability in these conditions. However, the growth rate was, by far, the most influential determinant. The respective influences of mRNA degradation and transcription on the regulation of intra-cellular transcript concentration were estimated. The role of degradation on mRNA homeostasis was clearly evidenced: for more than 90% of the mRNAs studied during continuous isoleucine-limited growth of L. lactis, degradation was antagonistic to transcription. Although both transcription and degradation had, opposite effects, the mRNA changes in response to growth rate were driven by transcription. Interestingly, degradation control increased during the dynamic adaptation of bacteria as the growth rate reduced due to progressive isoleucine starvation in batch cultures. This work shows that mRNA decay differs between gene transcripts and according to the growth rate. It demonstrates that mRNA degradation is an important regulatory process involved in bacterial adaptation. However, its impact on the regulation of mRNA levels is smaller than that of transcription in the conditions studied.

Published
2012

17. Bacterial translational regulations: high diversity between all mRNAs and major role in gene expression

Author

Flora Picard, Laurence Girbal, Pascal Loubière, Béatrice Laurent, Hélène Milhem, Muriel Cocaign-Bousquet, Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), Institut de Mathématiques de Toulouse UMR5219 (IMT), Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Université Toulouse 1 Capitole (UT1), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Université Toulouse - Jean Jaurès (UT2J)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS), French Ministry of Research, CNRS, INSA, INRA, Centre National de la Recherche Scientifique (CNRS)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Institut National de la Recherche Agronomique (INRA), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Université Toulouse 1 Capitole (UT1)-Université Toulouse - Jean Jaurès (UT2J)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Université Toulouse Capitole (UT Capitole), Université de Toulouse (UT)-Université de Toulouse (UT)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université Toulouse - Jean Jaurès (UT2J), Université de Toulouse (UT)-Université Toulouse III - Paul Sabatier (UT3), and Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)

Subjects
EFFICIENCY, lcsh:QH426-470, lcsh:Biotechnology, [SDV]Life Sciences [q-bio], SHINE-DALGARNO SEQUENCE, Translational regulation, mRNA Ribosome, Translatome, Statistical modeling, Lactococcus lactis, GENOME-WIDE ANALYSIS, LACTOCOCCUS-LACTIS, SECONDARY STRUCTURE, DESULFOVIBRIO-VULGARIS, QUANTITATIVE-ANALYSIS, PROTEIN TRANSLATION, ESCHERICHIA-COLI, FISSION YEAST, Biology, Ribosome, 03 medical and health sciences, lcsh:TP248.13-248.65, Polysome, Gene expression, Genetics, Protein biosynthesis, RNA, Messenger, 030304 developmental biology, Regulation of gene expression, 0303 health sciences, Messenger RNA, Models, Genetic, 030306 microbiology, Gene Expression Profiling, Computational Biology, Translation (biology), Gene Expression Regulation, Bacterial, Cell biology, lcsh:Genetics, Polyribosomes, Protein Biosynthesis, Research Article, Biotechnology
Abstract

Background In bacteria, the weak correlations at the genome scale between mRNA and protein levels suggest that not all mRNAs are translated with the same efficiency. To experimentally explore mRNA translational level regulation at the systemic level, the detailed translational status (translatome) of all mRNAs was measured in the model bacterium Lactococcus lactis in exponential phase growth. Results Results demonstrated that only part of the entire population of each mRNA species was engaged in translation. For transcripts involved in translation, the polysome size reached a maximum of 18 ribosomes. The fraction of mRNA engaged in translation (ribosome occupancy) and ribosome density were not constant for all genes. This high degree of variability was analyzed by bioinformatics and statistical modeling in order to identify general rules of translational regulation. For most of the genes, the ribosome density was lower than the maximum value revealing major control of translation by initiation. Gene function was a major translational regulatory determinant. Both ribosome occupancy and ribosome density were particularly high for transcriptional regulators, demonstrating the positive role of translational regulation in the coordination of transcriptional networks. mRNA stability was a negative regulatory factor of ribosome occupancy and ribosome density, suggesting antagonistic regulation of translation and mRNA stability. Furthermore, ribosome occupancy was identified as a key component of intracellular protein levels underlining the importance of translational regulation. Conclusions We have determined, for the first time in a bacterium, the detailed translational status for all mRNAs present in the cell. We have demonstrated experimentally the high diversity of translational states allowing individual gene differentiation and the importance of translation-level regulation in the complex process linking gene expression to protein synthesis.

Published
2012

18. Assessment of the Diversity of Dairy Lactococcus lactis subsp. lactis Isolates by an Integrated Approach Combining Phenotypic, Genomic, and Transcriptomic Analyses ▿ †

Author

Laurence Girbal, Pascal Loubière, Punthip Tan-a-ram, Muriel Cocaign-Bousquet, Sunthorn Kanchanatawee, Tamara Cardoso, Marie-Line Daveran-Mingot, Laboratoire de microbiologie et génétique moléculaires (LMGM), Centre de Biologie Intégrative (CBI), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Laboratoire d'Ingénierie des Systèmes Biologiques et des Procédés (LISBP), Institut National de la Recherche Agronomique (INRA)-Institut National des Sciences Appliquées - Toulouse (INSA Toulouse), Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-Centre National de la Recherche Scientifique (CNRS), Suranaree University of Technology (SUT), UMR 5504, Centre National de la Recherche Scientifique (CNRS), Université Fédérale Toulouse Midi-Pyrénées, French National Research Agency (ANR) [ANR-05-PNRA-020], French government, Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Institut National des Sciences Appliquées (INSA)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS), Laboratoire de microbiologie et génétique moléculaires - UMR5100 (LMGM), Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), and Université de Toulouse (UT)-Université de Toulouse (UT)-Centre National de la Recherche Scientifique (CNRS)

Subjects
EXPRESSION, Genotype, ACBACTERIUM, [SDV]Life Sciences [q-bio], Molecular Sequence Data, Genomics, Cheese ripening, Biology, Applied Microbiology and Biotechnology, Genome, SEQUENCE, Polymerase Chain Reaction, Microbiology, 03 medical and health sciences, CREMORIS, Species Specificity, Cheese, Genetic variation, [SDV.IDA]Life Sciences [q-bio]/Food engineering, [SPI.GPROC]Engineering Sciences [physics]/Chemical and Process Engineering, ADAPTATION, Gene, ComputingMilieux_MISCELLANEOUS, 030304 developmental biology, Oligonucleotide Array Sequence Analysis, Genetics, 0303 health sciences, Comparative Genomic Hybridization, Ecology, 030306 microbiology, STRAINS, Gene Expression Profiling, Lactococcus lactis, Genetic Variation, Hydrogen-Ion Concentration, biology.organism_classification, Bacterial Typing Techniques, Gene expression profiling, Phenotype, Food Microbiology, GROWTH, HYBRIDIZATION, Genome, Bacterial, Food Science, Biotechnology, RESPONSES
Abstract

The intrasubspecies diversity of six strains of Lactococcus lactis subsp. lactis was investigated at the genomic level and in terms of phenotypic and transcriptomic profiles in an ultrafiltration cheese model. The six strains were isolated from various sources, but all exhibited a dairy phenotype (growth in ultrafiltration cheese model and high acidification rate). The six strains exhibited similar behaviors in terms of growth during cheese ripening, while different acidification capabilities were detected. Even if all strains displayed large genomic similarities, sharing a large core genome of almost 2,000 genes, the expression of this core genome directly in the cheese matrix revealed major strain-specific differences that potentially could account for the observed different acidification capabilities. This work demonstrated that significant transcriptomic polymorphisms exist even among Lactococcus lactis subsp. lactis strains with the same dairy origin.

Published
2010

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