60 results on '"Laurence Girbal"'
Search Results
2. Attachment of the RNA degradosome to the bacterial inner cytoplasmic membrane prevents wasteful degradation of rRNA in ribosome assembly intermediates.
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Lydia Hadjeras, Marie Bouvier, Isabelle Canal, Leonora Poljak, Quentin Morin-Ogier, Carine Froment, Odile Burlet-Schlitz, Lina Hamouche, Laurence Girbal, Muriel Cocaign-Bousquet, and Agamemnon J Carpousis
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Biology (General) ,QH301-705.5 - Abstract
RNA processing and degradation shape the transcriptome by generating stable molecules that are necessary for translation (rRNA and tRNA) and by facilitating the turnover of mRNA, which is necessary for the posttranscriptional control of gene expression. In bacteria and the plant chloroplast, RNA degradosomes are multienzyme complexes that process and degrade RNA. In many bacterial species, the endoribonuclease RNase E is the central component of the RNA degradosome. RNase E-based RNA degradosomes are inner membrane proteins in a large family of gram-negative bacteria (β- and γ-Proteobacteria). Until now, the reason for membrane localization was not understood. Here, we show that a mutant strain of Escherichia coli, in which the RNA degradosome is localized to the interior of the cell, has high levels of 20S and 40S particles that are defective intermediates in ribosome assembly. These particles have aberrant protein composition and contain rRNA precursors that have been cleaved by RNase E. After RNase E cleavage, rRNA fragments are degraded to nucleotides by exoribonucleases. In vitro, rRNA in intact ribosomes is resistant to RNase E cleavage, whereas protein-free rRNA is readily degraded. We conclude that RNA degradosomes in the nucleoid of the mutant strain interfere with cotranscriptional ribosome assembly. We propose that membrane-attached RNA degradosomes in wild-type cells control the quality of ribosome assembly after intermediates are released from the nucleoid. That is, the compact structure of mature ribosomes protects rRNA against cleavage by RNase E. Turnover of a proportion of intermediates in ribosome assembly explains slow growth of the mutant strain. Competition between mRNA and rRNA degradation could be the cause of slower mRNA degradation in the mutant strain. We conclude that attachment of the RNA degradosome to the bacterial inner cytoplasmic membrane prevents wasteful degradation of rRNA precursors, thus explaining the reason for conservation of membrane-attached RNA degradosomes throughout the β- and γ-Proteobacteria.
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- 2023
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3. 5’UTR sequences influence protein levels in Escherichia coli by regulating translation initiation and mRNA stability
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Fan Chen, Muriel Cocaign-Bousquet, Laurence Girbal, and Sébastien Nouaille
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mRNA stability ,translation initiation ,post-transcriptional regulation ,gene expression regulation ,protein level regulation ,Escherichia coli ,Microbiology ,QR1-502 - Abstract
A set of 41 synthetic 5’UTRs with different theoretical translation initiation rates were generated to explore the role of 5’UTRs in the regulation of protein levels in Escherichia coli. The roles of the synthetic 5’UTRs in regulating the expression of different reporter genes were analyzed in vivo. Protein levels varied substantially between the different constructs but for most of the 5’UTRs, protein levels were not correlated with theoretical translation initiation rates. Large variations in mRNA concentrations were measured with the different 5’UTRs even though the same concentration of transcription inducer was used in each case. 5’UTRs were also found to strongly affect mRNA stability, and these changes in mRNA stability often contributed to observed differences in mRNA concentration. Unexpectedly, the effect of the 5’UTRs on mRNA half-lives was found to vary depending on the downstream reporter gene. These results clearly demonstrate that 5’UTRs contribute to gene expression regulation at the level of translation initiation and of mRNA stability, to an extent that depends on the nature of the downstream gene.
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- 2022
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4. Synergistic Regulation of Transcription and Translation in Escherichia coli Revealed by Codirectional Increases in mRNA Concentration and Translation Efficiency
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Huong Le Nguyen, Marie-Pierre Duviau, Sandrine Laguerre, Sébastien Nouaille, Muriel Cocaign-Bousquet, and Laurence Girbal
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Escherichia coli ,gene expression regulation ,mRNA concentration ,polysome profiling ,ribosome density ,ribosome occupancy ,Microbiology ,QR1-502 - Abstract
ABSTRACT Translational regulation was investigated at the genome-scale in Escherichia coli cells. Using the polysome profiling method, the ribosome occupancy (RO) and ribosome density (RD) of different mRNA copies were determined for several hundred mRNAs during the exponential- and stationary-phases, providing the most complete characterization of such regulation in E. coli. Although for most genes, nearly all mRNAs (>90%) were undergoing translation, they were loaded with far fewer than the theoretical maximum number of ribosomes, suggesting translation limitation at the initiation step. Multiple linear regression was used to identify key intrinsic factors involved in the genome-wide regulation of RO and RD (i.e., open reading frame GC%, protein function, and localization). Unexpectedly, mRNA concentration, a factor that depends on cell physiology, was predicted to positively regulate RO and RD during the exponential- and stationary-phases. Using a set of selected genes controlled by an inducible promoter, we confirmed that increasing the mRNA concentration upon transcription induction led to increases in both RO and ribosome load. The fact that this relationship between mRNA concentration and translation parameters was also effective when E. coli cells naturally adapted to carbon source changes demonstrates its physiological relevance. This work demonstrated that translation regulation is positively controlled by transcript availability. This new mechanism contributed to the codirectional regulation of transcription and translation with synergistic effects on gene expression and provided a systemic understanding of E. coli cell function. IMPORTANCE The process of gene expression is divided into translation and transcription. Considerable efforts have been made in bacteria to characterize the mechanisms underlying translational regulation and identify the regulatory factors for particular mRNAs. However, to understand bacterial physiology and adaptation, it is important to elucidate genome-wide translational regulation and examine its coordination with transcriptional regulation. Here, we provided a genome-wide picture of translational regulation in Escherichia coli. For most genes, nearly all mRNA copies were found to undergo translation but were loaded with a low number of ribosomes. We showed that mRNA concentration had a positive effect on translation regulation, linking translational regulation to transcriptional regulation as well as to cell physiology and growth conditions. The codirectional regulation of transcription and translation had synergistic effects on gene expression, contributing to E. coli cell function optimization. This finding could be used in biotechnology to optimize strategies for recombinant protein synthesis.
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- 2022
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5. PNPase is involved in the coordination of mRNA degradation and expression in stationary phase cells of Escherichia coli
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Clémentine Dressaire, Vânia Pobre, Sandrine Laguerre, Laurence Girbal, Cecilia Maria Arraiano, and Muriel Cocaign-Bousquet
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RNA decay ,PNPase ,RNase R ,E. coli ,Transcriptome ,Gene expression regulation ,Biotechnology ,TP248.13-248.65 ,Genetics ,QH426-470 - Abstract
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.
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- 2018
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6. Genomewide Stabilization of mRNA during a 'Feast-to-Famine' Growth Transition in Escherichia coli
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Manon Morin, Brice Enjalbert, Delphine Ropers, Laurence Girbal, and Muriel Cocaign-Bousquet
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mRNA stability ,Escherichia coli ,carbon starvation ,metabolic transition ,posttranscriptional regulation ,transcriptomic ,Microbiology ,QR1-502 - Abstract
ABSTRACT 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.
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- 2020
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7. Multiplexing polysome profiling experiments to study translation in Escherichia coli.
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Huong Le Nguyen, Marie-Pierre Duviau, Muriel Cocaign-Bousquet, Sébastien Nouaille, and Laurence Girbal
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Medicine ,Science - Abstract
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 fractionation 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.
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- 2019
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8. Escherichia coli under Ionic Silver Stress: An Integrative Approach to Explore Transcriptional, Physiological and Biochemical Responses.
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Claire Saulou-Bérion, Ignacio Gonzalez, Brice Enjalbert, Jean-Nicolas Audinot, Isabelle Fourquaux, Frédéric Jamme, Muriel Cocaign-Bousquet, Muriel Mercier-Bonin, and Laurence Girbal
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Medicine ,Science - Abstract
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 intracellular 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 microspectroscopy and a comprehensive transcriptome response was also determined. Using multivariate statistics, correlations between the physiological parameters, the extracellular concentration of AgNO3 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, ribosomal 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.
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- 2015
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9. A genome-scale integration and analysis of Lactococcus lactis translation data.
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Julien Racle, Flora Picard, Laurence Girbal, Muriel Cocaign-Bousquet, and Vassily Hatzimanikatis
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Biology (General) ,QH301-705.5 - 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.
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- 2013
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10. Role of mRNA stability during bacterial adaptation.
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Clémentine Dressaire, Flora Picard, Emma Redon, Pascal Loubière, Isabelle Queinnec, Laurence Girbal, and Muriel Cocaign-Bousquet
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Medicine ,Science - Abstract
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.
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- 2013
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11. When translation elongation is impaired, the mRNA is uniformly destabilized by the RNA degradosome, while the concentration of mRNA is altered along the molecule
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Marie-Pierre Duviau, Fan Chen, Anthony Emile, Muriel Cocaign-Bousquet, Laurence Girbal, Sébastien Nouaille, Toulouse Biotechnology Institute (TBI), 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)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE)
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[SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN] ,Genetics ,[SDV.MP.BAC]Life Sciences [q-bio]/Microbiology and Parasitology/Bacteriology - Abstract
mRNA sits at the crossroads of transcription, translation and mRNA degradation. Many questions remain about the coupling of these three processes in Escherichia coli and, in particular, how translation may have an effect on mRNA degradation and transcription. To characterize the interplay between mRNA degradation and translation while accounting for transcription, we altered the translation initiation or elongation and measured the effects on mRNA stability and concentration. Using a mapping method, we analysed mRNA concentration and stability at the local scale all along the transcript. We showed that a decrease in translation initiation efficiency destabilizes the mRNA and leads to a uniform decrease in mRNA concentration throughout the molecule. Prematurely terminating translation elongation by inserting a stop codon is associated with a drop in local mRNA concentration downstream of the stop codon, due to the uncoupling of transcription and translation. In contrast, this translation alteration uniformly destabilizes the coding and ribosome-free regions, in a process triggered by RNase E activity, and its ability to form the RNA degradome. These results demonstrate how ribosomes protect mRNA molecules and highlight how translation, mRNA degradation and transcription are deeply interconnected in the quality control process that avoids unproductive gene expression in cells.
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- 2023
12. Attachment of the RNA degradosome to the inner cytoplasmic membrane of Escherichia coli prevents wasteful degradation of rRNA intermediates in ribosome assembly
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Lydia Hadjeras, Marie Bouvier, Isabelle Canal, Leonora Poljak, Quentin Morin-Ogier, Carine Froment, Odile Burlet-Schlitz, Lina Hamouche, Laurence Girbal, Muriel Cocaign-Bousquet, and Agamemnon J. Carpousis
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BackgroundRNase E has crucial roles in the initiation of mRNA degradation, the processing of ‘stable’ transcripts such as rRNA and tRNA, and the quality control of ribosomes. With over 20’000 potential cleavage sites, RNase E is a low specificity endoribonuclease with the capacity to cleave multiple times nearly every transcript in the cell. A large noncatalytic region in the C-terminal half of RNase E is the scaffold for assembly of the multienzyme RNA degradosome. The components of the RNA degradosome cooperate in the degradation of mRNA to oligoribonucleotides, which are then degraded to nucleotides by oligoribonuclease. Over the past decade, compelling evidence has emerged that the RNA degradosome is attached to the phospholipid bilayer of the inner cytoplasmic membrane by the Membrane Targeting Sequence (MTS), which is a 15-residue amphipathic alpha-helix located in the noncatalytic region of RNase E. Systematic proteomic analyses have identified RNase E as an inner membrane protein that can only be solubilized by disrupting the phospholipid bilayer with detergent. Important components of the mRNA degradation machinery are therefore membrane-attached. The reason for this cellular localization has until now been a mystery.ResultsWe have constructed and characterized the rneΔMTS strain expressing ncRNase E (nucleo-cytoplasmic-RNase E), which is a soluble variant that is uniformly distributed in the interior of the cell. In the mutant strain, there is a slowdown in the rates of growth and mRNA degradation. Surprisingly, we have identified aberrant 20S and 40S ribosomal particles in the rneΔMTS strain that contain, respectively, precursors of 16S and 23S rRNA that have been ‘nicked’ by ncRNase E. Although intact ribosomes are resistant to RNase E cleavage in vitro, protein-free rRNA is readily degraded by RNase E. Partially unfolded ribosomes are susceptible to nicking by RNase E in vitro. We have mapped rRNA cleavage sites cRACE. In vivo and in vitro rRNA cleavages map to the same sites. The sequence of the cleavage sites matches the RNase E consensus sequence previously determined in a transcriptomic analysis that did not include rRNA. Construction of additional mutant strains demonstrated in vivo that fragments of 16S and 23S rRNA as well as a precursor of 5S rRNA are degraded in a pathway involving 3’ oligoadenylation and exonucleolytic digestion. A proteomic analysis showed that 17 small subunit proteins and 21 large subunit proteins are underrepresented in the 20S and 40S particles, respectively.ConclusionsRibosome biogenesis is a complex process involving co-transcriptional rRNA folding and r-protein binding in the nucleoid. Ribonucleoprotein intermediates are released from chromatin by RNase III cleavage. Maturation continues with the addition of ‘late’ proteins resulting in the compact rRNA structures found in mature 30S and 50S ribosomal subunits. Considering our experimental results, we propose that the physical separation of rRNA transcription in the nucleoid from the RNA degradosome on the inner cytoplasmic membrane protects intermediates in ribosome assembly from degradation. A corollary is that ribosome quality control normally occurs when defective ribosomal particles interact with the membrane-attached RNA degradosome. The rRNA degradation pathway described here is the same as described previously for RNase E-dependent degradation of mRNA. Since the pathway for rRNA degradation is the same as the pathway for mRNA degradation, the slowdown of mRNA degradation in the rneΔMTS strain could be due to competition by rRNA degradation. Since growth rate is limited by ribosome synthesis rate, the slow growth of the rneΔMTS strain is likely due to wasteful degradation of a proportion of newly synthesized rRNA. If r-proteins released by rRNA degradation are not recycled, then this would be an additional burden on cell growth. Avoiding a futile cycle in which rRNA intermediates in ribosome assembly are degraded likely explains why localization of RNase E homologues to the inner cytoplasmic membrane is conserved throughout the β- and γ-Proteobacteria.ImportanceIn E. coli, transcription in the nucleoid, translation in the cytoplasm and initiation of mRNA degradation on the inner cytoplasmic membrane are physically separated. Despite the lack of internal membranes, this separation can be viewed as a compartmentalization of the bacterial cell. Our work shows that the inner membrane localization of the RNA degradosome restricts access of RNase E to intermediates in ribosome assembly. Thus, as in the eukaryotic cell, the architecture of the bacterial cell has an important role in the organization of cellular processes such as ribosome biogenesis, ribosome quality control, and mRNA degradation.
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- 2022
13. Synergistic regulation of transcription and translation in Escherichia coli revealed by co-directional increases in mRNA concentration and translation efficiency
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Huong Le Nguyen, Marie-Pierre Duviau, Sandrine Laguerre, Sébastien Nouaille, Muriel Cocaign-Bousquet, Laurence Girbal, Toulouse Biotechnology Institute (TBI), 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 de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), and 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)
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Microbiology (medical) ,Physiology ,[SDV]Life Sciences [q-bio] ,translation regulation ,polysome profiling ,Open Reading Frames ,Genetics ,Escherichia coli ,RNA, Messenger ,General Immunology and Microbiology ,Ecology ,Escherichia coli Proteins ,ribosome density ,mRNA concentration ,Gene Expression Regulation, Bacterial ,Cell Biology ,gene expression regulation ,Carbon ,Infectious Diseases ,ribosome occupancy ,Polyribosomes ,Protein Biosynthesis ,Transcriptome ,transcription ,Ribosomes ,Genome, Bacterial - Abstract
International audience; Translational regulation was investigated at the genome scale in Escherichia coli cells. Using the polysome profiling method, the ribosome occupancy (RO) and ribosome density (RD) of different mRNA copies were determined for several hundred mRNAs during the exponential and stationary phases, providing the most complete characterisation of such regulation in E. coli. Although for most genes, nearly all mRNAs (>90%) were undergoing translation, they were loaded with far fewer than the theoretical maximum number of ribosomes, suggesting translation limitation at the initiation step. Multiple linear regression was used to identify key intrinsic factors involved in the genome-wide regulation of RO and RD (i.e., open reading frame GC%, protein function and localisation). Unexpectedly, mRNA concentration, a factor that depends on cell physiology, was predicted to positively regulate RO and RD during the exponential and stationary phases. Using a set of selected genes controlled by an inducible promoter, we confirmed that increasing the mRNA concentration upon transcription induction led to increases in both RO and ribosome load. The fact that this relationship between mRNA concentration and translation parameters was also effective when E. coli cells naturally adapted to carbon source changes demonstrates its physiological relevance. This work demonstrates that translation regulation is positively controlled by transcript availability. This new mechanism contributes to the co-directional regulation of transcription and translation with synergistic effects on gene expression, and provides a systemic understanding of E. coli cell function.
- Published
- 2022
14. Molecular characterization of the missing electron pathways for butanol synthesis in Clostridium acetobutylicum
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Céline Foulquier, Antoine Rivière, Mathieu Heulot, Suzanna Dos Reis, Caroline Perdu, Laurence Girbal, Mailys Pinault, Simon Dusséaux, Minyeong Yoo, Philippe Soucaille, Isabelle Meynial-Salles, Toulouse Biotechnology Institute (TBI), 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 de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), University of Nottingham, UK (UON), ANR acetoH2 PNRB 2006 ANR Bio6 BioE-001, ANR-08-BIOE-0012,BioButaFuel,Bioconversion d'hydrolysat de lignocellulose en Butanol, biocarburant de nouvelle génération de haute efficacité, à haut titre et rendement(2008), and ANR-14-CE05-0019,cellutanol,construction d'une souche d'E. coli à cellulosomes pour la conversion de la cellulose en butanol(2014)
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Clostridium ,Multidisciplinary ,[SDV.BIO]Life Sciences [q-bio]/Biotechnology ,[SDV]Life Sciences [q-bio] ,Butanols ,General Physics and Astronomy ,Electrons ,General Chemistry ,NAD ,General Biochemistry, Genetics and Molecular Biology ,Ferredoxin-NADP Reductase ,Fermentation ,Ferredoxins ,Clostridium acetobutylicum ,Oxidoreductases ,NADP - Abstract
International audience; Ferredoxin-NAD(P) + oxidoreductases are important enzymes for redox balancing in n-butanol production by Clostridium acetobutylicum, but the encoding genes remain unknown. Here, the authors identify the long sought-after genes and increase n-butanol production by optimizing the levels of the two enzymes.Clostridium acetobutylicum is a promising biocatalyst for the renewable production of n-butanol. Several metabolic strategies have already been developed to increase butanol yields, most often based on carbon pathway redirection. However, it has previously demonstrated that the activities of both ferredoxin-NADP(+) reductase and ferredoxin-NAD(+) reductase, whose encoding genes remain unknown, are necessary to produce the NADPH and the extra NADH needed for butanol synthesis under solventogenic conditions. Here, we purify, identify and partially characterize the proteins responsible for both activities and demonstrate the involvement of the identified enzymes in butanol synthesis through a reverse genetic approach. We further demonstrate the yield of butanol formation is limited by the level of expression of CA_C0764, the ferredoxin-NADP(+) reductase encoding gene and the bcd operon, encoding a ferredoxin-NAD(+) reductase. The integration of these enzymes into metabolic engineering strategies introduces opportunities for developing a homobutanologenic C. acetobutylicum strain.
- Published
- 2021
15. The essential role of mRNA degradation in understanding and engineering E. coli metabolism
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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)
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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.
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- 2021
16. Genomewide Stabilization of mRNA during a 'Feast-to-Famine' Growth Transition in Escherichia coli
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Laurence Girbal, Delphine Ropers, Manon Morin, Brice Enjalbert, and Muriel Cocaign-Bousquet
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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.
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- 2020
17. The stability of an mRNA is influenced by its concentration: a potential physical mechanism to regulate gene expression
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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
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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.
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- 2017
18. Multiplexing polysome profiling experiments to study translation in Escherichia coli
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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)
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[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.
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- 2019
19. 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
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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)
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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.
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- 2019
20. Large-Scale Measurement of mRNA Degradation in Escherichia coli: To Delay or Not to Delay
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Sandrine, Laguerre, Ignacio, González, Sébastien, Nouaille, Annick, Moisan, Nathalie, Villa-Vialaneix, Christine, Gaspin, Marie, Bouvier, Agamemnon J, Carpousis, Muriel, Cocaign-Bousquet, and Laurence, Girbal
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RNA, Bacterial ,Transcription, Genetic ,RNA Stability ,Escherichia coli ,RNA, Messenger - Abstract
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 34s. 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.
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- 2018
21. PNPase is involved in the coordination of mRNA degradation and expression in stationary phase cells of Escherichia coli
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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
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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.
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- 2018
22. Roles of the F-domain in [FeFe] hydrogenase
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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)
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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.
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- 2018
23. Large-Scale Measurement of mRNA Degradation in Escherichia coli: To Delay or Not to Delay
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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)
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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.
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- 2018
24. The Csr system regulates genome-wide mRNA stability and transcription and thus gene expression in Escherichia coli
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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
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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.
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- 2016
25. Dynamic Analysis of the Lactococcus lactis Transcriptome in Cheeses Made from Milk Concentrated by Ultrafiltration Reveals Multiple Strategies of Adaptation to Stresses
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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)
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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.
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- 2011
26. Examination of post-transcriptional regulations in prokaryotes by integrative biology
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Flora Picard, Laurence Girbal, Muriel Cocaign-Bousquet, and Clémentine Dressaire
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RNA, Untranslated ,RNA Stability ,Systems biology ,RNA-binding protein ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Structure-Activity Relationship ,Bacterial Proteins ,Translational regulation ,Escherichia coli ,Protein biosynthesis ,RNA, Messenger ,RNA Processing, Post-Transcriptional ,Genetics ,Regulation of gene expression ,Messenger RNA ,General Immunology and Microbiology ,Protein Stability ,Systems Biology ,Protein turnover ,RNA-Binding Proteins ,Translation (biology) ,Gene Expression Regulation, Bacterial ,General Medicine ,Cell biology ,Lactococcus lactis ,Systems Integration ,RNA, Bacterial ,Prokaryotic Cells ,Protein Biosynthesis ,General Agricultural and Biological Sciences ,Protein Processing, Post-Translational ,Ribosomes ,Bacillus subtilis - Abstract
In cells, mRNA and protein levels are fine-regulated to adjust continuously to cellular needs. Recently, several large-scale studies in prokaryotes showed weak correlations between mRNA and protein abundances highlighting the significant importance of post-transcriptional regulations. Post-transcriptional regulations involve dynamic adaptation of mRNA and protein turnover and also modulation of the efficiency of mRNA translation into protein. mRNA and protein stabilities are function of both sequence determinants and decay processes. Translation efficiency is mainly dependent on ribosome synthesis and activity. Conciliation through an integrative biology approach of large-scale data obtained for each level of regulation is now required to better understand global cell response to different environmental growth conditions. In this review, we report mechanisms involved in mRNA and protein stability and translation regulation in prokaryotes, and their dependence on growth phase and environmental growth conditions is particularly highlighted.
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- 2009
27. Optimized over-expression of [FeFe] hydrogenases with high specific activity in Clostridium acetobutylicum
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Thomas Happe, Philippe Soucaille, Alexey Silakov, Laurence Girbal, Gregory von Abendroth, Christian Croux, and Sven T. Stripp
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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.
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- 2008
28. PerR acts as a switch for oxygen tolerance in the strict anaerobe Clostridium acetobutylicum
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Laurence Girbal, Falk Hillmann, Florence Saint-Prix, Ralf-Jörg Fischer, and Hubert Bahl
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Clostridium acetobutylicum ,Rubrerythrin ,medicine.disease_cause ,Regulon ,Microbiology ,Article ,Clostridia ,Bacterial Proteins ,Heat shock protein ,medicine ,Anaerobiosis ,Molecular Biology ,biology ,fungi ,Obligate anaerobe ,Gene Expression Regulation, Bacterial ,Hydrogen Peroxide ,biology.organism_classification ,Aerobiosis ,Oxygen ,Repressor Proteins ,Oxidative Stress ,Biochemistry ,Genes, Bacterial ,Reactive Oxygen Species ,Gene Deletion ,Bacteria ,Oxidative stress ,Transcription Factors - Abstract
Clostridia belong to those bacteria which are considered as obligate anaerobe, e.g. oxygen is harmful or lethal to these bacteria. Nevertheless, it is known that they can survive limited exposure to air, and often eliminate oxygen or reactive derivatives via NAD(P)H-dependent reduction. This system does apparently contribute to survival after oxidative stress, but is insufficient to establish long-term tolerance of aerobic conditions. Here we show that manipulation of the regulatory mechanism of this defence mechanism can trigger aerotolerance in the obligate anaerobe Clostridium acetobutylicum. Deletion of a peroxide repressor (PerR)-homologous protein resulted in prolonged aerotolerance, limited growth under aerobic conditions and rapid consumption of oxygen from an aerobic environment. The mutant strain also revealed higher resistance to H 2 O 2 and activities of NADH-dependent scavenging of H 2 O 2 and organic peroxides in cell-free extracts increased by at least one order of magnitude. Several genes encoding the putative enzymes were upregulated and identified as members of the clostridial PerR regulon, including the heat shock protein Hsp21, a reverse rubrerythrin which was massively produced and became the most abundant protein in the absence of PerR. This multifunctional protein is proposed to play the crucial role in the oxidative stress defence.
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- 2008
29. Genome-wide investigation of mRNA lifetime determinants in Escherichia coli cells cultured at different growth rates
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Thomas Esquerré, Annick Moisan, Liisa Arike, Raivo Vilu, Christine Gaspin, Muriel Cocaign-Bousquet, Laurence Girbal, Hélène Chiapello, 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), 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), Unité de Biométrie et Intelligence Artificielle (UBIA), Institut National de la Recherche Agronomique (INRA), Competence Center of Food and Fermentation Technologies, Department of Chemistry, Tallinn University of Technology (TTÜ), Plate-forme Bio-informatique Genotoul, 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), Unité de Biométrie et Intelligence Artificielle (ancêtre de MIAT) (UBIA), Plateforme Bio-Informatique - Génotoul, Institut National des Sciences Appliquées (INSA)-Institut National des Sciences Appliquées (INSA)-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)
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Codon Adaptation Index ,RNA Stability ,[SDV]Life Sciences [q-bio] ,Biology ,medicine.disease_cause ,Models, Biological ,Open Reading Frames ,mRNA decay ,Genome-wide analysis ,medicine ,Genetics ,Escherichia coli ,RNA, Messenger ,Determinants ,Growth rate ,Codon ,Gene ,2. Zero hunger ,Base Composition ,Messenger RNA ,Base Sequence ,Escherichia coli Proteins ,RNA ,Cell biology ,Open reading frame ,DNA microarray ,5' Untranslated Regions ,Genome, Bacterial ,GC-content ,Research Article ,Half-Life ,Biotechnology - Abstract
Background Changes to mRNA lifetime adjust mRNA concentration, facilitating the adaptation of growth rate to changes in growth conditions. However, the mechanisms regulating mRNA lifetime are poorly understood at the genome-wide scale and have not been investigated in bacteria growing at different rates. Results We used linear covariance models and the best model selected according to the Akaike information criterion to identify and rank intrinsic and extrinsic general transcript parameters correlated with mRNA lifetime, using mRNA half-life datasets for E. coli, obtained at four growth rates. The principal parameter correlated with mRNA stability was mRNA concentration, the mRNAs most concentrated in the cells being the least stable. However, sequence-related features (codon adaptation index (CAI), ORF length, GC content, polycistronic mRNA), gene function and essentiality also affected mRNA lifetime at all growth rates. We also identified sequence motifs within the 5′UTRs potentially related to mRNA stability. Growth rate-dependent effects were confined to particular functional categories (e.g. carbohydrate and nucleotide metabolism). Finally, mRNA stability was less strongly correlated with the amount of protein produced than mRNA concentration and CAI. Conclusions This study provides the most complete genome-wide analysis to date of the general factors correlated with mRNA lifetime in E. coli. We have generalized for the entire population of transcripts or excluded determinants previously defined as regulators of stability for some particular mRNAs and identified new, unexpected general indicators. These results will pave the way for discussions of the underlying mechanisms and their interaction with the growth physiology of bacteria. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-1482-8) contains supplementary material, which is available to authorized users.
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- 2015
30. Escherichia coli under Ionic Silver Stress: An Integrative Approach to Explore Transcriptional, Physiological and Biochemical Responses
- Author
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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
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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
31. Metabolism of lactose by Clostridium thermolacticum growing in continuous culture
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Jean-Paul Schwitzguébel, Paul Péringer, Laurence Girbal, Philippe Soucaille, and Christophe Collet
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Pyruvate decarboxylation ,Pyruvate Synthase ,Lactose ,Dehydrogenase ,Biochemistry ,Microbiology ,chemistry.chemical_compound ,Adenosine Triphosphate ,Lactate dehydrogenase ,Genetics ,Glycolysis ,Biomass ,Molecular Biology ,Acetic Acid ,Clostridium ,Acetate kinase ,Ethanol ,L-Lactate Dehydrogenase ,Acetate Kinase ,Chemistry ,General Medicine ,Metabolism ,Carbon Dioxide ,NAD ,beta-Galactosidase ,Adenosine Diphosphate ,Fermentation ,Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+) ,Hydrogen - Abstract
The objective of the present study was to characterize the metabolism of Clostridium thermolacticum, a thermophilic anaerobic bacterium, growing continuously on lactose (10 g l(-1)) and to determine the enzymes involved in the pathways leading to the formation of the fermentation products. Biomass and metabolites concentration were measured at steady-state for different dilution rates, from 0.013 to 0.19 h(-1). Acetate, ethanol, hydrogen and carbon dioxide were produced at all dilution rates, whereas lactate was detected only for dilution rates below 0.06 h(-1). The presence of several key enzymes involved in lactose metabolism, including beta-galactosidase, glyceraldehyde-3-phosphate dehydrogenase, pyruvate:ferredoxin oxidoreductase, acetate kinase, ethanol dehydrogenase and lactate dehydrogenase, was demonstrated. Finally, the intracellular level of NADH, NAD+, ATP and ADP was also measured for different dilution rates. The production of ethanol and lactate appeared to be linked with the re-oxidation of NADH produced during glycolysis, whereas hydrogen produced should come from reduced ferredoxin generated during pyruvate decarboxylation. To produce more hydrogen or more acetate from lactose, it thus appears that an efficient H2 removal system should be used, based on a physical (membrane) or a biological approach, respectively, by cultivating C. thermolacticum with efficient H2 scavenging and acetate producing microorganisms.
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- 2006
32. Molecular Characterization and Transcriptional Analysis of adhE2 , the Gene Encoding the NADH-Dependent Aldehyde/Alcohol Dehydrogenase Responsible for Butanol Production in Alcohologenic Cultures of Clostridium acetobutylicum ATCC 824
- Author
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Isabelle Meynial-Salles, Laurence Girbal, Xinghong Yang, Philippe Soucaille, Christian Croux, and Lisa Fontaine
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Clostridium acetobutylicum ,Operon ,Butanol ,Mutant ,Alcohol oxidoreductase ,Biology ,biology.organism_classification ,Microbiology ,Molecular biology ,chemistry.chemical_compound ,Plasmid ,Clostridium ,Biochemistry ,chemistry ,biology.protein ,Molecular Biology ,Alcohol dehydrogenase - Abstract
The adhE2 gene of Clostridium acetobutylicum ATCC 824, coding for an aldehyde/alcohol dehydrogenase (AADH), was characterized from molecular and biochemical points of view. The 2,577-bp adhE2 codes for a 94.4-kDa protein. adhE2 is expressed, as a monocistronic operon, in alcohologenic cultures and not in solventogenic cultures. Primer extension analysis identified two transcriptional start sites 160 and 215 bp upstream of the adhE2 start codon. The expression of adhE2 from a plasmid in the DG1 mutant of C. acetobutylicum , a mutant cured of the pSOL1 megaplasmid, restored butanol production and provided elevated activities of NADH-dependent butyraldehyde and butanol dehydrogenases. The recombinant AdhE2 protein expressed in E. coli as a Strep -tag fusion protein and purified to homogeneity also demonstrated NADH-dependent butyraldehyde and butanol dehydrogenase activities. This is the second AADH identified in C. acetobutylicum ATCC 824, and to our knowledge this is the first example of a bacterium with two AADHs. It is noteworthy that the two corresponding genes, adhE and adhE2 , are carried by the pSOL1 megaplasmid of C. acetobutylicum ATCC 824.
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- 2002
33. Regulation of Carbon and Electron Flow in Clostridium butyricum VPI 3266 Grown on Glucose-Glycerol Mixtures
- Author
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Laurence Girbal, José Carlos Andrade, Kerstin Ahrens, Sylvie Saint-Amans, and Philippe Soucaille
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Glycerol ,Hydrogenase ,Physiology and Metabolism ,Glycerol dehydratase ,Electrons ,Dehydrogenase ,Microbiology ,Phosphates ,Electron Transport ,chemistry.chemical_compound ,Molecular Biology ,Clostridium butyricum ,Clostridium ,biology ,Chemiosmosis ,Proton-Motive Force ,Gene Expression Regulation, Bacterial ,Metabolism ,Hydrogen-Ion Concentration ,biology.organism_classification ,Carbon ,Culture Media ,Glucose ,chemistry ,Biochemistry ,Propylene Glycols ,Glycerol dehydrogenase - Abstract
The metabolism of Clostridium butyricum was manipulated at pH 6.5 and in phosphate-limited chemostat culture by changing the overall degree of reduction of the substrate using mixtures of glucose and glycerol. Cultures grown on glucose alone produced only acids (acetate, butyrate, and lactate) and a high level of hydrogen. In contrast, when glycerol was metabolized, 1,3-propanediol became the major product, the specific rate of acid formation decreased, and a low level of hydrogen was observed. Glycerol consumption was associated with the induction of (i) a glycerol dehydrogenase and a dihydroxyacetone kinase feeding glycerol into the central metabolism and (ii) an oxygen-sensitive glycerol dehydratase and an NAD-dependent 1,3-propanediol dehydrogenase involved in propanediol formation. The redirection of the electron flow from hydrogen to NADH formation was associated with a sharp decrease in the in vitro hydrogenase activity and the acetyl coenzyme A (CoA)/free CoA ratio that allows the NADH-ferredoxin oxidoreductase bidirectional enzyme to operate so as to reduce NAD in this culture. The decrease in acetate and butyrate formation was not explained by changes in the concentration of phosphotransacylases and acetate and butyrate kinases but by changes in in vivo substrate concentrations, as reflected by the sharp decrease in the acetyl-CoA/free CoA and butyryl-CoA/free CoA ratios and the sharp increase in the ATP/ADP ratio in the culture grown with glucose and glycerol compared with that in the culture grown with glucose alone. As previously reported for Clostridium acetobutylicum (L. Girbal, I. Vasconcelos, and P. Soucaille, J. Bacteriol. 176:6146–6147, 1994), the transmembrane pH of C. butyricum is inverted (more acidic inside) when the in vivo activity of hydrogenase is decreased (cultures grown on glucose-glycerol mixture). For both cultures, the stoichiometry of the H + ATPase was shown to remain constant and equal to 3 protons exported per molecule of ATP consumed.
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- 2001
34. A Genome-Scale Integration and Analysis of Lactococcus lactis Translation Data
- Author
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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.
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- 2013
35. Regulation of solvent production in Clostridium acetobutylicum
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Laurence Girbal and Philippe Soucaille
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Clostridium acetobutylicum ,biology ,Butanol ,Bioengineering ,equipment and supplies ,biology.organism_classification ,Chemical synthesis ,Solvent ,chemistry.chemical_compound ,Biochemistry ,chemistry ,Acetone ,Fermentation ,Clostridiaceae ,Bacteria ,Biotechnology - Abstract
The production of acetone and butanol by Clostridium acetobutylicum was once one of the largest fermentation processes but, once it was no longer competitive with chemical synthesis, it was discontinued. However, the combined efforts of several laboratories have increased our knowledge of the molecular basis of solvent production and this, combined with a better understanding of the regulation of the genes responsible for solvent formation, should enable a more pragmatic approach to the construction of C. acetobutylicum strains producing high yields of specific metabolites in a selective manner.
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- 1998
36. Why Does Clostridium acetireducens Not Use Interspecies Hydrogen Transfer for Growth on Leucine?
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Johann Orlygsson, Laurence Girbal, Jan C. Gottschal, and Bert J. Reinders
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chemistry.chemical_classification ,Alanine ,Methanobacterium ,biology ,Deamination ,Oxidative deamination ,General Medicine ,biology.organism_classification ,Applied Microbiology and Biotechnology ,Microbiology ,Amino acid ,Biochemistry ,chemistry ,Fermentation ,NAD+ kinase ,Leucine - Abstract
Clostridium acetireducens is the first reported anaerobic bacterium that is dependent on acetate as an electron acceptor for growth on branched-chain amino acids and alanine. The fermentation pathway of leucine and its deamination product α-ketoisocaproate were studied in this organism. Addition of Methanobacterium formicicum to pure cultures of C. acetireducens stimulated the degradation of α-ketoisocaproate but not the degradation of leucine, indicating that the electrons produced during the oxidative deamination of leucine were not transferred to hydrogen. This conclusion is supported by the observed low NAD(P)H ferredoxin reductase activity. Not only acetate but also crotonate proved to be an appropriate electron sink for the regeneration of NAD(P)+ in this bacterium. Interestingly, C. acetireducens was shown to form polyhydroxybutyrate during growth on leucine plus acetate.
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- 1997
37. Synchrotron FTIR microspectroscopy of Escherichia coli at single-cell scale under silver-induced stress conditions
- Author
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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 (
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- 2013
38. 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
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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
39. Regulation of metabolic shifts inClostridium acetobutylicumATCC 824
- Author
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Laurence Girbal, I. Vasconcelos, Philippe Soucaille, and Christian Croux
- Subjects
chemistry.chemical_classification ,Ethanol ,Clostridium acetobutylicum ,Hydrogenase ,biology ,Stereochemistry ,Butanol ,Intracellular pH ,Metabolic shift ,biology.organism_classification ,Microbiology ,chemistry.chemical_compound ,Alcohol formation ,Infectious Diseases ,Enzyme ,chemistry ,Biochemistry ,NADH ,NAD+ kinase ,Ferredoxin - Abstract
Alcohol formation was initiated in continuous cultures of Clostridium acetobutylicum under distinct steady‐state conditions: (i) in glucose‐limited cultures established at low operating pH with formation of butanol, ethanol and acetone (induction of the solventogenesis) in which cells contained normal levels of NADH and a high level of ATP and butyric acid; and (ii) by increasing the NADH pressure at neutral pH in glucose‐limited cultures after addition of Neutral red, or in glucose‐glycerol or glucose‐glycerol‐pyruvate grown cultures, with a strictly alcohologenic metabolism (no acetone produced) associated with high levels of intracellular NADH and various levels of ATP. These two different metabolic shift systems are correlated with the expression of different genes involved in the solvent‐forming pathways and the electron flow distribution. A high NADH level leading to butanol and ethanol formation was accompanied by increased activities of the NADH‐dependent alcohol and butyraldehyde dehydrogenases, and ferredoxin:NAD(P)+ reductases, and by decreased activities of the NADH:ferredoxin reductase. This last group of enzymes constitutes the key enzymes regulating electron flow, since no change in hydrogenase activity was observed. On the other hand, classical solventogenesis appears to be characterized by high levels of expression of the NADPH‐dependent alcohol and butyraldehyde dehydrogenases, and of the two enzymes involved in the acetone‐forming pathway, while the ferredoxin:NAD(P)+ reductases were not synthesized. A decrease of the in vitro hydrogenase activity explains the lower hydrogen generation. In addition, the regulation of the intracellular pH was different between the alcohologenic culture grown at neutral pH and the solventogenic cultures grown at low pH. An inversion of the transmembrane pH gradient was observed during the production of alcohol at neutral pH and was related to a lower in vivo specific rate of hydrogen production while in the cultures grown at low pH the transmembrane pH generation was not linked to the F1F0 ATPase activity.
- Published
- 1995
40. How neutral red modified carbon and electron flow inClostridium acetobutylicumgrown in chemostat culture at neutral pH
- Author
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I. Vasconcelos, Philippe Soucaille, Laurence Girbal, and Silvie Saint-Amans
- Subjects
chemistry.chemical_classification ,Clostridium acetobutylicum ,Pyruvate synthase ,Hydrogenase ,biology ,Chemistry ,biology.organism_classification ,Microbiology ,Infectious Diseases ,Biochemistry ,Oxidoreductase ,biology.protein ,Fermentation ,NAD+ kinase ,Ferredoxin ,Alcohol dehydrogenase - Abstract
The metabolism of Clostridium acetobutylicum was manipulated, at neutral pH and in chemostat culture, by the addition of Neutral red, a molecule that can replace ferredoxin in the oxido-reduction reactions catalysed by the enzymes involved in the distribution of the electron flow. Cultures grown on glucose alone produced mainly acids while cultures grown on glucose plus Neutral red produced mainly alcohols and butyrate and low levels of hydrogen. We demonstrated that just after addition of Neutral red to an acidogenic culture, the simultaneous utilizations of ferredoxin and dye deviate electron flow from hydrogen to NADH production initially by the enzymatic regulation of in vivo hydrogenase and ferredoxin NAD reductase activities. The higher NAD(P)H pool generated might, thereafter, be the signal for the setting up of a new metabolism. In the resulting steady-state, the NAD(P)H ‘pressure’ is maintained by high ferredoxin NAD and NADP reductases level associated to a low NADH ferredoxin reductase level. The regeneration of NAD is mainly achieved via the induced or increased NADH-dependent aldehyde and alcohol dehydrogenase activities.
- Published
- 1995
41. Role of mRNA stability during bacterial adaptation
- Author
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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
42. Bacterial translational regulations: high diversity between all mRNAs and major role in gene expression
- Author
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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
43. 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
44. Reductive Cleavage of Demeton- S -Methyl by Corynebacterium glutamicum in Cometabolism on More Readily Metabolizable Substrates
- Author
-
Laurence Girbal, Sébastien Leduc, Jean-Luc Rols, Nicholas D. Lindley, Didier Hilaire, and Laure Delery
- Subjects
Insecticides ,Corynebacterium ,Cometabolism ,Fructose ,Applied Microbiology and Biotechnology ,Xenobiotics ,Corynebacterium glutamicum ,Thiophosphate ,chemistry.chemical_compound ,Biotransformation ,Organophosphorus compound ,Organic chemistry ,Nuclear Magnetic Resonance, Biomolecular ,chemistry.chemical_classification ,Ecology ,biology ,Organothiophosphates ,Phosphorus Isotopes ,Substrate (chemistry) ,Physiology and Biotechnology ,biology.organism_classification ,Organophosphates ,Biodegradation, Environmental ,chemistry ,Oxidation-Reduction ,Food Science ,Biotechnology - Abstract
Corynebacterium glutamicum is able to biotransform demeton- S -methyl, an organophosphorus compound, during cometabolism with more readily metabolizable substrates. Among the cosubstrates used, fructose is the growth substrate that is most favorable for demeton- S -methyl biotransformation. The reaction mechanism of demeton- S -methyl biotransformation involves reductive cleavage of an S-C bond, which leads to accumulation of dimethyl thiophosphate in the culture medium.
- Published
- 2000
45. 'Hydrogen-activating enzymes: activity does not correlate with oxygen sensitivity'
- Author
-
Laurence Girbal, Christophe Léger, Carole Baffert, Bénédicte Burlat, Marie Demuez, Bruno Guigliarelli, Patrick Bertrand, Laurent Cournac, Biologie cellulaire et moléculaire des plantes et des bactéries (BCMPB), Centre National de la Recherche Scientifique (CNRS)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université de la Méditerranée - Aix-Marseille 2, Azzopardi, Laure, and Université de la Méditerranée - Aix-Marseille 2-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS)
- Subjects
Hydrogenase ,Time Factors ,Hydrogen ,Protein Conformation ,Kinetics ,Inorganic chemistry ,chemistry.chemical_element ,02 engineering and technology ,Photochemistry ,010402 general chemistry ,Oxygen ,01 natural sciences ,Sensitivity and Specificity ,Catalysis ,Electron transfer ,Sensitivity (control systems) ,Electrodes ,ComputingMilieux_MISCELLANEOUS ,chemistry.chemical_classification ,Binding Sites ,010405 organic chemistry ,General Chemistry ,General Medicine ,021001 nanoscience & nanotechnology ,0104 chemical sciences ,Enzyme Activation ,Enzyme ,chemistry ,Protein film voltammetry ,0210 nano-technology - Abstract
International audience
- Published
- 2008
46. Complete activity profile of Clostridium acetobutylicum [FeFe]-hydrogenase and kinetic parameters for endogenous redox partners
- Author
-
Laurence Girbal, Marie Demuez, Philippe Soucaille, Olivier Guerrini, and Laurent Cournac
- Subjects
Clostridium acetobutylicum ,Hydrogenase ,Flavodoxin ,Microbiology ,Redox ,Clostridium ,Genetics ,Escherichia coli ,Molecular Biology ,Ferredoxin ,Hydrogen production ,biology ,Chemistry ,fungi ,equipment and supplies ,biology.organism_classification ,Kinetics ,Biochemistry ,biology.protein ,Ferredoxins ,Hydrogen–deuterium exchange ,Oxidation-Reduction ,Hydrogen - Abstract
In Clostridium acetobutylicum, [FeFe]-hydrogenase is involved in hydrogen production in vivo by transferring electrons from physiological electron donors, ferredoxin and flavodoxin, to protons. In this report, by modifications of the purification procedure, the specific activity of the enzyme has been improved and its complete catalytic profile in hydrogen evolution, hydrogen uptake, proton/deuterium exchange and para-H2/ortho-H2 conversion has been determined. The major ferredoxin expressed in the solvent-producing C. acetobutylicum cells was purified and identified as encoded by ORF CAC0303. Clostridium acetobutylicum recombinant holoflavodoxin CAC0587 was also purified. The kinetic parameters of C. acetobutylicum [FeFe]-hydrogenase for both physiological partners, ferredoxin CAC0303 and flavodoxin CAC0587, are reported for hydrogen uptake and hydrogen evolution activities.
- Published
- 2007
47. Characterization of two 2[4Fe4S] ferredoxins from Clostridium acetobutylicum
- Author
-
Laurence Girbal, Olivier Guerrini, Christophe Léger, Bruno Guigliarelli, Philippe Soucaille, and Bénédicte Burlat
- Subjects
inorganic chemicals ,Iron-Sulfur Proteins ,Clostridium acetobutylicum ,Hydrogenase ,Stereochemistry ,Molecular Sequence Data ,Applied Microbiology and Biotechnology ,Microbiology ,Redox ,law.invention ,Electron Transport ,Electron transfer ,law ,Escherichia coli ,Amino Acid Sequence ,Cloning, Molecular ,Electron paramagnetic resonance ,Ferredoxin ,biology ,Chemistry ,Electron Spin Resonance Spectroscopy ,General Medicine ,Sequence Analysis, DNA ,biology.organism_classification ,Electron transport chain ,Biochemistry ,Ferredoxins ,Heterologous expression ,Oxidation-Reduction - Abstract
In vivo hydrogen production in Clostridium acetobutylicum involves electron transfer between ferredoxin and [FeFe]-hydrogenase. Five C. acetobutylicum open reading frames were annotated as coding for putative ferredoxins. We focused our biophysical and biochemical investigations on CAC0303 and CAC3527, which possess the sequence signature and length of classical 2[4Fe4S] clostridial ferredoxins but differ significantly in theoretical pI. After cloning, heterologous expression in E. coli followed by in vitro Fe-S incorporation and purification, CAC0303 was shown to have a regular electron paramagnetic resonance (EPR) signal for a classical 2[4Fe4S] clostridial ferredoxin, while CAC3527 displayed an unusual EPR signal and a quite low reduction potential. Both ferredoxins were reduced in vitro by C. acetobutylicum [FeFe]-hydrogenase, but the CAC3527 reduction rate was 10-fold lower than that of CAC0303. These results are consistent with the efficiency of intermolecular electron transfer being dictated by the redox thermodynamics, the contribution of the ferredoxin global charge being only minor. The physiological function of CAC3527 is discussed.
- Published
- 2007
48. Evolution of a Saccharomyces cerevisiae metabolic pathway in Escherichia coli
- Author
-
Philippe Soucaille, Nynne M. Forchhammer, Isabelle Meynial Salles, Laurence Girbal, and Christian Croux
- Subjects
Glycerol ,Saccharomyces cerevisiae ,Bioengineering ,Dehydrogenase ,Chemostat ,medicine.disease_cause ,Applied Microbiology and Biotechnology ,Evolution, Molecular ,chemistry.chemical_compound ,medicine ,Escherichia coli ,Glycerol-3-Phosphate Dehydrogenase (NAD+) ,biology ,Genetic Variation ,Metabolism ,biology.organism_classification ,Fusion protein ,Adaptation, Physiological ,Biological Evolution ,Phosphoric Monoester Hydrolases ,Metabolic pathway ,chemistry ,Biochemistry ,Genetic Engineering ,Biotechnology ,Signal Transduction - Abstract
The Saccharomyces cerevisiae glycerol pathway (GPD1 and GPP2) was evolved in vivo in Escherichia coli. The central metabolism of E. coli was engineered to link glucose consumption and glycerol production. The engineered strain was evolved in a chemostat culture and a high glycerol producer was rapidly obtained. The evolution of the strain was associated to a deletion between GPD1 and GPP2, resulting in the production of a fusion protein with both glycerol-3-P dehydrogenase and glycerol-3-P phosphatase activities. The higher efficiency of the fusion protein was due to partial glycerol-3-P channeling between the two active sites. The evolved strain produces glycerol from glucose at high yield, concentration and productivity.
- Published
- 2006
49. Homologous and Heterologous Overexpression in Clostridium acetobutylicum and Characterization of Purified Clostridial and Algal Fe-Only Hydrogenases with High Specific Activities
- Author
-
Laurence Girbal, Christian Croux, Isabelle Meynial-Salles, John W. Peters, Philippe Soucaille, Martin Winkler, Gregory von Abendroth, Paul M. C. Benton, and Thomas Happe
- Subjects
Iron-Sulfur Proteins ,Clostridium acetobutylicum ,Hydrogenase ,Ecology ,biology ,Electron Spin Resonance Spectroscopy ,Heterologous ,Chlamydomonas reinhardtii ,biology.organism_classification ,Applied Microbiology and Biotechnology ,Recombinant Proteins ,Biochemistry ,Hyda ,Chlorophyta ,Specific activity ,Clostridiaceae ,Enzymology and Protein Engineering ,Bacteria ,Food Science ,Biotechnology - Abstract
Clostridium acetobutylicum ATCC 824 was selected for the homologous overexpression of its Fe-only hydrogenase and for the heterologous expressions of the Chlamydomonas reinhardtii and Scenedesmus obliquus HydA1 Fe-only hydrogenases. The three Strep tag II-tagged Fe-only hydrogenases were isolated with high specific activities by two-step column chromatography. The purified algal hydrogenases evolve hydrogen with rates of around 700 μmol H 2 min −1 mg −1 , while HydA from C. acetobutylicum (HydA Ca ) shows the highest activity (5,522 μmol H 2 min −1 mg −1 ) in the direction of hydrogen uptake. Further, kinetic parameters and substrate specificity were reported. An electron paramagnetic resonance (EPR) analysis of the thionin-oxidized HydA Ca protein indicates a characteristic rhombic EPR signal that is typical for the oxidized H cluster of Fe-only hydrogenases.
- Published
- 2005
50. Insight into the mechanism of the B12-independent glycerol dehydratase from Clostridium butyricum: preliminary biochemical and structural characterization
- Author
-
Laurence Girbal, Christian Croux, Céline Raynaud, William N. Lanzilotta, Jessica R. O’Brien, and Philippe Soucaille
- Subjects
Glycerol ,Propanediol Dehydratase ,Molecular Sequence Data ,Glycerol dehydratase ,Crystallography, X-Ray ,Biochemistry ,Substrate Specificity ,Enzyme activator ,Structure-Activity Relationship ,Enzyme Reactivators ,Bacterial Proteins ,Acetyltransferases ,Structure–activity relationship ,Amino Acid Sequence ,Binding site ,Peptide sequence ,Clostridium butyricum ,Hydro-Lyases ,chemistry.chemical_classification ,Clostridium ,Binding Sites ,biology ,Chemistry ,biology.organism_classification ,Lyase ,Propylene Glycol ,Culture Media ,Enzyme Activation ,Vitamin B 12 ,Enzyme ,Crystallization - Abstract
The molecular characterization of a B12-independent glycerol dehydratase from Clostridium butyricum has recently been reported [Raynaud, C., et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 5010-5015]. In this work, we have further characterized this system by biochemical and crystallographic methods. Both the glycerol dehydratase (GD) and the GD-activating enzyme (GD-AE) could be purified to homogeneity under aerobic conditions. In this form, both the GD and GD-AE were inactive. A reconstitution procedure, similar to what has been reported for pyruvate formate lyase activating enzyme (PFL-AE), was employed to reconstitute the activity of the GD-AE. Subsequently, the reconstituted GD-AE could be used to reactivate the GD under strictly anaerobic conditions. We also report here the crystal structure of the inactive GD in the native (2.5 A resolution, Rcryst = 17%, Rfree = 20%), glycerol-bound (1.8 A resolution, Rcryst = 21%, Rfree = 24%), and 1,2-propanediol-bound (2.4 A resolution, Rcryst = 20%, Rfree = 24%) forms. The overall fold of the GD monomer was similar to what has been observed for pyruvate formate lyase (PFL) and anaerobic ribonucleotide reductase (ARNR), consisting of a 10-stranded beta/alpha barrel motif. Clear density was observed for both substrates, and a mechanism for the dehydration reaction is presented. This mechanism clearly supports a concerted pathway for migration of the OH group through a cyclic transition state that is stabilized by partial protonation of the migrating OH group. Finally, despite poor alignment (rmsd approximately 6.8 A) of the 10 core strands that comprise the barrel structure of the GD and PFL, the C-terminal domains of both proteins align well (rmsd approximately 0.7 A) and have structural properties consistent with this being the docking site for the activating enzyme. A single point mutation within this domain, at a strictly conserved arginine residue (R782K) in the GD, resulted in formation of a tight protein-protein complex between the GD and the GD-AE in vivo, thereby supporting this hypothesis.
- Published
- 2004
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