48 results on '"Maria Lluch‐Senar"'
Search Results
2. Engineered live bacteria suppress Pseudomonas aeruginosa infection in mouse lung and dissolve endotracheal-tube biofilms
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Rocco Mazzolini, Irene Rodríguez-Arce, Laia Fernández-Barat, Carlos Piñero-Lambea, Victoria Garrido, Agustín Rebollada-Merino, Anna Motos, Antoni Torres, Maria Jesús Grilló, Luis Serrano, and Maria Lluch-Senar
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Biologia sintètica ,Biotecnología ,Biomedical Engineering ,Molecular Medicine ,Bioengineering ,Biología sintética ,Applied Microbiology and Biotechnology ,Biotecnologia ,Synthetic biology ,Biotechnology - Abstract
Data de publicació electrònica: 19-01-2023 Engineered live bacteria could provide a new modality for treating lung infections, a major cause of mortality worldwide. In the present study, we engineered a genome-reduced human lung bacterium, Mycoplasma pneumoniae, to treat ventilator-associated pneumonia, a disease with high hospital mortality when associated with Pseudomonas aeruginosa biofilms. After validating the biosafety of an attenuated M. pneumoniae chassis in mice, we introduced four transgenes into the chromosome by transposition to implement bactericidal and biofilm degradation activities. We show that this engineered strain has high efficacy against an acute P. aeruginosa lung infection in a mouse model. In addition, we demonstrated that the engineered strain could dissolve biofilms formed in endotracheal tubes of patients with ventilator-associated pneumonia and be combined with antibiotics targeting the peptidoglycan layer to increase efficacy against Gram-positive and Gram-negative bacteria. We expect our M. pneumoniae-engineered strain to be able to treat biofilm-associated infections in the respiratory tract. This work was supported by the European Research Council under the European Union’s Horizon 2020 research and innovation program, under grant agreement no. 670216 (MYCOCHASSIS). We thank the Spanish Ministry of Economy, Industry and Competitiveness to the EMBL partnership, the Centro de Excelencia Severo Ochoa and the CERCA Program from the Generalitat de Catalunya, the European Union’s Horizon 2020 Research and Innovation Programme, grant no. 634942 (MycoSynVac), La Caixa Health (HR18-00058), CB 06/06/0028/CIBER de enfermedades respiratorias-Ciberes and ICREA Academy/Institució Catalana de Recerca i Estudis Avançats, 2.603/IDIBAPS, SGR/Generalitat de Catalunya for their support. M.L.-S. thanks the funder Instituto de Salud Carlos III (ISCIII, Acción Estratégica en Salud 2016, FEDER project, reference CP16/00094) for support of the research of this work. We also thank the staff of the CRG/UPF Proteomics Unit, which is part of the Spanish Infrastructure for Omics Technologies unit and a member of the ProteoRed PRB3 consortium, supported by grant no. PT17/0019 of the PE I+D+i 2013–2016 from the ISCIII and European Regional Development Fund.
- Published
- 2023
3. Bacterial expression of a designed single‐chain <scp>IL</scp> ‐10 prevents severe lung inflammation
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Ariadna Montero‐Blay, Javier Delgado Blanco, Irene Rodriguez‐Arce, Claire Lastrucci, Carlos Piñero‐Lambea, Maria Lluch‐Senar, and Luis Serrano
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Mycoplasma ,Computational Theory and Mathematics ,General Immunology and Microbiology ,Applied Mathematics ,Live biotherapeutics ,Protein engineering ,Interleukin ,Infection ,General Agricultural and Biological Sciences ,General Biochemistry, Genetics and Molecular Biology ,Information Systems - Abstract
Interleukin-10 (IL-10) is an anti-inflammatory cytokine that is active as a swapped domain dimer and is used in bacterial therapy of gut inflammation. IL-10 can be used as treatment of a wide range of pulmonary diseases. Here we have developed a non-pathogenic chassis (CV8) of the human lung bacterium Mycoplasma pneumoniae (MPN) to treat lung diseases. We find that IL-10 expression by MPN has a limited impact on the lung inflammatory response in mice. To solve these issues, we rationally designed a single-chain IL-10 (SC-IL10) with or without surface mutations, using our protein design software (ModelX and FoldX). As compared to the IL-10 WT, the designed SC-IL10 molecules increase the effective expression in MPN four-fold, and the activity in mouse and human cell lines between 10 and 60 times, depending on the cell line. The SC-IL10 molecules expressed in the mouse lung by CV8 in vivo have a powerful anti-inflammatory effect on Pseudomonas aeruginosa lung infection. This rational design strategy could be used to other molecules with immunomodulatory properties used in bacterial therapy. This work has been supported by the European Research Council (ERC) grant agreement 670216 (MYCOCHASSIS). We also acknowledge the support of the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) to the EMBL partnership, the Centro de Excelencia Severo Ochoa, the CERCA Program from the Generalitat de Catalunya, the European Union's Horizon 2020 research and innovation programme under grant agreement 634942 (MycoSynVac). ML-S acknowledges the support of the FEDER project from Instituto Carlos III (ISCIII, Acción Estratégica en Salud 2016; reference CP16/00094). We also acknowledge the staff of CRG/UPF Proteomics Unit who is part of the Spanish Infrastructure for Omics Technologies (ICTS OmicsTech) unit and is a member of the ProteoRed PRB3 consortium which is supported by grant PT17/0019 of the PE I + D + i 2013–2016 from the Instituto de Salud Carlos III (ISCIII) and ERDF.
- Published
- 2023
4. Exploring the adaptability and robustness of the central carbon metabolism of Mycoplasma pneumoniae
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Niels A. Zondervan, Eva Yus, Daniel C. Sévin, Sira Martinez, Carolina Gallo, Peter J. Schaap, Maria Lluch-Senar, Luis Serrano, Vitor A. P. Martins dos Santos, and Maria Suarez-Diez
- Abstract
In this study we explored the adaptability and robustness of glycolysis and pyruvate metabolism of Mycoplasma pneumoniae (MPN). We used a dual approach, we analysed metabolomics data collected for a large number of OE and KO mutants and perturbation samples. Furthermore, we trained a dynamic model of central carbon metabolism and tested the model’s capacity to predict these mutants and perturbation samples as well as identify key controlling factors in central carbon metabolism. Our analysis of metabolite data as well as our model analysis indicate MPN metabolism is inherently robust against perturbations due to its network structure. Two key control hubs of central carbon metabolism were identified.
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- 2022
5. MycoWiki: Functional annotation of the minimal model organism Mycoplasma pneumoniae
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Christoph Elfmann, Bingyao Zhu, Tiago Pedreira, Ben Hoßbach, Maria Lluch-Senar, Luis Serrano, and Jörg Stülke
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Database ,Microbiology (medical) ,MycoWiki ,Systems biology ,Essential genes ,Microbiology ,Genome annotation - Abstract
The human pathogen Mycoplasma pneumoniae is viable independently from host cells or organisms, despite its strongly reduced genome with only about 700 protein-coding genes. The investigation of M. pneumoniae can therefore help to obtain general insights concerning the basic requirements for cellular life. Accordingly, M. pneumoniae has become a model organism for systems biology in the past decade. To support the investigation of the components of this minimal bacterium, we have generated the database MycoWiki. (http://mycowiki.uni-goettingen.de) MycoWiki organizes data under a relational database and provides access to curated and state-of-the-art information on the genes and proteins of M. pneumoniae. Interestingly, M. pneumoniae has undergone an evolution that resulted in the limited similarity of many proteins to proteins of model organisms. To facilitate the analysis of the functions of M. pneumoniae proteins, we have integrated structure predictions from the AlphaFold Protein Structure Database for most proteins, structural information resulting from in vivo cross-linking, and protein-protein interactions based on a global in vivo study. MycoWiki is an important tool for the systems and synthetic biology community that will support the comprehensive understanding of a minimal organism and the functional annotation of so far uncharacterized proteins.
- Published
- 2022
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6. SURE editing: combining oligo-recombineering and programmable insertion/deletion of selection markers to efficiently edit the Mycoplasma pneumoniae genome
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Carlos Piñero-Lambea, Eva Garcia-Ramallo, Samuel Miravet-Verde, Raul Burgos, Margherita Scarpa, Luis Serrano, and Maria Lluch-Senar
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Biologia sintètica ,Assembly Cloning ,Synthetic biology and assembly cloning ,Recombinación ,Genetics ,Clonación de ensamblaje ,Synthetic Biology ,Biología sintética ,Recombinació ,Clonació de muntatges ,Recombination - Abstract
The development of advanced genetic tools is boosting microbial engineering which can potentially tackle wide-ranging challenges currently faced by our society. Here we present SURE editing, a multi-recombinase engineering rationale combining oligonucleotide recombineering with the selective capacity of antibiotic resistance via transient insertion of selector plasmids. We test this method in Mycoplasma pneumoniae, a bacterium with a very inefficient native recombination machinery. Using SURE editing, we can seamlessly generate, in a single step, a wide variety of genome modifications at high efficiencies, including the largest possible deletion of this genome (30 Kb) and the targeted complementation of essential genes in the deletion of a region of interest. Additional steps can be taken to remove the selector plasmid from the edited area, to obtain markerless or even scarless edits. Of note, SURE editing is compatible with different site-specific recombinases for mediating transient plasmid integration. This battery of selector plasmids can be used to select different edits, regardless of the target sequence, which significantly reduces the cloning load associated to genome engineering projects. Given the proven functionality in several microorganisms of the machinery behind the SURE editing logic, this method is likely to represent a valuable advance for the synthetic biology field. European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program [670216 (MYCOCHASSIS)]; Spanish Ministry of Economy, Industry and Competitiveness (MEIC) (to the EMBL partnership); Centro de Excelencia Severo Ochoa; CERCA Program from the Generalitat de Catalunya; European Union's Horizon 2020 Research; Innovation Program [634942 (MycoSynVac)]; LaCaixa Fundation [Livetherapeutics HR18-00058]; C.P.-L. acknowledges the support of ‘Programa Torres Quevedo’ grant [PTQ2020-011048] funded by MCIN/AEI/10.13039/501100011033; European Union ‘NextGenerationEU/PRTR’; M.L.-S. acknowledges the support from FEDER project from Instituto Carlos III (ISCIII, Acción Estratégica en Salud 2016) [CP16/00094].
- Published
- 2022
7. LoxTnSeq: random transposon insertions combined with cre/lox recombination and counterselection to generate large random genome reductions
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Luis Serrano, Maria Lluch-Senar, Daniel Shaw, Samuel Miravet-Verde, and Carlos Pinero
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Transposable element ,Mutant ,Material genético ,Cre recombinase ,Bioengineering ,Computational biology ,Biology ,Applied Microbiology and Biotechnology ,Biochemistry ,Genome ,Deep sequencing ,03 medical and health sciences ,Synthetic biology ,Material genètic ,Genetic material ,Gene ,Research Articles ,Genoma ,030304 developmental biology ,Recombination, Genetic ,Biologia sintètica ,0303 health sciences ,Integrases ,030306 microbiology ,Genomics ,Mutagenesis, Insertional ,Genòmica ,DNA Transposable Elements ,Transposon mutagenesis ,Cre-Lox recombination ,Biología sintética ,Genètica ,TP248.13-248.65 ,Research Article ,Biotechnology - Abstract
The removal of unwanted genetic material is a key aspect in many synthetic biology efforts and often requires preliminary knowledge of which genomic regions are dispensable. Typically, these efforts are guided by transposon mutagenesis studies, coupled to deepsequencing (TnSeq) to identify insertion points and gene essentiality. However, epistatic interactions can cause unforeseen changes in essentiality after the deletion of a gene, leading to the redundancy of these essentiality maps. Here, we present LoxTnSeq, a new methodology to generate and catalogue libraries of genome reduction mutants. LoxTnSeq combines random integration of lox sites by transposon mutagenesis, and the generation of mutants via Cre recombinase, catalogued via deep sequencing. When LoxTnSeq was applied to the naturally genome reduced bacterium Mycoplasma pneumoniae, we obtained a mutant pool containing 285 unique deletions. These deletions spanned from > 50 bp to 28 Kb, which represents 21% of the total genome. LoxTnSeq also highlighted large regions of non-essential genes that could be removed simultaneously, and other non-essential regions that could not, providing a guide for future genome reductions., Microbial Biotechnology, 14 (6), ISSN:1751-7915, ISSN:1751-7907
- Published
- 2021
8. CReasPy-Cloning: A Method for Simultaneous Cloning and Engineering of Megabase-Sized Genomes in Yeast Using the CRISPR-Cas9 System
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Yonathan Arfi, Vincent Talenton, Pascal Sirand-Pugnet, Franck Salin, Estelle Ruiz, Maria Lluch-Senar, Gabrielle Guesdon, Carole Lartigue, Marie-Pierre Dubrana, Biologie du fruit et pathologie (BFP), Université Bordeaux Segalen - Bordeaux 2-Institut National de la Recherche Agronomique (INRA)-Université Sciences et Technologies - Bordeaux 1, Barcelona Institute of Science and Technology (BIST), Biodiversité, Gènes & Communautés (BioGeCo), and Institut National de la Recherche Agronomique (INRA)-Université de Bordeaux (UB)
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DNA, Bacterial ,0106 biological sciences ,Saccharomyces cerevisiae ,Biomedical Engineering ,Genome cloning ,Computational biology ,Biology ,010402 general chemistry ,01 natural sciences ,Biochemistry, Genetics and Molecular Biology (miscellaneous) ,Genome ,Genome engineering ,03 medical and health sciences ,Mycoplasma ,Genome editing ,CRISPR-Associated Protein 9 ,010608 biotechnology ,CRISPR ,Cloning, Molecular ,DNA Cleavage ,Gene ,030304 developmental biology ,Gene Editing ,Genome transplantation ,0303 health sciences ,Cas9 ,Circular bacterial chromosome ,General Medicine ,Chromosomes, Bacterial ,biology.organism_classification ,Mycoplasma pneumoniae ,0104 chemical sciences ,Genetic Loci ,CRISPR-Cas Systems ,CRISPR-Cas9 ,Genetic Engineering ,Homologous recombination ,[SDV.MHEP]Life Sciences [q-bio]/Human health and pathology ,Genome, Bacterial ,Plasmids ,RNA, Guide, Kinetoplastida - Abstract
Over the past decade, a new strategy was developed to bypass the difficulties to genetically engineer some microbial species by transferring (or "cloning") their genome into another organism that is amenable to efficient genetic modifications and therefore acts as a living workbench. As such, the yeast Saccharomyces cerevisiae has been used to clone and engineer genomes from viruses, bacteria, and algae. The cloning step requires the insertion of yeast genetic elements in the genome of interest, in order to drive its replication and maintenance as an artificial chromosome in the host cell. Current methods used to introduce these genetic elements are still unsatisfactory, due either to their random nature (transposon) or the requirement for unique restriction sites at specific positions (TAR cloning). Here we describe the CReasPy-cloning, a new method that combines both the ability of Cas9 to cleave DNA at a user-specified locus and the yeast's highly efficient homologous recombination to simultaneously clone and engineer a bacterial chromosome in yeast. Using the 0.816 Mbp genome of Mycoplasma pneumoniae as a proof of concept, we demonstrate that our method can be used to introduce the yeast genetic element at any location in the bacterial chromosome while simultaneously deleting various genes or group of genes. We also show that CReasPy-cloning can be used to edit up to three independent genomic loci at the same time with an efficiency high enough to warrant the screening of a small (
- Published
- 2019
9. Engineering a genome-reduced bacterium to eliminate Staphylococcus aureus biofilms in vivo
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Eva Garcia-Ramallo, Carolina Gallo, Luis Serrano, María Collantes, Marc Weber, Victoria Garrido, Carlos Piñero-Lambea, Bernhard Paetzold, Irene Rodríguez-Arce, Tony Ferrar, Maria Lluch-Senar, Iván Peñuelas, María Jesús Grilló, European Research Council, European Commission, Generalitat de Catalunya, Ministerio de Economía, Industria y Competitividad (España), Fundación 'la Caixa', and Instituto de Salud Carlos III
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Medicine (General) ,Biopelícula ,medicine.disease_cause ,Bacteris ,biofilm ,Mice ,Mycoplasma ,Micoplasmosis ,News & Views ,Biology (General) ,Acterial therapy ,Pathogen ,Biologia sintètica ,bacterial therapy ,0303 health sciences ,Genome ,biology ,Biofilm ,Applied Mathematics ,in vivo ,Articles ,Microbiology, Virology & Host Pathogen Interaction ,Anti-Bacterial Agents ,Computational Theory and Mathematics ,Staphylococcus aureus ,General Agricultural and Biological Sciences ,Bacterias ,Information Systems ,616.9 ,Virulence Factors ,QH301-705.5 ,Microbial Sensitivity Tests ,Home -- Malalties ,General Biochemistry, Genetics and Molecular Biology ,Article ,Microbiology ,03 medical and health sciences ,R5-920 ,In vivo ,medicine ,Animals ,Humans ,Synthetic biology ,Genoma ,030304 developmental biology ,General Immunology and Microbiology ,Bacteria ,030306 microbiology ,Biotechnology & Synthetic Biology ,Pathogenic bacteria ,Bacteria Present ,biology.organism_classification ,Terapia bacteriana ,Mycoplasma pneumoniae ,Genòmica ,Biofilms ,Teràpia bacteriana ,Micoplasma ,Bacteris -- Tractament ,Biología sintética ,synthetic biology ,Ex vivo - Abstract
Bacteria present a promising delivery system for treating human diseases. Here, we engineered the genome-reduced human lung pathogen Mycoplasma pneumoniae as a live biotherapeutic to treat biofilm-associated bacterial infections. This strain has a unique genetic code, which hinders gene transfer to most other bacterial genera, and it lacks a cell wall, which allows it to express proteins that target peptidoglycans of pathogenic bacteria. We first determined that removal of the pathogenic factors fully attenuated the chassis strain in vivo. We then designed synthetic promoters and identified an endogenous peptide signal sequence that, when fused to heterologous proteins, promotes efficient secretion. Based on this, we equipped the chassis strain with a genetic platform designed to secrete antibiofilm and bactericidal enzymes, resulting in a strain capable of dissolving Staphylococcus aureus biofilms preformed on catheters in vitro, ex vivo, and in vivo. To our knowledge, this is the first engineered genome-reduced bacterium that can fight against clinically relevant biofilm-associated bacterial infections., This work has been supported by the European Research Council (ERC) underthe European Union’s Horizon2020research and innovation program, undergrant agreement 670216(MYCOCHASSIS). We also acknowledge the support ofthe Spanish Ministry of Economy, Industry and Competitiveness (MEIC) to theEMBL partnership, the Centro de Excelencia Severo Ochoa, the CERCA Programfrom the Generalitat de Catalunya, the European Union’s Horizon2020research and innovation program under grant agreement 634942 (Myco-SynVac), and the LaCaixa Fundation grant (Livetherapeutics HR18-00058).M.L.-S. acknowledges the support from FEDER project from Instituto Carlos III(ISCIII, Accion Estrategica en Salud2016) (reference CP16/00094). We alsoacknowledge the staff of CRG/UPF Proteomics Unit, which is part of the Span-ish Infrastructure for Omics Technologies (ICTS OmicsTech) unit is a memberof the ProteoRed PRB3consortium, which is supported by grant PT17/0019ofthe PE I+D+i2013-2016from the Instituto de Salud Carlos III (ISCIII) and ERDF. We also acknowledge Margarita Ecay for her assistance in the PETimages analysis and Samuel Miravet-Verde for the bioinformatic support.
- Published
- 2021
10. Functional characterization of the cell division gene cluster of the wall-less bacterium Mycoplasma genitalium
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Carlos Martínez-Torró, Sergi Torres-Puig, Marina Marcos-Silva, Marta Huguet-Ramón, Carmen Muñoz-Navarro, Maria Lluch-Senar, Luis Serrano, Enrique Querol, Jaume Piñol, and Oscar Q. Pich
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Microbiology (medical) ,cell division ,Cell division ,FtsZ localization ,Mutant ,mycoplasmas ,single cell analysis ,Mycoplasma genitalium ,macromolecular substances ,Cell cycle ,Bacteris ,physiological processes ,Microbiology ,Single-cell analysis ,Stable isotope labeling by amino acids in cell culture ,Micoplasmes ,Gene cluster ,FtsZ ,Gene ,Original Research ,biology ,Single cell analysis ,regulation ,biology.organism_classification ,QR1-502 ,Cell biology ,Mycoplasmas ,biology.protein ,bacteria ,cell cycle ,biological phenomena, cell phenomena, and immunity ,Genètica ,Regulation - Abstract
It is well-established that FtsZ drives peptidoglycan synthesis at the division site in walled bacteria. However, the function and conservation of FtsZ in wall-less prokaryotes such as mycoplasmas are less clear. In the genome-reduced bacterium Mycoplasma genitalium, the cell division gene cluster is limited to four genes: mraZ, mraW, MG_223, and ftsZ. In a previous study, we demonstrated that ftsZ was dispensable for growth of M. genitalium under laboratory culture conditions. Herein, we show that the entire cell division gene cluster of M. genitalium is non-essential for growth in vitro. Our analyses indicate that loss of the mraZ gene alone is more detrimental for growth of M. genitalium than deletion of ftsZ or the entire cell division gene cluster. Transcriptional analysis revealed a marked upregulation of ftsZ in the mraZ mutant. Stable isotope labeling by amino acids in cell culture (SILAC)-based proteomics confirmed the overexpression of FtsZ in MraZ-deprived cells. Of note, we found that ftsZ expression was upregulated in non-adherent cells of M. genitalium, which arise spontaneously at relatively high rates. Single cell analysis using fluorescent markers showed that FtsZ localization varied throughout the cell cycle of M. genitalium in a coordinated manner with the chromosome and the terminal organelle (TMO). In addition, our results indicate a possible role for the RNA methyltransferase MraW in the regulation of FtsZ expression at the post-transcriptional level. Altogether, this study provides an extensive characterization of the cell division gene cluster of M. genitalium and demonstrates the existence of regulatory elements controlling FtsZ expression at the temporal and spatial level in mycoplasmas.
- Published
- 2021
11. Lox’d in translation: contradictions in the nomenclature surrounding common lox-site mutants and their implications in experiments
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Luis Serrano, Daniel Shaw, and Maria Lluch-Senar
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DNA repair ,Mutant ,Computational biology ,Biology ,Microbiology ,DNA sequencing ,Insert (molecular biology) ,03 medical and health sciences ,chemistry.chemical_compound ,Terminology as Topic ,Recombinase ,Enginyeria genètica ,Seqüència de nucleòtids ,030304 developmental biology ,Recombination, Genetic ,0303 health sciences ,Bacteria ,Base Sequence ,Llocs loxP ,030306 microbiology ,Mechanism (biology) ,Molecular Sequence Annotation ,Translation (biology) ,Mycoplasma pneumoniae ,chemistry ,Mutation ,Genetic Engineering ,DNA - Abstract
The Cre-Lox system is a highly versatile and powerful DNA recombinase mechanism, mainly used in genetic engineering to insert or remove desired DNA sequences. It is widely utilized across multiple fields of biology, with applications ranging from plants, to mammals, to microbes. A key feature of this system is its ability to allow recombination between mutant lox sites. Two of the most commonly used mutant sites are named lox66 and lox71, which recombine to create a functionally inactive double mutant lox72 site. However, a large portion of the published literature has incorrectly annotated these mutant lox sites, which in turn can lead to difficulties in replication of methods, design of proper vectors and confusion over the proper nomenclature. Here, we demonstrate common errors in annotations, the impacts they can have on experimental viability, and a standardized naming convention. We also show an example of how this incorrect annotation can induce toxic effects in bacteria that lack optimal DNA repair systems, exemplified by Mycoplasma pneumoniae. This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement 634942 (MycoSynVac) and was also financed by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme, under grant agreement 670216 (MYCOCHASSIS). We also acknowledge support of the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) to the EMBL partnership, the Centro de Excelencia Severo Ochoa and the CERCA Programme/Generalitat de Catalunya
- Published
- 2021
12. Protein quality control and regulated proteolysis in the genome‐reduced organism Mycoplasma pneumoniae
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Luis Serrano, Marc Weber, Maria Lluch-Senar, Raul Burgos, and Sira Martínez
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Medicine (General) ,Protein Folding ,proteomic approach ,Transcription, Genetic ,regulated proteolysis ,Protein aggregation ,Substrate Specificity ,0302 clinical medicine ,ATP‐dependent protease ,Biology (General) ,Cellular localization ,0303 health sciences ,Applied Mathematics ,Articles ,Microbiology, Virology & Host Pathogen Interaction ,Cell biology ,Phenotype ,Computational Theory and Mathematics ,Proteome ,protein degradation ,Protein folding ,General Agricultural and Biological Sciences ,Information Systems ,Quality Control ,Proteases ,QH301-705.5 ,Biology ,Protein degradation ,Models, Biological ,General Biochemistry, Genetics and Molecular Biology ,Article ,03 medical and health sciences ,R5-920 ,Bacterial Proteins ,Amino Acid Sequence ,Shotgun proteomics ,mycoplasma ,030304 developmental biology ,General Immunology and Microbiology ,Post-translational Modifications, Proteolysis & Proteomics ,Reproducibility of Results ,Gene Expression Regulation, Bacterial ,Proteòlisi ,Mycoplasma pneumoniae ,Genòmica ,Membrane protein ,Mutation ,Proteolysis ,Proteïnes ,030217 neurology & neurosurgery ,Genome, Bacterial ,Peptide Hydrolases - Abstract
Protein degradation is a crucial cellular process in all‐living systems. Here, using Mycoplasma pneumoniae as a model organism, we defined the minimal protein degradation machinery required to maintain proteome homeostasis. Then, we conditionally depleted the two essential ATP‐dependent proteases. Whereas depletion of Lon results in increased protein aggregation and decreased heat tolerance, FtsH depletion induces cell membrane damage, suggesting a role in quality control of membrane proteins. An integrative comparative study combining shotgun proteomics and RNA‐seq revealed 62 and 34 candidate substrates, respectively. Cellular localization of substrates and epistasis studies supports separate functions for Lon and FtsH. Protein half‐life measurements also suggest a role for Lon‐modulated protein decay. Lon plays a key role in protein quality control, degrading misfolded proteins and those not assembled into functional complexes. We propose that regulating complex assembly and degradation of isolated proteins is a mechanism that coordinates important cellular processes like cell division. Finally, by considering the entire set of proteases and chaperones, we provide a fully integrated view of how a minimal cell regulates protein folding and degradation., A minimal protein degradation machinery required for maintaining proteome homeostasis is defined in the genome‐reduced bacterium M. pneumoniae. Genetic and high‐throughput analyses identify substrates and pathways regulated by degradation.
- Published
- 2020
13. The role of clonal communication and heterogeneity in breast cancer
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Ana Martín-Pardillos, Stefan Hümmer, Victor Sebastian, Roberto Piñeiro Cid, Luis Serrano, Samuel Miravet-Verde, Gemma Bande Vargas, Aitor Rodriguez-Casanova, Josep Castellví, Ana Bribián, Angel Diaz-Lagares, Santiago Ramón y Cajal, Pablo Hurtado Blanco, Pedro J. Guijarro, Laura López-Mascaraque, Ángeles Valls Chiva, Eva Bejar Serrano, Maria Lluch-Senar, Rafael López-López, Generalitat de Catalunya, Instituto de Salud Carlos III, and European Commission
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0301 basic medicine ,Cancer Research ,MDA-MB-231 ,Cell ,Gene Expression ,Apoptosis ,Cell Communication ,Metastasis ,Mice ,0302 clinical medicine ,Cell Movement ,Breast ,Zebrafish ,Cancer ,Tumor ,Communication ,lcsh:Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,3. Good health ,Cell biology ,medicine.anatomical_structure ,Oncology ,030220 oncology & carcinogenesis ,Cytokines ,Heterografts ,Female ,Clone (B-cell biology) ,Research Article ,Cell Survival ,Clone ,Mice, Nude ,Breast Neoplasms ,Biology ,lcsh:RC254-282 ,Genetic Heterogeneity ,03 medical and health sciences ,Cell Line, Tumor ,Genetics ,medicine ,Animals ,Humans ,Epigenetics ,Genetic heterogeneity ,medicine.disease ,Coculture Techniques ,Clone Cells ,Cooperation ,030104 developmental biology ,Cell culture ,Tumor progression ,Cancer cell ,DNA Transposable Elements ,Heterogeneity ,Neoplasm Transplantation - Abstract
[Background] Cancer is a rapidly evolving, multifactorial disease that accumulates numerous genetic and epigenetic alterations. This results in molecular and phenotypic heterogeneity within the tumor, the complexity of which is further amplified through specific interactions between cancer cells. We aimed to dissect the molecular mechanisms underlying the cooperation between different clones., [Methods] We produced clonal cell lines derived from the MDA-MB-231 breast cancer cell line, using the UbC-StarTrack system, which allowed tracking of multiple clones by color: GFP C3, mKO E10 and Sapphire D7. Characterization of these clones was performed by growth rate, cell metabolic activity, wound healing, invasion assays and genetic and epigenetic arrays. Tumorigenicity was tested by orthotopic and intravenous injections. Clonal cooperation was evaluated by medium complementation, co-culture and co-injection assays., [Results] Characterization of these clones in vitro revealed clear genetic and epigenetic differences that affected growth rate, cell metabolic activity, morphology and cytokine expression among cell lines. In vivo, all clonal cell lines were able to form tumors; however, injection of an equal mix of the different clones led to tumors with very few mKO E10 cells. Additionally, the mKO E10 clonal cell line showed a significant inability to form lung metastases. These results confirm that even in stable cell lines heterogeneity is present. In vitro, the complementation of growth medium with medium or exosomes from parental or clonal cell lines increased the growth rate of the other clones. Complementation assays, co-growth and co-injection of mKO E10 and GFP C3 clonal cell lines increased the efficiency of invasion and migration., [Conclusions] These findings support a model where interplay between clones confers aggressiveness, and which may allow identification of the factors involved in cellular communication that could play a role in clonal cooperation and thus represent new targets for preventing tumor progression., SRYC acknowledges support from Fondo de Investigaciones Sanitarias (FIS; PI17/02247 and PI14/01320), Centro de Investigación Biomédica en Red de Cáncer (CIBERONC; CB16/12/00363) and Generalitat de Catalunya (AGAUR; 2017 SGR 1799 and 2014 SGR 1131). AMP is funded by a FP7 Marie Sklodowska-Curie COFUND program under Grant Agreement n° 267128 (INCOMED program). ADL is funded by a “Juan Rodés” contract (JR17/00016) from ISCIII.
- Published
- 2020
14. A RAGE Based Strategy for the Genome Engineering of the Human Respiratory Pathogen
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Luis, Garcia-Morales, Estelle, Ruiz, Géraldine, Gourgues, Fabien, Rideau, Carlos, Piñero-Lambea, Maria, Lluch-Senar, Alain, Blanchard, and Carole, Lartigue
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DNA, Bacterial ,Gene Editing ,Virulence ,Virulence Factors ,Saccharomyces cerevisiae ,Mycoplasma pneumoniae ,Recombinases ,Escherichia coli ,Humans ,Chromosomes, Artificial ,Synthetic Biology ,Cloning, Molecular ,Genetic Engineering ,Genome, Bacterial - Abstract
Genome engineering of microorganisms has become a standard in microbial biotechnologies. Several efficient tools are available for the genetic manipulation of model bacteria such as
- Published
- 2020
15. Lox’d in translation: Contradictions in the nomenclature surrounding common lox site mutants and their implications in experiments
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Samuel Miravet-Verde, Daniel Shaw, Carlos Piñero-Lambea, Maria Lluch-Senar, and Luis Serrano
- Subjects
Synthetic biology ,Mutant ,Rational engineering ,Epistasis ,Cre recombinase ,Transposon mutagenesis ,Computational biology ,Biology ,Gene ,Genome - Abstract
The Cre-Lox system is a highly versatile and powerful DNA recombinase mechanism, mainly used in genetic engineering to insert or remove desired DNA sequences. It is widely utilised across multiple fields of biology, with applications ranging from plants, to mammals, to microbes. A key feature of this system is its ability to allow recombination between mutant lox sites, traditionally named lox66 and lox71, to create a functionally inactive double mutant lox72 site. However, a large portion of the published literature has incorrectly annotated these mutant lox sites, which in turn can lead to difficulties in replication of methods, design of proper vectors, and confusion over the proper nomenclature. Here, we demonstrate common errors in annotations, the impacts they can have on experimental viability, and a standardised naming convention. We also show an example of how this incorrect annotation can induce toxic effects in bacteria that lack optimal DNA repair systems, exemplified byMycoplasma pneumoniae.Data SummaryThe authors confirm all supporting data, code and protocols have been provided within the article or through supplementary data files.
- Published
- 2020
16. Characterization of different alginate lyases for dissolving Pseudomonas aeruginosa biofilms
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Tony Ferrar, Núria Blanco-Cabra, Luis Serrano, Bernhard Paetzold, Eduard Torrents, Rocco Mazzolini, and Maria Lluch-Senar
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0301 basic medicine ,Alginates ,medicine.drug_class ,030106 microbiology ,Antibiotics ,lcsh:Medicine ,Lyases ,Complex Mixtures ,medicine.disease_cause ,Article ,Substrate Specificity ,law.invention ,Applied microbiology ,03 medical and health sciences ,Bacterial Proteins ,Tandem Mass Spectrometry ,law ,Pseudomonas ,medicine ,Humans ,Pseudomonas Infections ,Sphingobacterium ,Cloning, Molecular ,lcsh:Science ,Sugar ,Dissolution ,chemistry.chemical_classification ,Multidisciplinary ,biology ,Pseudomonas aeruginosa ,Chemistry ,lcsh:R ,Biofilm ,Drug Synergism ,Sphingomonas ,biology.organism_classification ,Anti-Bacterial Agents ,3. Good health ,Biological Therapy ,Immune system ,030104 developmental biology ,Enzyme ,Biochemistry ,Biofilms ,Sistema immunitari ,Recombinant DNA ,lcsh:Q - Abstract
Aggregates of Pseudomonas aeruginosa form a protective barrier against antibiotics and the immune system. These barriers, known as biofilms, are associated with several infectious diseases. One of the main components of these biofilms is alginate, a homo- and hetero-polysaccharide that consists of β-D-mannuronate (M) and α-L-guluronate (G) units. Alginate lyases degrade this sugar and have been proposed as biotherapeutic agents to dissolve P. aeruginosa biofilms. However, there are contradictory reports in the literature regarding the efficacy of alginate lyases against biofilms and their synergistic effect with antibiotics. We found that most positive reports used a commercial crude extract from Flavobacterium multivorum as the alginate lyase source. By using anion exchange chromatography coupled to nano LC MS/MS, we identified two distinct enzymes in this extract, one has both polyM and polyG (polyM/G) degradation activities and it is similar in sequence to a broad-spectrum alginate lyase from Flavobacterium sp. S20 (Alg2A). The other enzyme has only polyG activity and it is similar in sequence to AlyA1 from Zobellia galactanivorans. By characterizing both of these enzymes together with three recombinant alginate lyases (a polyM, a polyG and a polyM/G), we showed that only enzymes with polyM/G activity such as Alg2A and A1-II' (alginate lyase from Sphingomonas sp.) are effective in dissolving biofilms. Furthermore, both activities are required to have a synergistic effect with antibiotics. We acknowledge support of the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program under agreement No 670216 (MYCOCHASSIS), the Spanish Ministry of Economy and Competitiveness and Fondo Europeo de Desarrollo Regional (MINECO-FEDER) (BIO2015-63557-R), ‘Centro de Excelencia Severo Ochoa 2013-2017’, FEDER project from Instituto Carlos III (ISCIII, Acción Estratégica en Salud 2016) (reference CP16/00094) and “Secretaria d’Universitats i Recerca del Departament d’Economia i Coneixement de la Generalitat de Catalunya / CERCA programme” (2014SGR678 and 2017SGR1079). The CRG/UPF Proteomics Unit is part of the “Plataforma de Recursos Biomoleculares y Bioinformáticos (ProteoRed)” supported by grant PT13/0001 of Instituto de Salud Carlos III from the Spanish Government.
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- 2020
17. Immunodominant proteins P1 and P40/P90 from human pathogen Mycoplasma pneumoniae
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Rosa Pérez-Luque, Paula Bierge, Maria Lluch-Senar, Oscar Q. Pich, Keiichi Namba, Isabel Sanfeliu, Tsuyoshi Kenri, Akihiro Kawamoto, Jesús Martín, Mateu Espasa, Rocco Mazzolini, Achilleas S. Frangakis, Keigo Shibayama, Ramiro Illanes, Ignacio Fita, Jaume Pinyol, Miguel Fernández-Huerta, U Matsumoto, Makoto Miyata, David Aparicio, Juliana Esperalba, David Vizarraga, Takayuki Kato, Shigetarou Mori, Margot P. Scheffer, Ministerio de Ciencia, Innovación y Universidades (España), Agencia Estatal de Investigación (España), Instituto de Salud Carlos III, and Osaka University
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0301 basic medicine ,Mycoplasma pneumoniae ,電子顕微鏡 ,Science ,030106 microbiology ,Immunology ,General Physics and Astronomy ,Human pathogen ,medicine.disease_cause ,Crystallography, X-Ray ,Microbiology ,General Biochemistry, Genetics and Molecular Biology ,Bacterial Adhesion ,Article ,Serology ,03 medical and health sciences ,chemistry.chemical_compound ,Protein Domains ,Pneumonia, Mycoplasma ,medicine ,Electron microscopy ,lcsh:Science ,Adhesins, Bacterial ,X-ray crystallography ,Infectivity ,Multidisciplinary ,biology ,Cryoelectron Microscopy ,hemic and immune systems ,General Chemistry ,マイコプラズマ・ニューモニエ ,medicine.disease ,eye diseases ,Sialic acid ,Bacterial adhesin ,030104 developmental biology ,chemistry ,Atypical pneumonia ,Polyclonal antibodies ,biology.protein ,lcsh:Q ,sense organs ,Structural biology - Abstract
Mycoplasma pneumoniae is a bacterial human pathogen that causes primary atypical pneumonia. M. pneumoniae motility and infectivity are mediated by the immunodominant proteins P1 and P40/P90, which form a transmembrane adhesion complex. Here we report the structure of P1, determined by X-ray crystallography and cryo-electron microscopy, and the X-ray structure of P40/P90. Contrary to what had been suggested, the binding site for sialic acid was found in P40/P90 and not in P1. Genetic and clinical variability concentrates on the N-terminal domain surfaces of P1 and P40/P90. Polyclonal antibodies generated against the mostly conserved C-terminal domain of P1 inhibited adhesion of M. pneumoniae, and serology assays with sera from infected patients were positive when tested against this C-terminal domain. P40/P90 also showed strong reactivity against human infected sera. The architectural elements determined for P1 and P40/P90 open new possibilities in vaccine development against M. pneumoniae infections., This work was supported by grants BFU2018-101265-B-100 (MINECO) to I.F., BIO2017-84166-R (MINECO) to J.P., and BES-2015-076104 (MINECO) to M.L.S., JSPS KAKENHI Grant Number JP25000013 to K.N., and a FEDER project from Instituto de Salud Carlos III (ISCIII, Acción Estratégica en Salud 2016). This work has also been funded by the Platform Project for Supporting Drug Discovery and Life Science Research (BINDS) from AMED under Grant Number JP19am0101117 to K.N. (support number 1282), by the Cyclic Innovation for Clinical Empowerment (CiCLE) from AMED under Grant Number JP17pc0101020 to K.N., and by JEOL YOKOGUSHI Research Alliance Laboratories of Osaka University to K.N. This work was supported by a Grant-in-Aid for Scientific Research on the Innovative Area “Harmonized Supramolecular Motility Machinery and Its Diversity” (MEXT KAKENH, JP24117002 to M.M., JP25117530 and JP15H01337 to T.K.), Grants-in-Aid for Scientific Research (B) and (A) (MEXT KAKENHI, JP24390107, JP17H01544), JST CREST (JPMJCR19S5), Osaka City University (OCU) Strategic Research Grant 2018 for top priority researches to M.M. D.A. acknowledges a María de Maeztu Unit of Excellence grant MDM-2014-0435.
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- 2020
18. Comparative Gene Essentiality across the Bacterial Domain
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Antonio Hermoso, Daniel Shaw, Luis Serrano, and Maria Lluch-Senar
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Comparative genomics ,0303 health sciences ,biology ,030306 microbiology ,Computational biology ,biology.organism_classification ,Genome ,Housekeeping gene ,Biological pathway ,03 medical and health sciences ,Transcription (biology) ,Gene ,Genome size ,Bacteria ,030304 developmental biology - Abstract
Comparative genomics among bacteria has been used to gain insight into the minimal number of conserved genes in biological pathways. Essentiality studies have provided information regarding which genes are non-dispensable (essential, E) for cell growth. Here, we integrated studies of gene conservation, essentiality and function, performed in 47 diverse bacterial species. We showed there is a modest positive correlation between genome size and number of essential genes. Interestingly, we observed a clear shift in the functions assigned to these essential genes as genome size increases. For instance, essential genes related to transcription and translation dominate small genomes. In contrast, in large genomes functions of essential genes are related with cellular processing and metabolism. Finally, and most intriguing, we found a group of genes that while being highly conserved are also typically non-essential. This suggests that some housekeeping genes confer a significant survival benefit in nature while being non-essential in vitro.
- Published
- 2020
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19. Mycoplasma pneumoniae genome editing based on oligo recombineering and Cas9-mediated counterselection
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Javier Delgado, Luis Serrano, Carlos Piñero-Lambea, Sira Martínez, Maria Lluch-Senar, and Eva Garcia-Ramallo
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0106 biological sciences ,Biomedical Engineering ,Computational biology ,Biology ,medicine.disease_cause ,01 natural sciences ,Biochemistry, Genetics and Molecular Biology (miscellaneous) ,Genome ,Recombineering ,03 medical and health sciences ,Synthetic biology ,Biopolymers ,Genome editing ,010608 biotechnology ,medicine ,Recombinase ,Genetics ,CRISPR ,030304 developmental biology ,0303 health sciences ,Bacteria ,Cas9 ,Sensors ,General Medicine ,Mycoplasma ,Genomics ,3. Good health ,Research Article - Abstract
Mycoplasma species share a set of features, such as lack of a cell wall, streamlined genomes, simplified metabolism, and the use of a deviant genetic code, that make them attractive approximations of what a chassis strain should ideally be. Among them, Mycoplasma pneumoniae arises as a candidate for synthetic biology projects, as it is one of the most deeply characterized bacteria. However, the historical paucity of tools for editing Mycoplasma genomes has precluded the establishment of M. pneumoniae as a suitable chassis strain. Here, we developed an oligonucleotide recombineering method for this strain based on GP35, a ssDNA recombinase originally encoded by a Bacillus subtilis-associated phage. GP35-mediated oligo recombineering is able to carry out point mutations in the M. pneumoniae genome with an efficiency as high as 2.7 × 10-2, outperforming oligo recombineering protocols developed for other bacteria. Gene deletions of different sizes showed a decreasing power trend between efficiency and the scale of the attempted edition. However, the editing rates for all modifications increased when CRISPR/Cas9 was used to counterselect nonedited cells. This allowed edited clones carrying chromosomal deletions of up to 1.8 kb to be recovered with little to no screening of survivor cells. We envision this technology as a major step toward the use of M. pneumoniae, and possibly other Mycoplasmas, as synthetic biology chassis strains. This project has received funding from the European Union’s Horizon 2020 research and innovation program under Grant 634942 (MycoSynVac) and was also financed by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program under Grant 670216 (MYCOCHASSIS) and the FEDER project from Instituto Carlos III (ISCIII, Acción Estratégica en Salud 2016) (reference CP16/00094). We also acknowledge support of the Spanish Ministry of Science and Innovation, to the EMBL partnership, the Centro de Excelencia Severo Ochoa, and the CERCA Programme/Generalitat de Catalunya. Finally, we would like to thank Dr. Sarah A. Head for critical manuscript revision.
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- 2020
20. A RAGE Based Strategy for the Genome Engineering of the Human Respiratory Pathogen Mycoplasma pneumoniae
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Carlos Piñero-Lambea, Luis Garcia-Morales, Estelle Ruiz, Carole Lartigue, Alain Blanchard, Géraldine Gourgues, Fabien Rideau, Maria Lluch-Senar, Biologie du fruit et pathologie (BFP), Université de Bordeaux (UB)-Institut National de Recherche pour l’Agriculture, l’Alimentation et l’Environnement (INRAE), Barcelona Institute of Science and Technology (BIST), European Union’s Horizon 2020 research and innovationprogram under Grant Agreement [No. 634942], and the European MiniCell project selected by ANR, in the frame of the ERASynBio second Joint Call for Transnational ResearchProjects [No. ANR-15-SYNB-0001-04], ANR-15-SYNB-0001,MiniCell,A model-driven approach to minimal cell engineering for medical therapy(2015), and European Project: 634942,H2020,H2020-LEIT-BIO-2014-1,MycoSynVac(2015)
- Subjects
[SDV]Life Sciences [q-bio] ,Biomedical Engineering ,Computational biology ,Human artificial chromosome ,Bacterial genome size ,Saccharomyces cerevisiae ,Biology ,Biochemistry, Genetics and Molecular Biology (miscellaneous) ,Genome ,transformation-associated recombination-cloning (TAR-cloning) ,Genome engineering ,03 medical and health sciences ,Synthetic biology ,Genome editing ,recombinase-assisted genomic engineering (RAGE) ,Genetics ,genome editing ,Gene ,Mycoplasma pneumoniae (MPN) transformation-associated recombination-cloning (TAR-cloning) recombinase-assisted genomic engineering (RAGE) Saccharomyces cerevisiae genome editing phage recombinases ,Mycoplasma pneumoniae (MPN) ,030304 developmental biology ,0303 health sciences ,[SDV.BA.MVSA]Life Sciences [q-bio]/Animal biology/Veterinary medicine and animal Health ,Bacteria ,030306 microbiology ,[SDV.BA]Life Sciences [q-bio]/Animal biology ,Fungi ,phage recombinases ,General Medicine ,Genomics ,Recombination ,3. Good health ,Transplantation ,[SDV.MHEP]Life Sciences [q-bio]/Human health and pathology - Abstract
Genome engineering of microorganisms has become a standard in microbial biotechnologies. Several efficient tools are available for the genetic manipulation of model bacteria such as Escherichia coli and Bacillus subtilis, or the yeast Saccharomyces cerevisiae. Difficulties arise when transferring these tools to nonmodel organisms. Synthetic biology strategies relying on genome transplantation (GT) aim at using yeast cells for engineering bacterial genomes cloned as artificial chromosomes. However, these strategies remain unsuccessful for many bacteria, including Mycoplasma pneumoniae (MPN), a human pathogen infecting the respiratory tract that has been extensively studied as a model for systems biology of simple unicellular organisms. Here, we have designed a novel strategy for genome engineering based on the recombinase-assisted genomic engineering (RAGE) technology for editing the MPN genome. Using this strategy, we have introduced a 15 kbp fragment at a specific locus of the MPN genome and replaced 38 kbp from its genome by engineered versions modified either in yeast or in E. coli. A strain harboring a synthetic version of this fragment cleared of 13 nonessential genes could also be built and propagated in vitro. These strains were depleted of known virulence factors aiming at creating an avirulent chassis for SynBio applications. Such a chassis and technology are a step forward to build vaccines or deliver therapeutic compounds in the lungs to prevent or cure respiratory diseases in humans. The authors thank Clara Blanchard for English proofreading. We also thank Dr. Don L. Court for providing biological material and advice. We acknowledge the financial support by the European Union’s Horizon 2020 research and innovation program under Grant Agreement [No. 634942]; and the European MiniCell project selected by ANR, in the frame of the ERASynBio second Joint Call for Transnational Research Projects [No. ANR-15-SYNB-0001-04].
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- 2020
21. Cryo-electron tomography analyses of terminal organelle mutants suggest the motility mechanism of Mycoplasma genitalium
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Achilleas S. Frangakis, Anja Seybert, Franziska Matthaeus, Ana M. Mariscal, Enrique Querol, Luis González-González, Jaume Piñol, Maria Lluch-Senar, and Margot P. Scheffer
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0301 basic medicine ,030106 microbiology ,Motility ,Adhesion ,Biology ,Microbiology ,Cell biology ,Cell membrane ,03 medical and health sciences ,medicine.anatomical_structure ,Cytoplasm ,Organelle ,Ultrastructure ,medicine ,Cryo-electron tomography ,Cell adhesion ,Molecular Biology - Abstract
The terminal organelle of Mycoplasma genitalium is responsible for bacterial adhesion, motility and pathogenicity. Localized at the cell tip, it comprises an electron-dense core that is anchored to the cell membrane at its distal end and to the cytoplasm at its proximal end. The surface of the terminal organelle is also covered with adhesion proteins. We performed cellular cryoelectron tomography on deletion mutants of eleven proteins that are implicated in building the terminal organelle, to systematically analyze the ultrastructural effects. These data were correlated with microcinematographies, from which the motility patterns can be quantitatively assessed. We visualized diverse phenotypes, ranging from mild to severe cell adhesion, motility and segregation defects. Based on our observations, we propose a double-spring ratchet model for the motility mechanism that explains our current and previous observations. Our model, which expands and integrates the previously suggested inchworm model, allocates specific functions to each of the essential components of this unique bacterial motility system.
- Published
- 2018
22. Impact of C-terminal amino acid composition on protein expression in bacteria
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Jae-Seong Yang, Raul Burgos, Maria Lluch-Senar, Luis Serrano, Marc Weber, and Eva Yus
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Medicine (General) ,bias ,Arginine ,Lysine ,Mutant ,Bacteris ,0302 clinical medicine ,Cluster Analysis ,Amino Acids ,Biology (General) ,Threonine ,Codon Usage ,bacteria ,Phylogeny ,degradation ,chemistry.chemical_classification ,0303 health sciences ,biology ,Applied Mathematics ,Bacterial taxonomy ,Articles ,Protein Biosynthesis & Quality Control ,Amino acid ,Computational Theory and Mathematics ,Biochemistry ,Codon, Terminator ,General Agricultural and Biological Sciences ,Hydrophobic and Hydrophilic Interactions ,Information Systems ,C‐terminal ,QH301-705.5 ,Protein degradation ,Article ,General Biochemistry, Genetics and Molecular Biology ,Evolution, Molecular ,Amino Acids, Aromatic ,03 medical and health sciences ,Residue (chemistry) ,R5-920 ,Bacterial Proteins ,Protein Domains ,expression ,Amino Acid Sequence ,030304 developmental biology ,General Immunology and Microbiology ,Computational Biology ,biology.organism_classification ,Mycoplasma pneumoniae ,chemistry ,Genes, Bacterial ,Protein Processing, Post-Translational ,Proteïnes ,030217 neurology & neurosurgery ,Bacteria - Abstract
The C‐terminal sequence of a protein is involved in processes such as efficiency of translation termination and protein degradation. However, the general relationship between features of this C‐terminal sequence and levels of protein expression remains unknown. Here, we identified C‐terminal amino acid biases that are ubiquitous across the bacterial taxonomy (1,582 genomes). We showed that the frequency is higher for positively charged amino acids (lysine, arginine), while hydrophobic amino acids and threonine are lower. We then studied the impact of C‐terminal composition on protein levels in a library of Mycoplasma pneumoniae mutants, covering all possible combinations of the two last codons. We found that charged and polar residues, in particular lysine, led to higher expression, while hydrophobic and aromatic residues led to lower expression, with a difference in protein levels up to fourfold. We further showed that modulation of protein degradation rate could be one of the main mechanisms driving these differences. Our results demonstrate that the identity of the last amino acids has a strong influence on protein expression levels., Large‐scale genomics analyses combined with high‐throughput experimental assays reveal that the C‐terminal amino acid composition has a strong influence on protein expression levels in bacteria.
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- 2019
23. Unraveling the hidden universe of small proteins in bacterial genomes
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Eduard Sabidó, Maria Lluch-Senar, Samuel Miravet-Verde, Rocco Mazzolini, Anas Gharrab, Luis Serrano, Tony Ferrar, and Guadalupe Espadas-García
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Proteomics ,Medicine (General) ,Proteome ,QH301-705.5 ,European Regional Development Fund ,mycoplasmas ,random forest classifier ,small proteins ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Mass Spectrometry ,Article ,03 medical and health sciences ,0302 clinical medicine ,R5-920 ,Mycoplasma ,Bacterial Proteins ,media_common.cataloged_instance ,European union ,Biology (General) ,030304 developmental biology ,media_common ,0303 health sciences ,Bacteriologia ,General Immunology and Microbiology ,Applied Mathematics ,European research ,Computational Biology ,Post-translational Modifications, Proteolysis & Proteomics ,Articles ,protein prediction ,Microbiology, Virology & Host Pathogen Interaction ,Genòmica ,Computational Theory and Mathematics ,mass spectroscopy ,Christian ministry ,General Agricultural and Biological Sciences ,Peptides ,Humanities ,Proteïnes ,030217 neurology & neurosurgery ,Genome, Bacterial ,Information Systems - Abstract
Identification of small open reading frames (smORFs) encoding small proteins (≤ 100 amino acids; SEPs) is a challenge in the fields of genome annotation and protein discovery. Here, by combining a novel bioinformatics tool (RanSEPs) with "-omics" approaches, we were able to describe 109 bacterial small ORFomes. Predictions were first validated by performing an exhaustive search of SEPs present in Mycoplasma pneumoniae proteome via mass spectrometry, which illustrated the limitations of shotgun approaches. Then, RanSEPs predictions were validated and compared with other tools using proteomic datasets from different bacterial species and SEPs from the literature. We found that up to 16 ± 9% of proteins in an organism could be classified as SEPs. Integration of RanSEPs predictions with transcriptomics data showed that some annotated non-coding RNAs could in fact encode for SEPs. A functional study of SEPs highlighted an enrichment in the membrane, translation, metabolism, and nucleotide-binding categories. Additionally, 9.7% of the SEPs included a N-terminus predicted signal peptide. We envision RanSEPs as a tool to unmask the hidden universe of small bacterial proteins. We thank Dr. Luca Cozzuto from the Bioinformatics Unit at CRG for providing valuable guidance for the conservation studies. Also, we would like to thank Dr. Carolina Gallo, Dr. Eva Yus, and Dr. Raul Burgos for providing MS data. Finally, we thank Dr. Marc Weber for his recommendation about how to analyze Ribo-Seq data. We acknowledge support of the Spanish Ministry of Economy, Industry and Competitiveness (MEIC) to the EMBL partnership, the Spanish Ministry of Economy and Competitiveness, “Centro de Excelencia Severo Ochoa”, the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program under agreement No 670216 (MYCOCHASSIS), the CERCA Programme/Generalitat de Catalunya, the European Regional Development Fund (ERDF) project from Instituto Carlos III (ISCIII, Acción Estratégica en Salud 2016; reference CP16/00094), and “Secretaria d’Universitats i Recerca del Departament d’Economia i Coneixement de la Generalitat de Catalunya” (2014SGR678). The CRG/UPF Proteomics Unit is part of the “Plataforma de Recursos Biomoleculares y Bioinformáticos (ProteoRed)” supported by grant PT13/0001 of Instituto de Salud Carlos III from the Spanish Government.
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- 2019
24. Determination of the gene regulatory network of a genome-reduced bacterium highlights alternative regulation independent of transcription factors
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Maria Lluch-Senar, Carolina Gallo, Cedric Blötz, Hinnerk Eilers, Eva Yus, Luis Serrano, Sira Martínez, Verónica Lloréns-Rico, and Jörg Stülke
- Subjects
EXPRESSION ,Biochemistry & Molecular Biology ,STRESS ,Histology ,Transcription, Genetic ,PROTEINS ,Systems biology ,Gene regulatory network ,gene regulatory network ,MYCOPLASMA-PNEUMONIAE ,Computational biology ,METABOLISM ,Biology ,Genome ,Article ,BACILLUS-SUBTILIS ,Pathology and Forensic Medicine ,03 medical and health sciences ,0302 clinical medicine ,Transcription (biology) ,DNA TOPOLOGY ,transcription factors ,Transcriptional regulation ,Gene Regulatory Networks ,Mycoplasma pneumoniae ,systems biology ,transcription ,transcription regulation ,Gene ,Transcription factor ,030304 developmental biology ,0303 health sciences ,Science & Technology ,Models, Genetic ,Gene Expression Profiling ,Robustness (evolution) ,Gene Expression Regulation, Bacterial ,Cell Biology ,Gene Expression Regulation ,NONCODING RNAS ,GROWTH ,(P)PPGPP ,Life Sciences & Biomedicine ,Genome, Bacterial ,030217 neurology & neurosurgery ,Signal Transduction - Abstract
Summary Here, we determined the relative importance of different transcriptional mechanisms in the genome-reduced bacterium Mycoplasma pneumoniae, by employing an array of experimental techniques under multiple genetic and environmental perturbations. Of the 143 genes tested (21% of the bacterium’s annotated proteins), only 55% showed an altered phenotype, highlighting the robustness of biological systems. We identified nine transcription factors (TFs) and their targets, representing 43% of the genome, and 16 regulators that indirectly affect transcription. Only 20% of transcriptional regulation is mediated by canonical TFs when responding to perturbations. Using a Random Forest, we quantified the non-redundant contribution of different mechanisms such as supercoiling, metabolic control, RNA degradation, and chromosome topology to transcriptional changes. Model-predicted gene changes correlate well with experimental data in 95% of the tested perturbations, explaining up to 70% of the total variance when also considering noise. This analysis highlights the importance of considering non-TF-mediated regulation when engineering bacteria., Graphical Abstract, Highlights • Full comprehensive reconstruction of a bacterial gene regulatory network achieved • Genome-reduced bacterium Mycoplasma pneumoniae is robust to genetic perturbations • Large part of transcription regulation in bacteria is transcription-factor independent • Transcription-factor-independent regulation has a smaller dynamic range, We have achieved a comprehensive reconstruction of a gene regulatory network in a genome-reduced bacterium, Mycoplasma pneumoniae. With this network, we observed that a large part of transcription regulation is determined by non-canonical factors such as DNA supercoiling, riboswitches, and genome organization or RNA-mediated regulation. This analysis highlights the importance of considering non-transcription-factor (TF)-mediated regulation when engineering bacteria.
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- 2019
25. FASTQINS and ANUBIS: two bioinformatic tools to explore facts and artifacts in transposon sequencing and essentiality studies
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Samuel Miravet-Verde, Luis Serrano, Javier Delgado, Raul Burgos, and Maria Lluch-Senar
- Subjects
AcademicSubjects/SCI00010 ,Sequence analysis ,Computational biology ,Biology ,Genome ,Cellular life ,Set (abstract data type) ,03 medical and health sciences ,Synthetic biology ,Genetics ,Narese/7 ,030304 developmental biology ,Recombination, Genetic ,0303 health sciences ,Training set ,030302 biochemistry & molecular biology ,Genomics ,Sequence Analysis, DNA ,Transposon Sequencing ,Mycoplasma pneumoniae ,3. Good health ,Narese/24 ,DNA Transposable Elements ,Methods Online ,Anubis ,Software - Abstract
Transposon sequencing is commonly applied for identifying the minimal set of genes required for cellular life; a major challenge in fields such as evolutionary or synthetic biology. However, the scientific community has no standards at the level of processing, treatment, curation and analysis of this kind data. In addition, we lack knowledge about artifactual signals and the requirements a dataset has to satisfy to allow accurate prediction. Here, we have developed FASTQINS, a pipeline for the detection of transposon insertions, and ANUBIS, a library of functions to evaluate and correct deviating factors known and uncharacterized until now. ANUBIS implements previously defined essentiality estimate models in addition to new approaches with advantages like not requiring a training set of genes to predict general essentiality. To highlight the applicability of these tools, and provide a set of recommendations on how to analyze transposon sequencing data, we performed a comprehensive study on artifacts corrections and essentiality estimation at a 1.5-bp resolution, in the genome-reduced bacterium Mycoplasma pneumoniae. We envision FASTQINS and ANUBIS to aid in the analysis of Tn-seq procedures and lead to the development of accurate genome essentiality estimates to guide applications such as designing live vaccines or growth optimization., Nucleic Acids Research, 48 (17), ISSN:1362-4962, ISSN:0301-5610
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- 2020
26. Inferring Active Metabolic Pathways from Proteomics and Essentiality Data
- Author
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Carlos Piñero-Lambea, Ariadna Montero-Blay, Samuel Miravet-Verde, Luis Serrano, and Maria Lluch-Senar
- Subjects
Proteomics ,0301 basic medicine ,Transposable element ,Proteome ,Mycoplasma agalactiae ,Quantitative proteomics ,ved/biology.organism_classification_rank.species ,Computational biology ,Biology ,Mass Spectrometry ,Article ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,Mycoplasma ,0302 clinical medicine ,Bacterial Proteins ,Transposon ,lcsh:QH301-705.5 ,Gene ,Chromatography, High Pressure Liquid ,chemistry.chemical_classification ,Genes, Essential ,Bacteria ,ved/biology ,Active pathways ,metabolism, bacteria, transposon, active pathways, mycoplasma, proteomics, essentiality ,Metabolism ,Carbon ,Mycoplasma pneumoniae ,Essentiality ,Metabolic pathway ,Glucose ,030104 developmental biology ,Enzyme ,lcsh:Biology (General) ,chemistry ,Glycolysis ,Metabolic Networks and Pathways ,030217 neurology & neurosurgery - Abstract
Summary Here, we propose an approach to identify active metabolic pathways by integrating gene essentiality analysis and protein abundance. We use two bacterial species (Mycoplasma pneumoniae and Mycoplasma agalactiae) that share a high gene content similarity yet show significant metabolic differences. First, we build detailed metabolic maps of their carbon metabolism, the most striking difference being the absence of two key enzymes for glucose metabolism in M. agalactiae. We then determine carbon sources that allow growth in M. agalactiae, and we introduce glucose-dependent growth to show the functionality of its remaining glycolytic enzymes. By analyzing gene essentiality and performing quantitative proteomics, we can predict the active metabolic pathways connected to carbon metabolism and show significant differences in use and direction of key pathways despite sharing the large majority of genes. Gene essentiality combined with quantitative proteomics and metabolic maps can be used to determine activity and directionality of metabolic pathways., Graphical Abstract, Highlights • Active metabolic bacterial pathways are identified • Integration of gene essentiality and proteomics allow prediction of active pathways • Glucose-dependent growth is restored in Mycoplasma agalactiae • Two Mycoplasma species show different usage of metabolic pathways, Montero-Blay et al. identify active metabolic pathways in bacteria by integrating gene essentiality data and quantitative proteomics. Predictions agree with experimental information and show substantial differences in usage and directionality of metabolic pathways in bacteria with high degree of gene similarity.
- Published
- 2020
27. Cryo-electron tomography analyses of terminal organelle mutants suggest the motility mechanism of Mycoplasma genitalium
- Author
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Anja, Seybert, Luis, Gonzalez-Gonzalez, Margot P, Scheffer, Maria, Lluch-Senar, Ana M, Mariscal, Enrique, Querol, Franziska, Matthaeus, Jaume, Piñol, and Achilleas S, Frangakis
- Subjects
Organelles ,Electron Microscope Tomography ,Bacterial Proteins ,Mutation ,Cell Adhesion ,Electrons ,Mycoplasma genitalium ,Adhesins, Bacterial ,Bacterial Adhesion ,Mycoplasma pneumoniae - Abstract
The terminal organelle of Mycoplasma genitalium is responsible for bacterial adhesion, motility and pathogenicity. Localized at the cell tip, it comprises an electron-dense core that is anchored to the cell membrane at its distal end and to the cytoplasm at its proximal end. The surface of the terminal organelle is also covered with adhesion proteins. We performed cellular cryoelectron tomography on deletion mutants of eleven proteins that are implicated in building the terminal organelle, to systematically analyze the ultrastructural effects. These data were correlated with microcinematographies, from which the motility patterns can be quantitatively assessed. We visualized diverse phenotypes, ranging from mild to severe cell adhesion, motility and segregation defects. Based on our observations, we propose a double-spring ratchet model for the motility mechanism that explains our current and previous observations. Our model, which expands and integrates the previously suggested inchworm model, allocates specific functions to each of the essential components of this unique bacterial motility system.
- Published
- 2018
28. Reconstruction of the Regulatory Network in a Minimal Bacterium Reveals Extensive Non-Transcription Factor Dependent Regulation
- Author
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Cedric Blötz, Hinnerk Eilers, Luis Serrano, Carolina Gallo, Jörg Stülke, Eva Yus, Maria Lluch-Senar, Sira Martínez, and Verónica Lloréns-Rico
- Subjects
0303 health sciences ,030306 microbiology ,Gene regulatory network ,Computational biology ,Biology ,Chromatin ,03 medical and health sciences ,Sigma factor ,Transcription (biology) ,Gene expression ,Transcriptional regulation ,Gene ,Transcription factor ,030304 developmental biology - Abstract
Determining the gene regulatory network of an organism is fundamental for achieving a global understanding of cellular behaviour. Here, we determine the relative importance of different transcription regulators in the genome-reduced bacterium Mycoplasma pneumoniae, by integrating DNA affinity chromatography, chromatin isolation, ChIP-seq, transcriptomics, proteomics and growth curves experiments for 143 genes (20.8% of the bacteria´s NCBI annotated proteins). In 55% of the cases, gene overexpression or mutation does not result in transcriptional nor growth phenotypes, highlighting the robustness of even very simple cells. We identified nine TFs (besides the sigma factor) and their targets, representing 33% of the genome. Nevertheless, we find that the majority of transcription regulation in M. pneumoniae is not mediated by canonical TFs, but rather occurs at the level of supercoiling, metabolic control, RNA degradation, and chromosome topology. For the first time, we quantify the non-redundant contribution of each of these mechanisms in various environmental perturbations using a random forest model, showing that cells use different mechanisms to respond to distinct stresses. The gene changes predicted by our model correlate with the actual changes in 95% of the tested perturbations, and after accounting for experimental and gene expression noise, we can explain over 70% of the total variance. This comprehensive analysis highlights the importance of non-TF-mediated regulation in bacteria, an ancient type of regulation that allows adaptation to mild environmental changes.
- Published
- 2018
29. Tuning gene activity by inducible and targeted regulation of gene expression in minimal bacterial cells
- Author
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Adam J. Moore, Yo Suzuki, Jaume Piñol, J. Craig Venter, Lijie Sun, Kazuki Tanaka, Alicia Broto, Carlos Piñero-Lambea, Gavin Tse, Clyde A. Hutchison, Enrique Querol, Luis González-González, Maria Lluch-Senar, Chuck Merryman, John I. Glass, Hamilton O. Smith, Shigeyuki Kakizawa, Masaru Tomita, Kim S. Wise, Ana M. Mariscal, Philip D. Weyman, and Jonathan Y. Hsu
- Subjects
0301 basic medicine ,Transposable element ,Systems biology ,030106 microbiology ,Biomedical Engineering ,Inducible promoters ,Biochemistry, Genetics and Molecular Biology (miscellaneous) ,03 medical and health sciences ,Mycoplasma ,Bacterial Proteins ,Gene expression ,Clustered Regularly Interspaced Short Palindromic Repeats ,Gene Regulatory Networks ,Gene ,Gene knockout ,Regulation of gene expression ,biology ,Functional genomics ,General Medicine ,Gene Expression Regulation, Bacterial ,Methyltransferases ,Tetracycline ,biology.organism_classification ,Cell biology ,Clustered regularly interspaced short palindromic repeats (CRISPR) ,Luminescent Proteins ,030104 developmental biology ,Aminoglycosides ,Tetracycline-mediated repression ,Riboswitch ,CRISPR-Cas Systems ,Microorganisms, Genetically-Modified ,Mycoplasma mycoides ,Genetic Engineering - Abstract
Functional genomics studies in minimal mycoplasma cells enable unobstructed access to some of the most fundamental processes in biology. Conventional transposon bombardment and gene knockout approaches often fail to reveal functions of genes that are essential for viability, where lethality precludes phenotypic characterization. Conditional inactivation of genes is effective for characterizing functions central to cell growth and division, but tools are limited for this purpose in mycoplasmas. Here we demonstrate systems for inducible repression of gene expression based on clustered regularly interspaced short palindromic repeats-mediated interference (CRISPRi) in Mycoplasma pneumoniae and synthetic Mycoplasma mycoides, two organisms with reduced genomes actively used in systems biology studies. In the synthetic cell, we also demonstrate inducible gene expression for the first time. Time-course data suggest rapid kinetics and reversible engagement of CRISPRi. Targeting of six selected endogenous genes with this system results in lowered transcript levels or reduced growth rates that agree with lack or shortage of data in previous transposon bombardment studies, and now produces actual cells to analyze. The ksgA gene encodes a methylase that modifies 16S rRNA, rendering it vulnerable to inhibition by the antibiotic kasugamycin. Targeting the ksgA gene with CRISPRi removes the lethal effect of kasugamycin and enables cell growth, thereby establishing specific and effective gene modulation with our system. The facile methods for conditional gene activation and inactivation in mycoplasmas open the door to systematic dissection of genetic programs at the core of cellular life. This work was supported by internal funding from the J. Craig Venter Institute to H.O.S. and C.A.H., as well as grants BIO2013-4870R and BIO2013-50176EXP from the Ministerio de Economía y Competitividad to E.Q. and J.P., respectively, and Japan Society for the Promotion of Science KAKENHI grants JP26710015, JP15KK0266, and JP26106004 to S.K.A.M.M. is a recipient of a predoctoral fellowship from the Generalitat de Catalunya (FI-DGR 2014).
- Published
- 2018
30. Defined chromosome structure in the genome-reduced bacterium Mycoplasma pneumoniae
- Author
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Steven P. Djordjevic, Marc A. Marti-Renom, Davide Baù, Thomas Pengo, Marie Trussart, Eva Yus, Michael Widjaja, Lynne Turnbull, Sira Martínez, Luis Serrano, Simon Kretschmer, Yuhei O Tahara, Cynthia B. Whitchurch, Jim Swoger, Maria Lluch-Senar, and Makoto Miyata
- Subjects
DNA, Bacterial ,0301 basic medicine ,Science ,030106 microbiology ,Molecular Conformation ,General Physics and Astronomy ,Bacterial genome size ,Biology ,Genome ,Article ,General Biochemistry, Genetics and Molecular Biology ,Microbiology ,Bacterial genetics ,03 medical and health sciences ,chemistry.chemical_compound ,Gene ,Genetics ,Microscopy ,Multidisciplinary ,DNA, Superhelical ,Chromosome structures ,Chromosome ,Gene Expression Regulation, Bacterial ,Pneumonia ,General Chemistry ,Chromosomes, Bacterial ,Mycoplasma pneumoniae ,Attachment organelle ,030104 developmental biology ,Chromosome Structures ,chemistry ,Alveolar rhabdomyosarcoma ,Nucleic Acid Conformation ,DNA supercoil ,Molecular modelling ,Genome, Bacterial ,DNA - Abstract
DNA-binding proteins are central regulators of chromosome organization; however, in genome-reduced bacteria their diversity is largely diminished. Whether the chromosomes of such bacteria adopt defined three-dimensional structures remains unexplored. Here we combine Hi-C and super-resolution microscopy to determine the structure of the Mycoplasma pneumoniae chromosome at a 10 kb resolution. We find a defined structure, with a global symmetry between two arms that connect opposite poles, one bearing the chromosomal Ori and the other the midpoint. Analysis of local structures at a 3 kb resolution indicates that the chromosome is organized into domains ranging from 15 to 33 kb. We provide evidence that genes within the same domain tend to be co-regulated, suggesting that chromosome organization influences transcriptional regulation, and that supercoiling regulates local organization. This study extends the current understanding of bacterial genome organization and demonstrates that a defined chromosomal structure is a universal feature of living systems., The three-dimensional architecture of genome-reduced bacteria is poorly understood. Here the authors combine Hi-C with super-resolution microscopy in Mycoplasma pneumoniae and provide evidence of how supercoiling and local organization influences gene regulation.
- Published
- 2017
31. From systems to synthetic biology: Mycoplasma pneumoniae as minimal chassis
- Author
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Maria Lluch-Senar, Jonathan Karr, Carlos Pinero, Daniel Shawn, Samuel Miravet, Verónica Lloréns-Rico, Tony Ferrar, Sira Martinez, Eva Yus, Marie Tussart, Alain Blanchard, Pascal SIRAND-PUGNET, Jörg Stülke, Carole Lartigue, Luis Serrano, Centro de Regulación Genómica (CRG), Universitat Pompeu Fabra [Barcelona] (UPF), Icahn School of Medicine at Mount Sinai [New York] (MSSM), Biologie du fruit et pathologie (BFP), Université Bordeaux Segalen - Bordeaux 2-Institut National de la Recherche Agronomique (INRA)-Université Sciences et Technologies - Bordeaux 1, University Medical Center Göttingen (UMG), and ProdInra, Migration
- Subjects
[SDV.MHEP] Life Sciences [q-bio]/Human health and pathology ,ComputingMilieux_MISCELLANEOUS ,[SDV.MHEP]Life Sciences [q-bio]/Human health and pathology - Abstract
International audience
- Published
- 2016
32. Rescuing discarded spectra: full comprehensive analysis of a minimal proteome
- Author
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Francesco M. Mancuso, Marcia I. Peña-Paz, Héctor Climente-González, Luis Serrano, Maria Lluch-Senar, and Eduard Sabidó
- Subjects
0301 basic medicine ,Proteomics ,Proteome ,PTM ,Unassigned spectra ,Biology ,Proteòmica ,Biochemistry ,03 medical and health sciences ,Protein sequencing ,Bacterial Proteins ,Tandem Mass Spectrometry ,Pneumonia, Mycoplasma ,Humans ,ORFS ,Deamidation ,Databases, Protein ,Molecular Biology ,Peptide sequence ,chemistry.chemical_classification ,Translational errors ,030102 biochemistry & molecular biology ,Protein Identification ,MS ,Amino acid ,Mycoplasma pneumoniae ,030104 developmental biology ,Proteome coverage ,chemistry ,Transfer RNA ,Transcriptome ,Systems biology ,Protein Processing, Post-Translational ,Proteïnes ,Genome, Bacterial - Abstract
A common problem encountered when performing large-scale MS proteome analysis is the loss of information due to the high percentage of unassigned spectra. To determine the causes behind this loss we have analyzed the proteome of one of the smallest living bacteria that can be grown axenically, Mycoplasma pneumoniae (729 ORFs). The proteome of M. pneumoniae cells, grown in defined media, was analyzed by MS. An initial search with both Mascot and a species-specific NCBInr database with common contaminants (NCBImpn), resulted in around 79% of the acquired spectra not having an assignment. The percentage of non-assigned spectra was reduced to 27% after re-analysis of the data with the PEAKS software, thereby increasing the proteome coverage of M. pneumoniae from the initial 60% to over 76%. Nonetheless, 33 413 spectra with assigned amino acid sequences could not be mapped to any NCBInr database protein sequence. Approximately, 1% of these unassigned peptides corresponded to PTMs and 4% to M. pneumoniae protein variants (deamidation and translation inaccuracies). The most abundant peptide sequence variants (Phe-Tyr and Ala-Ser) could be explained by alterations in the editing capacity of the corresponding tRNA synthases. About another 1% of the peptides not associated to any protein had repetitions of the same aromatic/hydrophobic amino acid at the N-terminus, or had Arg/Lys at the C-terminus. Thus, in a model system, we have maximized the number of assigned spectra to 73% (51 453 out of the 70 040 initial acquired spectra). This work was sup-ported by the European Research Council (ERC), the Fundación Marcelino Botín, the Spanish Ministerio de Economía y Com-petitividad BIO2007-61762 and the ISCIII (PI10/01702). TheCRG/UPF Proteomics Unit is part of the “Plataforma de Re-cursos Biomoleculares y Bioinform´aticos (ProteoRed)” supportedby grant PT13/0001 of Instituto de Salud Carlos III (ISCIII).We acknowledge the support of the Spanish Ministry of Economyand Competitiveness, ‘Centro de Excelencia Severo Ochoa 2013–2017’, SEV-2012-0208. This project has received funding fromthe European Union’s Horizon 2020 research and innovationprogram under grant agreement No 634942
- Published
- 2016
33. Integration of multi-omics data of a genome-reduced bacterium: Prevalence of post-transcriptional regulation and its correlation with protein abundances
- Author
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Guglielmo Roma, Judith A. H. Wodke, Christina Pesavento, Vera van Noort, Peer Bork, Maria Lluch-Senar, Marco L. Hennrich, Luis Serrano, Eva Yus, Andreu Alibés, Daniel R. Mende, Athanasios Typas, Anne-Claude Gavin, and Wei-Hua Chen
- Subjects
Proteomics ,0301 basic medicine ,RNA, Untranslated ,Proteome ,Molecular Sequence Data ,Bacterial/genetics ,Proteome/genetics/metabolism ,Untranslated/genetics ,NcRNA transcription ,Genomics ,Biology ,Genome ,03 medical and health sciences ,Bacterial Proteins ,Genomics/methods/statistics & numerical data ,Genetics ,Cluster Analysis ,Proteomics/methods/statistics & numerical data ,Amino Acid Sequence ,Genome size ,Protein Processing ,2. Zero hunger ,Regulation of gene expression ,Mycoplasma pneumoniae/genetics/metabolism ,Base Sequence ,Systems Biology/methods/statistics & numerical data ,Gene Expression Profiling ,Systems Biology ,Post-Translational ,Molecular Sequence Annotation ,Mycoplasma pneumoniae ,Gene expression profiling ,030104 developmental biology ,Gene Expression Regulation ,Cardiovascular and Metabolic Diseases ,Bacterial Proteins/genetics/metabolism ,Gene Expression Profiling/methods/statistics & numerical data ,RNA ,Protein Processing, Post-Translational ,Genome, Bacterial - Abstract
We developed a comprehensive resource for the genome-reduced bacterium Mycoplasma pneumoniae comprising 1748 consistently generated '-omics' data sets, and used it to quantify the power of antisense non-coding RNAs (ncRNAs), lysine acetylation, and protein phosphorylation in predicting protein abundance (11%, 24% and 8%, respectively). These factors taken together are four times more predictive of the proteome abundance than of mRNA abundance. In bacteria, post-translational modifications (PTMs) and ncRNA transcription were both found to increase with decreasing genomic GC-content and genome size. Thus, the evolutionary forces constraining genome size and GC-content modify the relative contributions of the different regulatory layers to proteome homeostasis, and impact more genomic and genetic features than previously appreciated. Indeed, these scaling principles will enable us to develop more informed approaches when engineering minimal synthetic genomes. The research leading to these results has received funding from the European Research Council under the European Union's Seventh Framework Programme (FP7/2007–2013)/ERC Grant Agreement No. 232913. We acknowledge support from the Spanish Ministry of Economy and Competitiveness, ‘Centro de Excelencia Severo Ochoa 2013–2017’, SEV-2012–0208. This project has received funding from the European Union's Horizon 2020 research and innovation program under Grant Agreement No. 634942. Funding for open access charge: European Research council (ERC) advanced Grant Agreement No. 232913, the Fundación Marcelino Botin, the Spanish Ministerio de Economía y Competitividad BIO2007-61762, the ISCIII, Subdirección General de evaluación y fomento de la investigación PI10/01702 to the ICREA researcher L.S.
- Published
- 2016
34. Cell division in a minimal bacterium in the absence of ftsZ
- Author
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Jaume Piñol, Maria Lluch-Senar, and Enrique Querol
- Subjects
0303 health sciences ,Cell division ,biology ,030306 microbiology ,Cell growth ,Mycoplasma ,medicine.disease_cause ,biology.organism_classification ,Microbiology ,Cell biology ,03 medical and health sciences ,medicine ,biology.protein ,Cytoskeleton ,Mycoplasma genitalium ,FtsZ ,Molecular Biology ,Gene ,Cytokinesis ,030304 developmental biology - Abstract
Mycoplasma genomes exhibit an impressively low amount of genes involved in cell division and some species even lack the ftsZ gene, which is found widespread in the microbial world and is considered essential for cell division by binary fission. We constructed a Mycoplasma genitalium ftsZ null mutant by gene replacement to investigate the role of this gene and the presence of alternative cell division mechanisms in this minimal bacterium. Our results demonstrate that ftsZ is non-essential for cell growth and reveal that, in the absence of the FtsZ protein, M. genitalium can manage feasible cell divisions and cytokinesis using the force generated by its motile machinery. This is an alternative mechanism, completely independent of the FtsZ protein, to perform cell division by binary fission in a microorganism. We also propose that the mycoplasma cytoskeleton, a complex network of proteins involved in many aspects of the biology of these microorganisms, may have taken over the function of many genes involved in cell division, allowing their loss in the regressive evolution of the streamlined mycoplasma genomes.
- Published
- 2010
35. A new promoterless reporter vector reveals antisense transcription in Mycoplasma genitalium
- Author
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Maria Lluch-Senar, Jaume Piñol, Enrique Querol, and Miquel Vallmitjana
- Subjects
Transposable element ,Genetics ,Transcription, Genetic ,biology ,Genetic Vectors ,Mycoplasma genitalium ,beta-Galactosidase ,biology.organism_classification ,Microbiology ,Molecular biology ,Artificial Gene Fusion ,Genes, Reporter ,Transcription (biology) ,Gene expression ,Sense (molecular biology) ,DNA Transposable Elements ,RNA, Antisense ,ORFS ,Promoter Regions, Genetic ,Repeated sequence ,Gene - Abstract
The mechanisms that promote and regulate transcription in mycoplasmas are poorly understood. Here, a promoter-probe vector based on the pMTnTetM438 minitransposon and containing a promoterless lacZ reporter gene was constructed to analyse Mycoplasma genitalium transcription in vivo. Recovered transposon insertions were in monocopy, with 16 % expressing enough beta-galactosidase (beta-Gal) to yield colonies exhibiting a detectable blue colour. A sample of 52 blue colonies was propagated and selected for further analyses. The beta-Gal activity of the corresponding cultures was measured to quantify, in a reproducible way, the transcription levels of the interrupted ORFs. Several insertions were found in sense with the interrupted ORF, but surprisingly there was also a number of insertions in non-coding regions, many of them in repetitive DNA regions known as MgPa islands. Moreover, 30 % of the analysed transposon insertions had the lacZ gene in the opposite orientation to the coding frame, suggesting the existence of antisense transcripts that may be involved in the control of gene expression in M. genitalium.
- Published
- 2007
36. MyMpn: a database for the systems biology model organism Mycoplasma pneumoniae
- Author
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Eva Yus, Luca Cozzuto, Luis Serrano, Judith A. H. Wodke, Antonio Hermoso, Maria Lluch-Senar, Guglielmo Roma, and Andreu Alibés
- Subjects
Mycoplasma pneumoniae ,Proteome ,Systems biology ,ved/biology.organism_classification_rank.species ,Biology ,medicine.disease_cause ,computer.software_genre ,Mycoplasma pneumoniae -- Aspectes genètics ,Bases de dades ,Databases, Genetic ,Genetics ,medicine ,Profiling (information science) ,Database Issue ,Model organism ,Organism ,Comparative genomics ,Internet ,Genètica bacteriana ,Database ,ved/biology ,Systems Biology ,Genomes bacterians ,Gene expression profiling ,Mycoplasma pneumoniae -- Metabolisme ,Metabolome ,Transcriptome ,computer ,Genome, Bacterial - Abstract
MyMpn (http://mympn.crg.eu) is an online resource devoted to studying the human pathogen Mycoplasma pneumoniae, a minimal bacterium causing lower respiratory tract infections. Due to its small size, its ability to grow in vitro, and the amount of data produced over the past decades, M. pneumoniae is an interesting model organisms for the development of systems biology approaches for unicellular organisms. Our database hosts a wealth of omics-scale datasets generated by hundreds of experimental and computational analyses. These include data obtained from gene expression profiling experiments, gene essentiality studies, protein abundance profiling, protein complex analysis, metabolic reactions and network modeling, cell growth experiments, comparative genomics and 3D tomography. In addition, the intuitive web interface provides access to several visualization and analysis tools as well as to different data search options. The availability and--even more relevant--the accessibility of properly structured and organized data are of up-most importance when aiming to understand the biology of an organism on a global scale. Therefore, MyMpn constitutes a unique and valuable new resource for the large systems biology and microbiology community. European Union through the European Research Council (ERC) [‘Celldoctor’ GA 232913]; Spanish Ministerio de Economía y Competitividad Plan Nacional [BIO2007-61762]; Fundaci´on Marcelino Botin [to L.S.]. laCaixa-CRG Fellowship, Fundació laCaixa [to J.W.]; Spanish Ministry of Economy and Competitiveness No [PTA2010-4446-I to A.H.; PTA2011-6729-I to L.C.]. Funding for open access charge: ERC [to L.S.].
- Published
- 2015
37. Comparative ?-omics? in Mycoplasma pneumoniae Clinical Isolates Reveals Key Virulence Factors
- Author
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Sabine Pereyre, Maria Lluch-Senar, Jaime González Cano, Verónica Lloréns-Rico, Luca Cozzuto, Javier Delgado, Luis Serrano, Cécile Bébéar, Centro de Regulación Genómica (CRG), Universitat Pompeu Fabra [Barcelona] (UPF), USC EA3671, Infections humaines à mycoplasmes et à chlamydiae, Institut National de la Recherche Agronomique (INRA), Laboratoire de Bactériologie, Université Joseph Fourier - Grenoble 1 (UJF)-CHU Grenoble, and Institució Catalana de Recerca i Estudis Avançats (ICREA)
- Subjects
Mycoplasma pneumoniae ,Tunisia ,Proteome ,Gene prediction ,[SDV]Life Sciences [q-bio] ,Bacterial Toxins ,Molecular Sequence Data ,Virulence ,lcsh:Medicine ,Biology ,medicine.disease_cause ,Polymorphism, Single Nucleotide ,Genome ,Microbiology ,Open Reading Frames ,Bacterial Proteins ,INDEL Mutation ,Japan ,Pneumonia, Mycoplasma ,medicine ,Antigenic variation ,Humans ,Adhesins, Bacterial ,lcsh:Science ,Gene ,Pathogen ,Genetics ,Antigens, Bacterial ,Genètica bacteriana ,Multidisciplinary ,Base Sequence ,lcsh:R ,Gene Expression Regulation, Bacterial ,Antigenic Variation ,3. Good health ,Europe ,Bacterial adhesin ,Mutation ,Bacteris patògens ,lcsh:Q ,Transcriptome ,Genome, Bacterial ,Research Article - Abstract
The human respiratory tract pathogen M. pneumoniae is one of the best characterized minimal bacterium. Until now, two main groups of clinical isolates of this bacterium have been described (types 1 and 2), differing in the sequence of the P1 adhesin gene. Here, we have sequenced the genomes of 23 clinical isolates of M. pneumoniae. Studying SNPs, non-synonymous mutations, indels and genome rearrangements of these 23 strains and 4 previously sequenced ones, has revealed new subclasses in the two main groups, some of them being associated with the country of isolation. Integrative analysis of in vitro gene essentiality and mutation rates enabled the identification of several putative virulence factors and antigenic proteins; revealing recombination machinery, glycerol metabolism and peroxide production as possible factors in the genetics and physiology of these pathogenic strains. Additionally, the transcriptomes and proteomes of two representative strains, one from each of the two main groups, have been characterized to evaluate the impact of mutations on RNA and proteins levels. This study has revealed that type 2 strains show higher expression levels of CARDS toxin, a protein recently shown to be one of the major factors of inflammation. Thus, we propose that type 2 strains could be more toxigenic than type 1 strains of M. pneumoniae. This work was supported by the European Research Council (ERC). This project has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No 634942.
- Published
- 2015
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38. Assessing the hodgepodge of non-mapped reads in bacterial transcriptomes: real or artifactual RNA chimeras?
- Author
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Verónica Lloréns-Rico, Maria Lluch-Senar, and Luis Serrano
- Subjects
Chimeric RNAs ,Fusion transcripts ,Statistics as Topic ,RNA-Seq ,Biology ,Proteomics ,Transcriptome ,Chimera (genetics) ,RNA Isoforms ,Genetics ,Genètica bacteriana ,Bacteria ,Base Sequence ,Sequence Analysis, RNA ,Gene Expression Profiling ,Methodology Article ,RNA ,Library preparation protocols ,Gene expression profiling ,RNA, Bacterial ,DNA microarray ,RNA-seq ,Artifacts ,Software ,Biotechnology - Abstract
Background: RNA sequencing methods have already altered our view of the extent and complexity of bacterial and eukaryotic transcriptomes, revealing rare transcript isoforms (circular RNAs, RNA chimeras) that could play an important role in their biology./nResults: We performed an analysis of chimera formation by four different computational approaches, including a custom designed pipeline, to study the transcriptomes of M. pneumoniae and P. aeruginosa, as well as mixtures of both. We found that rare transcript isoforms detected by conventional pipelines of analysis could be artifacts of the experimental procedure used in the library preparation, and that they are protocol-dependent. Conclusion: By using a customized pipeline we show that optimal library preparation protocol and the pipeline to analyze the results are crucial to identify real chimeric RNAs. Keywords: Chimeric RNAs, Fusion transcripts, RNA-seq, Library preparation protocols The research leading to these results has received funding from the European Union Seventh Framework Programme (FP7/2007-2013), through the European Research Council, under grant agreement Nr. 232913, the Fundación Botín, the Spanish Ministry of Economy and Competitiveness (BIO2007-61762), the National Plan of R + D + i, the ISCIII -Subdirección General de Evaluación y/nFomento de la Investigación- (PI10/01702), and the European Regional Development Fund (ERDF) to the ICREA Research Professor LS. We acknowledge support from the Spanish Ministry of Economy and Competitiveness, ‘Centro de Excelencia Severo Ochoa 2013-2017’ (SEV-2012-0208)
- Published
- 2014
39. In situ overlap and sequence synthesis during DNA assembly
- Author
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Luis Serrano, Carlo Carolis, Maria Lluch-Senar, Bernhard Paetzold, and Tony Ferrar
- Subjects
Genetic Vectors ,Biomedical Engineering ,Cloning vector ,cloning ,Computational biology ,Molecular cloning ,Biology ,de novo synthesis ,Polymerase cycling assembly ,Biochemistry, Genetics and Molecular Biology (miscellaneous) ,overlaps ,03 medical and health sciences ,chemistry.chemical_compound ,Multiple cloning site ,Escherichia coli ,Technical Note ,Cloning, Molecular ,030304 developmental biology ,Genetics ,0303 health sciences ,oligonucleotides ,Oligonucleotide ,030302 biochemistry & molecular biology ,fungi ,food and beverages ,General Medicine ,DNA ,in situ DNA assembly ,Restriction enzyme ,chemistry ,In vitro recombination - Abstract
Modern cloning methods are independent from restriction enzyme recognition sites. However, nearly all current cloning methods still require the introduction of overlaps by PCR, which can introduce undesired mutations. Here, we investigated whether overlaps needed for DNA assembly can be synthesized in situ and we tested if the de novo synthesis of sequences can be simultaneously combined with the assembly of larger double-stranded DNA fragments. We showed in a set of 44 cloning experiments that overlaps of 20 bp needed for DNA assembly can be synthesized in situ from single-stranded oligonucleotides. Short sequences of 30–255 bp can be synthesized from single-stranded oligonucleotides concurrently with DNA assembly, and both techniques can be combined. The assembly of similar constructs by state-of-the-art techniques would have required multiple rounds of cloning or tedious sample preparations, whereas our approach is a one-step reaction.
- Published
- 2013
40. Dissecting the energy metabolism in Mycoplasma pneumoniae through genome‐scale metabolic modeling
- Author
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Edda Klipp, Josep Marcos, Miguel Godinho, Judith A. H. Wodke, Maria Lluch-Senar, Jacek Puchałka, Luis Serrano, Eva Yus, Ricardo Gutiérrez-Gallego, Vítor A. P. Martins dos Santos, Tobias Maier, and EMBL/CRG Systems Biology Research Unit, Centre for Genomic Regulation (CRG), Barcelona, Spain.
- Subjects
reduced bacterium ,ved/biology.organism_classification_rank.species ,Metabolic network ,Bioinformatics ,invivo measurement ,Genome ,metabolic modeling ,Microorganismes patògens ,energy metabolism ,Systems and Synthetic Biology ,genetic-analysis ,bacillus-subtilis ,Mycoplasma pneumonia ,Systeem en Synthetische Biologie ,0303 health sciences ,Applied Mathematics ,Phenotype ,Computational Theory and Mathematics ,General Agricultural and Biological Sciences ,Metabolic Networks and Pathways ,Information Systems ,Metabolisme energètic ,optimal selection ,biochemical networks ,Systems biology ,In silico ,membrane-lipids ,Computational biology ,Biology ,Models, Biological ,Article ,General Biochemistry, Genetics and Molecular Biology ,flux balance models ,03 medical and health sciences ,Computer Simulation ,cobra toolbox extension ,biomass composition ,Model organism ,VLAG ,030304 developmental biology ,in silico knock-outs ,General Immunology and Microbiology ,030306 microbiology ,ved/biology ,Gene Expression Regulation, Bacterial ,Mycoplasma pneumoniae ,Genòmica ,Mutation ,escherichia-coli ,Minimal genome ,Energy Metabolism ,Flux (metabolism) ,Genome, Bacterial ,Genètica - Abstract
A new genome-scale metabolic reconstruction of M. pneumonia is used in combination with external metabolite measurement and protein abundance measurements to quantitatively explore the energy metabolism of this genome-reduce human pathogen., We established a detailed biomass composition for M. pneumoniae, thus allowing for growth simulations. Using our metabolic model, we corrected the metabolic network topology and the functional annotation of key metabolic enzymes. M. pneumoniae, unlike other laboratory-grown bacteria, uses a high fraction of energy (up to 89%) for cellular maintenance and not for growth. Simulating different growth conditions as well as single and double mutant phenotypes, we analyzed pathway connectivity and the impact of gene deletions on the growth performance of M. pneumoniae, highlighting the limited adaptive capabilities of this minimal model organism., Mycoplasma pneumoniae, a threatening pathogen with a minimal genome, is a model organism for bacterial systems biology for which substantial experimental information is available. With the goal of understanding the complex interactions underlying its metabolism, we analyzed and characterized the metabolic network of M. pneumoniae in great detail, integrating data from different omics analyses under a range of conditions into a constraint-based model backbone. Iterating model predictions, hypothesis generation, experimental testing, and model refinement, we accurately curated the network and quantitatively explored the energy metabolism. In contrast to other bacteria, M. pneumoniae uses most of its energy for maintenance tasks instead of growth. We show that in highly linear networks the prediction of flux distributions for different growth times allows analysis of time-dependent changes, albeit using a static model. By performing an in silico knock-out study as well as analyzing flux distributions in single and double mutant phenotypes, we demonstrated that the model accurately represents the metabolism of M. pneumoniae. The experimentally validated model provides a solid basis for understanding its metabolic regulatory mechanisms.
- Published
- 2013
41. Bacterial transcriptomics: what is beyond the RNA horiz-ome?
- Author
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Eva Yus, Luis Serrano, Maria Lluch-Senar, and Marc Güell
- Subjects
Regulation of gene expression ,Genetics ,General Immunology and Microbiology ,Bacteria ,Transcription, Genetic ,Gene Expression Profiling ,RNA ,Gene Expression Regulation, Bacterial ,Biology ,Microbiology ,Bacterial genetics ,Epigenesis, Genetic ,Transcriptome ,Gene expression profiling ,RNA, Bacterial ,Infectious Diseases ,Transcription (biology) ,Operon ,Epigenetics ,Post-transcriptional regulation - Abstract
Over the past 3 years, bacterial transcriptomics has undergone a massive revolution. Increased sequencing capacity and novel tools have made it possible to explore the bacterial transcriptome to an unprecedented depth, which has revealed that the transcriptome is more complex and dynamic than expected. Alternative transcripts within operons challenge the classic operon definition, and many small RNAs involved in the regulation of transcription, translation and pathogenesis have been discovered. Furthermore, mRNAs may localize to specific areas in the cell, and the spatial organization and dynamics of the chromosome have been shown to be important for transcription. Epigenetic modifications of DNA also affect transcription, and RNA processing affects translation. Therefore, transcription in bacteria resembles that in eukaryotes in terms of complexity more closely than was previously thought. Here we will discuss the contribution of 'omics' approaches to these discoveries as well as the possible impact that they are expected to have in the future.
- Published
- 2011
42. A trigger enzyme in Mycoplasma pneumoniae: impact of the glycerophosphodiesterase GlpQ on virulence and gene expression
- Author
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Dörte Becher, Andreas Otto, Julia Busse, Jaume Piñol, Sebastian R. Schmidl, Jörg Stülke, Maria Lluch-Senar, and Flynn, JoAnne L.
- Subjects
Glycerol ,Proteome ,Cytotoxicity ,Mutant ,Enzyme Metabolism ,Biochemistry ,Transcription (biology) ,Gene Duplication ,Microbial Physiology ,Gene expression ,Coding region ,lcsh:QH301-705.5 ,0303 health sciences ,Virulence ,Phenotype ,3. Good health ,Enzymes ,Bacterial Pathogens ,Bacterial Biochemistry ,Complementation ,Host-Pathogen Interaction ,Medical Microbiology ,Research Article ,lcsh:Immunologic diseases. Allergy ,Cell Survival ,Immunology ,Glycerolphosphate Dehydrogenase ,Biology ,Microbiology ,Molecular Genetics ,03 medical and health sciences ,Virology ,Genetics ,Humans ,HeLa cells ,Mutant strains ,Molecular Biology ,Gene ,030304 developmental biology ,Gram Positive ,030306 microbiology ,Phosphoric Diester Hydrolases ,Proteins ,Bacteriology ,Gene Expression Regulation, Bacterial ,Hydrogen Peroxide ,Cycoplasma pneumoniae ,Hydrogen peroxide ,Lipid Metabolism ,Molecular biology ,Mycoplasma pneumoniae ,Glucose ,Metabolism ,lcsh:Biology (General) ,Trigger ,Enzyme ,Glycerophosphodiesterase ,GlpQ ,Gene Expression ,Parasitology ,lcsh:RC581-607 ,HeLa Cells - Abstract
Mycoplasma pneumoniae is a causative agent of atypical pneumonia. The formation of hydrogen peroxide, a product of glycerol metabolism, is essential for host cell cytotoxicity. Phosphatidylcholine is the major carbon source available on lung epithelia, and its utilization requires the cleavage of deacylated phospholipids to glycerol-3-phosphate and choline. M. pneumoniae possesses two potential glycerophosphodiesterases, MPN420 (GlpQ) and MPN566. In this work, the function of these proteins was analyzed by biochemical, genetic, and physiological studies. The results indicate that only GlpQ is an active glycerophosphodiesterase. MPN566 has no enzymatic activity as glycerophosphodiesterase and the inactivation of the gene did not result in any detectable phenotype. Inactivation of the glpQ gene resulted in reduced growth in medium with glucose as the carbon source, in loss of hydrogen peroxide production when phosphatidylcholine was present, and in a complete loss of cytotoxicity towards HeLa cells. All these phenotypes were reverted upon complementation of the mutant. Moreover, the glpQ mutant strain exhibited a reduced gliding velocity. A comparison of the proteomes of the wild type strain and the glpQ mutant revealed that this enzyme is also implicated in the control of gene expression. Several proteins were present in higher or lower amounts in the mutant. This apparent regulation by GlpQ is exerted at the level of transcription as determined by mRNA slot blot analyses. All genes subject to GlpQ-dependent control have a conserved potential cis-acting element upstream of the coding region. This element overlaps the promoter in the case of the genes that are repressed in a GlpQ-dependent manner and it is located upstream of the promoter for GlpQ-activated genes. We may suggest that GlpQ acts as a trigger enzyme that measures the availability of its product glycerol-3-phosphate and uses this information to differentially control gene expression., Author Summary Mycoplasma pneumoniae serves as a model organism for bacteria with very small genomes that are nonetheless independently viable. These bacteria infect the human lung and cause an atypical pneumonia. The major virulence determinant of M. pneumoniae is hydrogen peroxide that is generated during the utilization of glycerol-3-phosphate, which might be derived from free glycerol or from the degradation of phospholipids. Indeed, lecithin is the by far most abundant carbon source on lung epithelia. In this study, we made use of the recent availability of methods to isolate mutants of M. pneumoniae and characterized the enzyme that generates glycerol-3-phosphate from deacylated lecithin (glycerophosphocholine). This enzyme, called GlpQ, is essential for the formation of hydrogen peroxide when the bacteria are incubated with glycerophosphocholine. Moreover, M. pneumoniae is unable to cause any detectable damage to the host cells in the absence of GlpQ. This underlines the important role of phospholipid metabolism for the virulence of M. pneumoniae. We observed that GlpQ in addition to its enzymatic activity is also involved in the control of expression of several genes, among them the glycerol transporter. Thus, GlpQ is central to the normal physiology and to pathogenicity of the minimal pathogen M. pneumoniae.
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- 2011
43. Cell division in a minimal bacterium in the absence of ftsZ
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Maria, Lluch-Senar, Enrique, Querol, and Jaume, Piñol
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Cytoskeletal Proteins ,Transformation, Genetic ,Bacterial Proteins ,Genes, Bacterial ,Genetic Complementation Test ,Mycoplasma genitalium ,Bacterial Adhesion ,Cell Division ,Gene Deletion ,Cytokinesis - Abstract
Mycoplasma genomes exhibit an impressively low amount of genes involved in cell division and some species even lack the ftsZ gene, which is found widespread in the microbial world and is considered essential for cell division by binary fission. We constructed a Mycoplasma genitalium ftsZ null mutant by gene replacement to investigate the role of this gene and the presence of alternative cell division mechanisms in this minimal bacterium. Our results demonstrate that ftsZ is non-essential for cell growth and reveal that, in the absence of the FtsZ protein, M. genitalium can manage feasible cell divisions and cytokinesis using the force generated by its motile machinery. This is an alternative mechanism, completely independent of the FtsZ protein, to perform cell division by binary fission in a microorganism. We also propose that the mycoplasma cytoskeleton, a complex network of proteins involved in many aspects of the biology of these microorganisms, may have taken over the function of many genes involved in cell division, allowing their loss in the regressive evolution of the streamlined mycoplasma genomes.
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- 2010
44. Comprehensive Methylome Characterization of Mycoplasma genitalium and Mycoplasma pneumoniae at Single-Base Resolution
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Luis Serrano, Stephen Turner, Javier Delgado, Maria Lluch-Senar, Verónica Lloréns-Rico, Eric E. Schadt, Gang Fang, Kristi E. Spittle, Khai Luong, Tyson A. Clark, and Jonas Korlach
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Cancer Research ,lcsh:QH426-470 ,DNA repair ,DNA transcription ,Mycoplasma genitalium ,Biology ,Microbiology ,Genome ,03 medical and health sciences ,Micoplasmes ,Genetics ,Humans ,Genome Sequencing ,Epigenetics ,Nucleotide Motifs ,Microbial Pathogens ,Molecular Biology ,Genetics (clinical) ,Ecology, Evolution, Behavior and Systematics ,030304 developmental biology ,Regulation of gene expression ,0303 health sciences ,Chromosome Biology ,030306 microbiology ,Systems Biology ,Methyltransferases ,Genomics ,Methylation ,DNA Methylation ,Comparative Genomics ,biology.organism_classification ,Mycoplasma pneumoniae ,3. Good health ,lcsh:Genetics ,DNA methylation ,Gene expression ,Gene Expression Regulation, Archaeal ,Metilació ,DNA modification ,Sequence motif ,Genètica ,Genome, Bacterial ,Research Article - Abstract
In the bacterial world, methylation is most commonly associated with restriction-modification systems that provide a defense mechanism against invading foreign genomes. In addition, it is known that methylation plays functionally important roles, including timing of DNA replication, chromosome partitioning, DNA repair, and regulation of gene expression. However, full DNA methylome analyses are scarce due to a lack of a simple methodology for rapid and sensitive detection of common epigenetic marks (ie N6-methyladenine (6 mA) and N4-methylcytosine (4 mC)), in these organisms. Here, we use Single-Molecule Real-Time (SMRT) sequencing to determine the methylomes of two related human pathogen species, Mycoplasma genitalium G-37 and Mycoplasma pneumoniae M129, with single-base resolution. Our analysis identified two new methylation motifs not previously described in bacteria: a widespread 6 mA methylation motif common to both bacteria (5′-CTAT-3′), as well as a more complex Type I m6A sequence motif in M. pneumoniae (5′-GAN7TAY-3′/3′-CTN7 ATR-5′). We identify the methyltransferase responsible for the common motif and suggest the one involved in M. pneumoniae only. Analysis of the distribution of methylation sites across the genome of M. pneumoniae suggests a potential role for methylation in regulating the cell cycle, as well as in regulation of gene expression. To our knowledge, this is one of the first direct methylome profiling studies with single-base resolution from a bacterial organism., Author Summary DNA methylation in bacteria plays important roles in cell division, DNA repair, regulation of gene expression, and pathogenesis. Here, we use a novel sequencing technique, Single-Molecule Real-Time (SMRT) sequencing, to determine the methylomes of two related human pathogen species, Mycoplasma genitalium G-37 and Mycoplasma pneumoniae M129. Our analysis identified two novel methylation motifs, one of them present uniquely in M. pneumoniae and the other common to both bacteria. We also identify the methyltransferase responsible for the common methylation motif and suggest the one associated with the M. pneumoniae unique motif. Functional analysis of the data suggests a potential role for methylation in regulating the cell cycle of M. pneumoniae, as well as in regulation of gene expression. To our knowledge, this is one of the first genome-wide approaches to study the biological role of methylation in a bacterial organism.
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- 2013
45. Defining a minimal cell: essentiality of small ORFs and ncRNAs in a genome-reduced bacterium
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Jörg Stülke, Eva Yus, Robert J. Nichols, Javier Delgado, Sira Martínez, Judith A. H. Wodke, Anne-Claude Gavin, Maria Lluch-Senar, Arne Schmeisky, Peer Bork, Francis J. O’Reilly, Verónica Lloréns-Rico, E. Besray Unal, Wei-Hua Chen, Vera van Noort, Luis Serrano, Ana Vivancos, and Tony Ferrar
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DNA, Bacterial ,RNA, Untranslated ,Transcription, Genetic ,Protein Conformation ,Biology ,Genome ,General Biochemistry, Genetics and Molecular Biology ,Non-coding RNAs ,Open Reading Frames ,chemistry.chemical_compound ,Minimal genome ,ORFS ,Gene ,minimal genome ,non-coding RNAs ,small proteins ,Genetics ,Genes, Essential ,General Immunology and Microbiology ,Applied Mathematics ,RNA ,Sequence Analysis, DNA ,biology.organism_classification ,Small proteins ,Mycoplasma pneumoniae ,RNA no missatgers ,Computational Theory and Mathematics ,chemistry ,Cardiovascular and Metabolic Diseases ,General Agricultural and Biological Sciences ,Genome, Bacterial ,DNA ,Function (biology) ,Bacteria ,Interaccions RNA-proteïna ,Reports ,Information Systems - Abstract
Identifying all essential genomic components is critical for the assembly of minimal artificial life. In the genome-reduced bacterium Mycoplasma pneumoniae, we found that small ORFs (smORFs; < 100 residues), accounting for 10% of all ORFs, are the most frequently essential genomic components (53%), followed by conventional ORFs (49%). Essentiality of smORFs may be explained by their function as members of protein and/or DNA/RNA complexes. In larger proteins, essentiality applied to individual domains and not entire proteins, a notion we could confirm by expression of truncated domains. The fraction of essential non-coding RNAs (ncRNAs) non-overlapping with essential genes is 5% higher than of non-transcribed regions (0.9%), pointing to the important functions of the former. We found that the minimal essential genome is comprised of 33% (269,410 bp) of the M. pneumoniae genome. Our data highlight an unexpected hidden layer of smORFs with essential functions, as well as non-coding regions, thus changing the focus when aiming to define the minimal essential genome. ispartof: MOLECULAR SYSTEMS BIOLOGY vol:11 issue:1 ispartof: location:England status: published
46. Distinguishing between productive and abortive promoters using a random forest classifier in Mycoplasma pneumoniae
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Luis Serrano, Verónica Lloréns-Rico, and Maria Lluch-Senar
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Mycoplasma pneumoniae ,Bacterial genome size ,Biology ,medicine.disease_cause ,Genome ,Mycoplasma pneumoniae -- Aspectes genètics ,Transcriptome ,03 medical and health sciences ,Transcription (biology) ,Pneumonia, Mycoplasma ,Genetics ,medicine ,Promoter Regions, Genetic ,Gene ,030304 developmental biology ,0303 health sciences ,Genètica bacteriana ,030302 biochemistry & molecular biology ,Computational Biology ,Promoter ,Genomes bacterians ,Models, Theoretical ,biology.organism_classification ,Mycoplasma pneumoniae -- Metabolisme ,Genes, Bacterial ,Bacteria - Abstract
Distinguishing between promoter-like sequences in bacteria that belong to true or abortive promoters, or to those that do not initiate transcription at all, is one of the important challenges in transcriptomics. To address this problem, we have studied the genome-reduced bacterium Mycoplasma pneumoniae, for which the RNAs associated with transcriptional start sites have been recently experimentally identified. We determined the contribution to transcription events of different genomic features: the -10, extended -10 and -35 boxes, the UP element, the bases surrounding the -10 box and the nearest-neighbor free energy of the promoter region. Using a random forest classifier and the aforementioned features transformed into scores, we could distinguish between true, abortive promoters and non-promoters with good -10 box sequences. The methods used in this characterization of promoters can be extended to other bacteria and have important applications for promoter design in bacterial genome engineering. European Union Seventh Framework Programme (FP7/2007–2013), through the European Research Council [232913]; Fundación Botín, the Spanish Ministry of Economy and Competitiveness [BIO2007-61762]; National Plan of R + D + i; ISCIII – Subdirección General de Evaluación y Fomento de la Investigación [PI10/01702]; European Regional Development Fund (ERDF) (to the ICREA Research Professor L.S.]; Spanish Ministry of Economy and Competitiveness, ‘Centro de Excelencia Severo Ochoa 2013–2017 [SEV-2012-0208]. Funding for open access charge: European Union Seventh Framework Programme (FP7/2007–2013), through the European Research Council [232913]; Fundaci´on Bot´ın, the Spanish Ministry of Economy and Competitiveness [BIO2007-61762]; National Plan of R + D + i; ISCIII – Subdirección General de Evaluación y Fomento de la Investigación [PI10/01702]; European Regional Development Fund (ERDF) (to the ICREA Research Professor L.S.]; Spanish Ministry of Economy and Competitiveness, ‘Centro de Excelencia Severo Ochoa 2013–2017 [SEV-2012-0208].
47. Bacterial antisense RNAs are mainly the product of transcriptional noise
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Amparo Latorre, Luis Serrano, John I. Glass, Verónica Lloréns-Rico, Maria Lluch-Senar, Tjerko Kamminga, Jaime González Cano, Wei-Hua Chen, Rosario Gil, and Peer Bork
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0301 basic medicine ,Transcription, Genetic ,Bacterial antisense RNAs ,030106 microbiology ,information science ,Biology ,Genome ,Transcriptome ,03 medical and health sciences ,Species Specificity ,Transcription (biology) ,medicine ,Life Science ,natural sciences ,RNA, Antisense ,Systems and Synthetic Biology ,Research Articles ,Genetics ,Biomolecules ,Messenger RNA ,Systeem en Synthetische Biologie ,Multidisciplinary ,RNA ,SciAdv r-articles ,Promoter ,social sciences ,medicine.disease ,equipment and supplies ,health care quality, access, and evaluation ,Chloroplast ,RNA, Bacterial ,Cardiovascular and Metabolic Diseases ,bacterial antisense RNAs ,Transcriptional noise ,Research Article - Abstract
Most of the antisense transcripts in bacteria are the product of transcriptional noise derived from spurious promoters., cis-Encoded antisense RNAs (asRNAs) are widespread along bacterial transcriptomes. However, the role of most of these RNAs remains unknown, and there is an ongoing discussion as to what extent these transcripts are the result of transcriptional noise. We show, by comparative transcriptomics of 20 bacterial species and one chloroplast, that the number of asRNAs is exponentially dependent on the genomic AT content and that expression of asRNA at low levels exerts little impact in terms of energy consumption. A transcription model simulating mRNA and asRNA production indicates that the asRNA regulatory effect is only observed above certain expression thresholds, substantially higher than physiological transcript levels. These predictions were verified experimentally by overexpressing nine different asRNAs in Mycoplasma pneumoniae. Our results suggest that most of the antisense transcripts found in bacteria are the consequence of transcriptional noise, arising at spurious promoters throughout the genome.
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48. Widespread ribosome stalling in a genome-reduced bacterium and the need for translational quality control
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Luis Serrano, Carolina Gallo, Marc Weber, Maria Lluch-Senar, and Raul Burgos
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medicine.medical_treatment ,Proteolysis ,Science ,Mutant ,Ribosome ,Genome ,Article ,03 medical and health sciences ,transcriptomics ,cell biology ,medicine ,molecular biology ,030304 developmental biology ,0303 health sciences ,Multidisciplinary ,Protease ,Bacteriologia ,medicine.diagnostic_test ,biology ,030306 microbiology ,molecular mechanism of gene regulation ,biological sciences ,RNA ,Cell biology ,Genòmica ,Proteostasis ,Chaperone (protein) ,biology.protein ,Proteïnes ,Genètica - Abstract
Summary Trans-translation is a ubiquitous bacterial mechanism of ribosome rescue mediated by a transfer-messenger RNA (tmRNA) that adds a degradation tag to the truncated nascent polypeptide. Here, we characterize this quality control system in a genome-reduced bacterium, Mycoplasma pneumoniae (MPN), and perform a comparative analysis of protein quality control components in slow and fast-growing prokaryotes. We show in vivo that in MPN the sole quality control cytoplasmic protease (Lon) degrades efficiently tmRNA-tagged proteins. Analysis of tmRNA-mutants encoding a tag resistant to proteolysis reveals extensive tagging activity under normal growth. Unlike knockout strains, these mutants are viable demonstrating the requirement of tmRNA-mediated ribosome recycling. Chaperone and Lon steady-state levels maintain proteostasis in these mutants suggesting a model in which co-evolution of Lon and their substrates offer simple mechanisms of regulation without specialized degradation machineries. Finally, comparative analysis shows relative increase in Lon/Chaperone levels in slow-growing bacteria suggesting physiological adaptation to growth demand., Graphical abstract, Highlights • Lon degrades efficiently tmRNA-tagged proteins in a genome-reduced bacterium • tmRNA-tag mutants are viable and reveal extensive tagging activity in M. pneumoniae • Co-evolution of Lon and their substrates offer simple mechanisms of regulation • Chaperone and Lon relative levels correlate with bacterial growth rates, Biological sciences; Molecular biology; Molecular mechanism of gene regulation; Cell biology; Transcriptomics
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