A Trigger Enzyme in Mycoplasma pneumoniae: Impact of the Glycerophosphodiesterase GlpQ on Virulence and Gene Expression

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

Estudio de la Expresión Génica y la División Celular en Mycoplasma genitalium

Los micoplasmas son bacterias sin pared celular que pertenecen a la clase Mollicutes. Estos microorganismos se caracterizan por su pequeño tamaño y por tener un genoma reducido con un bajo porcentaje de C+G. Muchas especies de mycoplasma actúan como parásitos y patógenos de un amplio rango de huéspedes, causando enfermedades comunes en humanos. Por ejemplo, Mycoplasma genitalium, objeto de esta tesis, es el causante de la uretritis no gonocócica. La secuenciación del genoma de algunos micoplasmas ha favorecido el conocimiento de la fisiología y genética de estos microorganismos, pero algunos mecanismos como la regulación de la expresión génica y la división celular siguen siendo aún un misterio por desvelar. M. genitalium, con tan sólo 525 genes, es considerado un modelo de célula mínima. Posee el genoma más pequeño de entre todas las bacterias que se pueden cultivar axénicamente, siendo así un candidato ideal para el estudio de los procesos biológicos con el mínimo número de genes implicados. El trabajo presentado en esta tesis se divide en tres capítulos independientes. En el primero se estudia la regulación de la expresión génica en M. genitalium y M. pneumoniae. El trabajo con M. pneumoniae fue llevado a cabo durante una estancia en el laboratorio del Prof. J. Stülke (Göttingen, Alemania). Este primer trabajo dio lugar a un artículo publicado en la revista Microbiology. La obtención de un mutante por transposición de M. genitalium que deleccionaba el gen ftsZ, descrito como esencial para la división celular en la mayor parte de bacterias, derivó en el segundo capítulo de esta tesis. En este segundo trabajo se investiga la división celular en micoplasma en ausencia de este gen. Este estudio ha sido publicado recientemente en la revista Molecular Microbiology. Por último, con el objetivo de profundizar en el tema de la división celular en micoplasma, en el tercer capítulo se analiza la funcionalidad del gen mraZ, que junto con mraW, mg223 y ftsZ conforma el operón de división celular de M. genitalium.Mycoplasmas are the smallest and simplest free-living microorganisms. They are members of the Mollicutes class which is characterized by the absence of cell wall and by having genomes with a low G+C content. Despite this apparent simplicity, some mycoplasma species are parasites and pathogens of a wide range of hosts. For instance, Mycoplasma genitalium, which is the object of this thesis, is the agent of non-gonococcal and non-chlamydial urethritis. Genome sequencing has advanced the knowledge concerning the physiology and genetics of these microorganisms, but some mechanisms such as the regulation of gene expression and the cell division remain unclear. With only 525 genes M. genitalium is considered a model of minimal cell, since it has the smallest genome of any microorganism that can be grown in a pure or axenic culture. It is considered a perfect candidate to study biological processes with the minimal set of genes. This thesis is divided into three independent chapters. In the first one we study the regulation of gene expression in M. genitalium and M. pneumoniae. This work was published in 2007 in the journal "Microbiology". A minitransposon insertion was found in the ftsZ gene of M. genitalium, indicating that this gene was not essential for cell division in this microorganism. In the second chapter of this thesis we investigate the cell division process in M. genitalium in the absence of ftsZ. This work was recently published in the journal "Molecular Microbiology". Finally, to gain insight into the cell division process in mycoplasma, in the third chapter we analyze the function of the mraZ gene, which together with mraW, mg223 and ftsZ comprise the cell division operon of M. genitalium

Lox'd in translation: contradictions in the nomenclature surrounding common lox-site mutants and their implications in experiments

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 Cataluny

La biologia de sistemes: la biologia del segle XXI?

Al final del segle xx un nou món va emergir dins la ciència, la biologia de sistemes. Aquest terme, que molts consideren una paraula de moda, està ben establert en la comunitat científica. Fins i tot disposem de diversos instituts o departaments a Europa i els Estats Units amb aquest nom. La definició de biologia de sistemes ha esdevingut un dels grans dilemes per als científics. L?ampli ventall de definicions va des de col·leccions de dades fisiològiques amb llistes de parts moleculars quantificades (p. ex., gens, nivells d?expressió, localitzacions), fins a la modelització matemàtica abstracta de processos biològics. L?escala en què se centra la biologia del sistemes és també un afer discutit; una proteïna minúscula pot ser un sistema biològic tan complex (encara no sabem com es plega) com un ecosistema sencer amb milers d?espècies. El terme biologia del sistemes probablement augmentarà les seves accepcions fins a arribar a un primer pla i les oportunitats de finançament haurien de ser agafades seriosament per les comunitats científiques. En principi la biologia de sistemes ofereix l?oportunitat d?entendre processos biològics i obre la possibilitat de modificar-los i fer enginyeria d?una manera racional (biologia sintètica). La biologia de sistemes és aquí per quedar-se i obrir camí a teràpies noves, a la medicina individualitzada i, en combinació amb la biologia sintètica, a l?enginyeria racional dels sistemes vius.Systems biology: The biology of the XXI century? At the end of the 20th century a new world emerged in science, systems biology. This term that many consider a buzzword is now well established in the scientific community and we even have several institutes or departments in Europe and the United States bearing this name. However, scientists remain in a quandary about defining Systems Biology. Definitions range from collections of physiological data with quantified molecular parts lists (e.g. genes, expression levels, localizations) to abstract mathematical modelling of biological processes. The scale at which Systems Biology focuses is also a matter of contention: a tiny protein can be a complicated biological system (we still do not know how it folds) as is obviously an entire ecosystem with thousands of species. The term «Systems Biology» will probably broaden its meaning even further as it is now under the limelight and funding opportunities have to be taken seriously by very diverse scientific communities. In principle Systems Biology offers an opportunity to understand biological processes in a way in which we could modify and engineer them in a rational manner (Synthetic Biology). Systems Biology is here to stay and will open the way to new therapies, individualized medicine and, in combination with synthetic biology, to the rational engineering of living systems

FASTQINS and ANUBIS: two bioinformatic tools to explore facts and artifacts in transposon sequencing and essentiality studies

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.ISSN:1362-4962ISSN:0301-561

Assessing the hodgepodge of non-mapped reads in bacterial transcriptomes: real or artifactual RNA chimeras?

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 protocolsThe 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

La biologia de sistemes: la biologia del segle XXI?

Al final del segle xx un nou món va emergir dins la ciència, la biologia de sistemes. Aquest terme, que molts consideren una paraula de moda, està ben establert en la comunitat científica. Fins i tot disposem de diversos instituts o departaments a Europa i els Estats Units amb aquest nom. La definició de biologia de sistemes ha esdevingut un dels grans dilemes per als científics. L?ampli ventall de definicions va des de col·leccions de dades fisiològiques amb llistes de parts moleculars quantificades (p. ex., gens, nivells d?expressió, localitzacions), fins a la modelització matemàtica abstracta de processos biològics. L?escala en què se centra la biologia del sistemes és també un afer discutit; una proteïna minúscula pot ser un sistema biològic tan complex (encara no sabem com es plega) com un ecosistema sencer amb milers d?espècies. El terme biologia del sistemes probablement augmentarà les seves accepcions fins a arribar a un primer pla i les oportunitats de finançament haurien de ser agafades seriosament per les comunitats científiques. En principi la biologia de sistemes ofereix l?oportunitat d?entendre processos biològics i obre la possibilitat de modificar-los i fer enginyeria d?una manera racional (biologia sintètica). La biologia de sistemes és aquí per quedar-se i obrir camí a teràpies noves, a la medicina individualitzada i, en combinació amb la biologia sintètica, a l?enginyeria racional dels sistemes vius.Systems biology: The biology of the XXI century? At the end of the 20th century a new world emerged in science, systems biology. This term that many consider a buzzword is now well established in the scientific community and we even have several institutes or departments in Europe and the United States bearing this name. However, scientists remain in a quandary about defining Systems Biology. Definitions range from collections of physiological data with quantified molecular parts lists (e.g. genes, expression levels, localizations) to abstract mathematical modelling of biological processes. The scale at which Systems Biology focuses is also a matter of contention: a tiny protein can be a complicated biological system (we still do not know how it folds) as is obviously an entire ecosystem with thousands of species. The term «Systems Biology» will probably broaden its meaning even further as it is now under the limelight and funding opportunities have to be taken seriously by very diverse scientific communities. In principle Systems Biology offers an opportunity to understand biological processes in a way in which we could modify and engineer them in a rational manner (Synthetic Biology). Systems Biology is here to stay and will open the way to new therapies, individualized medicine and, in combination with synthetic biology, to the rational engineering of living systems

Distinguishing between productive and abortive promoters using a random forest classifier in Mycoplasma pneumoniae

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]

LoxTnSeq: random transposon insertions combined with cre/lox recombination and counterselection to generate large random genome reductions

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.ISSN:1751-7915ISSN:1751-790

Assessing the hodgepodge of non-mapped reads in bacterial transcriptomes: real or artifactual RNA chimeras?

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 protocolsThe 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