Integrative understanding of transcription in a minimal cell model

One of the major challenges of biology is to understand how entire cells or organisms behave in homeostasis and in response to perturbations. Whole-cell modeling promotes this understanding by integrating different cellular processes in a single model that is able to predict emergent cellular behaviors. In this thesis, we have developed the first whole-cell model of the genome-reduced bacterium Mycoplasma pneumoniae, which encodes for less than 700 protein-coding genes. This model follows the structure of the previously described model in Mycoplasma genitalium. However, the lack of comprehensive knowledge of even these simple organisms limits the predictive power of these models. To address this problem and improve the model, we have focused in the process of transcription regulation, and we have studied the major determinants of transcript abundance in this bacterium. Therefore, we have characterized promoters and the role of small RNAs. We have also reconstructed the gene regulatory network, revealing that non-transcription factor regulation may have a large impact in coordinating RNA levels in M. pneumoniae. Furthermore, by analyzing the ‘omics’ data used to investigate the process of transcription and to fit the whole-cell model, we have found different biases of high-throughput profiling experiments, and we have described that chimeric RNAs identified in these datasets may be artifacts generated in RNA-sequencing experiments.Uno de los mayores retos de la biología es entender cómo células u organismos completos se comportan tanto en homeostasis como en respuesta a perturbaciones. El campo del modelado de células completas pretende comprender esto integrando diferentes procesos celulares en un único modelo capaz de predecir comportamientos celulares emergentes. En esta tesis, hemos desarrollado el primer modelo de célula completa de la bacteria de genoma reducido Mycoplasma pneumoniae, que codifica para menos de 700 proteínas. Este modelo sigue la estructura del descrito previamente en Mycoplasma genitalium. Sin embargo, la falta de conocimientos exhaustivos incluso para estos organismos simples limita el poder predictivo de estos modelos. Para hacer frente a este problema y mejorar el modelo, nos hemos centrado en el proceso de regulación de la transcripción, y hemos estudiando los principales factores que determinan la abundancia de tránscritos en esta bacteria. Así, hemos caracterizado los promotores y el papel de ARNs pequeños. También hemos reconstruido la red de regulación génica, observando que la regulación no debida a factores de transcripción puede tener un gran impacto en la coordinación de los niveles de ARN en M. pneumoniae. Además, analizando los datos procedentes de experimentos ómicos usados para investigar el proceso de la transcripción y ajustar el modelo, hemos encontrado diferentes sesgos en estos experimentos a gran escala u ‘ómicos’, y hemos descrito que ARNs quiméricos que se identifican en estos datos pueden ser artefactos generados en experimentos de secuenciación de ARN

Alternative transcriptional regulation in genome-reduced bacteria

Transcription is a core process of bacterial physiology, and as such it must be tightly controlled, so that bacterial cells maintain steady levels of each RNA molecule in homeostasis and modify them in response to perturbations. The major regulators of transcription in bacteria (and in eukaryotes) are transcription factors. However, in genome-reduced bacteria, the limited number of these proteins is insufficient to explain the variety of responses shown upon changes in their environment. Thus, alternative regulators may play a central role in orchestrating RNA levels in these microorganisms. These alternative mechanisms rely on intrinsic features within DNA and RNA molecules, suggesting they are ancestral mechanisms shared among bacteria that could have an increased relevance on transcriptional regulation in minimal cells. In this review, we summarize the alternative elements that can regulate transcript abundance in genome-reduced bacteria and how they contribute to the RNA homeostasis at different levels.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) and the Spanish Ministry of Economy and Competitiveness, 'Centro de Excelencia Severo Ochoa 2013-2017′

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

Alternative transcriptional regulation in genome-reduced bacteria

Transcription is a core process of bacterial physiology, and as such it must be tightly controlled, so that bacterial cells maintain steady levels of each RNA molecule in homeostasis and modify them in response to perturbations. The major regulators of transcription in bacteria (and in eukaryotes) are transcription factors. However, in genome-reduced bacteria, the limited number of these proteins is insufficient to explain the variety of responses shown upon changes in their environment. Thus, alternative regulators may play a central role in orchestrating RNA levels in these microorganisms. These alternative mechanisms rely on intrinsic features within DNA and RNA molecules, suggesting they are ancestral mechanisms shared among bacteria that could have an increased relevance on transcriptional regulation in minimal cells. In this review, we summarize the alternative elements that can regulate transcript abundance in genome-reduced bacteria and how they contribute to the RNA homeostasis at different levels.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) and the Spanish Ministry of Economy and Competitiveness, 'Centro de Excelencia Severo Ochoa 2013-2017′

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

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]

Insights into the Mechanisms of Basal Coordination of Transcription Using a Genome-Reduced Bacterium

Coordination of transcription in bacteria occurs at supra-operonic scales, but the extent, specificity, and mechanisms of such regulation are poorly understood. Here, we tackle this problem by profiling the transcriptome of the model organism Mycoplasma pneumoniae across 115 growth conditions. We identify three qualitatively different levels of co-expression corresponding to distinct relative orientations and intergenic properties of adjacent genes. We reveal that the degree of co-expression between co-directional adjacent operons, and more generally between genes, is tightly related to their capacity to be transcribed en bloc into the same mRNA. We further show that this genome-wide pervasive transcription of adjacent genes and operons is specifically repressed by DNA regions preferentially bound by RNA polymerases, by intrinsic terminators, and by large intergenic distances. Taken together, our findings suggest that the basal coordination of transcription is mediated by the physical entities and mechanical properties of the transcription process itself, and that operon-like behaviors may strongly vary from condition to condition.This work was supported by Fundación Marcelino Botin and the Spanish Ministerio de Economía y Competitividad (BIO2007-61762). This project was financed by Instituto de Salud Carlos III and co-financed by Federación Española de Enfermedades Raras under grant agreement PI10/01702 and the European Research Council and European Union’s Horizon 2020 research and innovation program under grant agreements 634942 (MycoSynVac) and 670216 (MYCOCHASSIS). The Centre for Genomic Regulation acknowledges the support of the Spanish Ministry of Economy and Competitiveness, “Centro de Excelencia Severo Ochoa 2013-2017,” SEV-2012-0208

Correction: Integrated culturing, modeling and transcriptomics uncovers complex interactions and emergent behavior in a three-species synthetic gut community.

status: Published onlin

Comparative "-omics" in mycoplasma pneumoniae clinical isolates reveals key virulence factors

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