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1.
Mol Syst Biol ; 19(10): e11301, 2023 Oct 12.
Artículo en Inglés | MEDLINE | ID: mdl-37642167

RESUMEN

Translation efficiency has been mainly studied by ribosome profiling, which only provides an incomplete picture of translation kinetics. Here, we integrated the absolute quantifications of tRNAs, mRNAs, RNA half-lives, proteins, and protein half-lives with ribosome densities and derived the initiation and elongation rates for 475 genes (67% of all genes), 73 with high precision, in the bacterium Mycoplasma pneumoniae (Mpn). We found that, although the initiation rate varied over 160-fold among genes, most of the known factors had little impact on translation efficiency. Local codon elongation rates could not be fully explained by the adaptation to tRNA abundances, which varied over 100-fold among tRNA isoacceptors. We provide a comprehensive quantitative view of translation efficiency, which suggests the existence of unidentified mechanisms of translational regulation in Mpn.

2.
Mol Syst Biol ; 19(1): e11037, 2023 01.
Artículo en Inglés | MEDLINE | ID: mdl-36598022

RESUMEN

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.


Asunto(s)
Interleucina-10 , Neumonía , Ratones , Humanos , Animales , Interleucina-10/genética , Pulmón , Neumonía/prevención & control , Neumonía/patología , Citocinas , Inflamación/patología , Bacterias , Pseudomonas aeruginosa
3.
Nucleic Acids Res ; 50(22): e127, 2022 12 09.
Artículo en Inglés | MEDLINE | ID: mdl-36215032

RESUMEN

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.


Asunto(s)
Edición Génica , Mycoplasma pneumoniae , Sistemas CRISPR-Cas , Mycoplasma pneumoniae/genética , Plásmidos/genética
4.
Mol Syst Biol ; 17(10): e10145, 2021 10.
Artículo en Inglés | MEDLINE | ID: mdl-34612607

RESUMEN

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.


Asunto(s)
Biopelículas , Staphylococcus aureus , Antibacterianos , Humanos , Pruebas de Sensibilidad Microbiana , Staphylococcus aureus/genética , Factores de Virulencia
5.
Nucleic Acids Res ; 48(17): e102, 2020 09 25.
Artículo en Inglés | MEDLINE | ID: mdl-32813015

RESUMEN

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.


Asunto(s)
Elementos Transponibles de ADN , Genómica/métodos , Análisis de Secuencia de ADN/métodos , Programas Informáticos , Genómica/normas , Mycoplasma pneumoniae , Recombinación Genética , Análisis de Secuencia de ADN/normas
6.
Microbiology (Reading) ; 167(1)2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-33284099

RESUMEN

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.


Asunto(s)
Bacterias/genética , Anotación de Secuencia Molecular , Mycoplasma pneumoniae/genética , Recombinación Genética , Secuencia de Bases , Ingeniería Genética , Mutación , Terminología como Asunto
7.
Mol Syst Biol ; 16(12): e9530, 2020 12.
Artículo en Inglés | MEDLINE | ID: mdl-33320415

RESUMEN

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.


Asunto(s)
Proteínas Bacterianas/metabolismo , Genoma Bacteriano , Mycoplasma pneumoniae/genética , Proteolisis , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Modelos Biológicos , Mutación/genética , Mycoplasma pneumoniae/enzimología , Péptido Hidrolasas/metabolismo , Fenotipo , Pliegue de Proteína , Control de Calidad , Reproducibilidad de los Resultados , Especificidad por Sustrato , Transcripción Genética
8.
Mol Syst Biol ; 16(5): e9208, 2020 05.
Artículo en Inglés | MEDLINE | ID: mdl-32449593

RESUMEN

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.


Asunto(s)
Aminoácidos/química , Bacterias/química , Bacterias/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Genes Bacterianos , Procesamiento Proteico-Postraduccional , Secuencia de Aminoácidos , Aminoácidos/metabolismo , Aminoácidos Aromáticos/química , Aminoácidos Aromáticos/metabolismo , Arginina/química , Arginina/metabolismo , Bacterias/genética , Proteínas Bacterianas/clasificación , Proteínas Bacterianas/genética , Análisis por Conglomerados , Uso de Codones/genética , Codón de Terminación/genética , Biología Computacional , Evolución Molecular , Interacciones Hidrofóbicas e Hidrofílicas , Lisina/química , Lisina/metabolismo , Mycoplasma pneumoniae/química , Mycoplasma pneumoniae/genética , Mycoplasma pneumoniae/metabolismo , Filogenia , Dominios Proteicos , Procesamiento Proteico-Postraduccional/genética
9.
Mol Syst Biol ; 15(2): e8290, 2019 02 22.
Artículo en Inglés | MEDLINE | ID: mdl-30796087

RESUMEN

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.


Asunto(s)
Proteínas Bacterianas/genética , Biología Computacional , Péptidos/genética , Proteoma/genética , Proteínas Bacterianas/aislamiento & purificación , Genoma Bacteriano/genética , Espectrometría de Masas , Mycoplasma/genética , Proteómica
11.
Mol Microbiol ; 108(3): 319-329, 2018 05.
Artículo en Inglés | MEDLINE | ID: mdl-29470847

RESUMEN

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.


Asunto(s)
Mycoplasma genitalium/genética , Mycoplasma genitalium/fisiología , Orgánulos/genética , Adhesinas Bacterianas/genética , Adhesinas Bacterianas/metabolismo , Adhesión Bacteriana/genética , Proteínas Bacterianas/metabolismo , Adhesión Celular , Tomografía con Microscopio Electrónico/métodos , Electrones , Mutación , Mycoplasma pneumoniae/genética , Orgánulos/metabolismo
12.
BMC Cancer ; 19(1): 666, 2019 Jul 05.
Artículo en Inglés | MEDLINE | ID: mdl-31277602

RESUMEN

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.


Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Células Clonales/metabolismo , Heterogeneidad Genética , Animales , Apoptosis , Comunicación Celular , Línea Celular Tumoral , Movimiento Celular , Supervivencia Celular , Células Clonales/patología , Técnicas de Cocultivo , Citocinas/análisis , Elementos Transponibles de ADN/genética , Femenino , Expresión Génica , Xenoinjertos , Humanos , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Pez Cebra
13.
Nucleic Acids Res ; 44(3): 1192-202, 2016 Feb 18.
Artículo en Inglés | MEDLINE | ID: mdl-26773059

RESUMEN

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.


Asunto(s)
Genoma Bacteriano/genética , Genómica/métodos , Mycoplasma pneumoniae/genética , Mycoplasma pneumoniae/metabolismo , Proteómica/métodos , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Análisis por Conglomerados , Perfilación de la Expresión Génica/métodos , Perfilación de la Expresión Génica/estadística & datos numéricos , Regulación de la Expresión Génica , Genómica/estadística & datos numéricos , Anotación de Secuencia Molecular , Datos de Secuencia Molecular , Procesamiento Proteico-Postraduccional , Proteoma/genética , Proteoma/metabolismo , Proteómica/estadística & datos numéricos , ARN no Traducido/genética , Biología de Sistemas/métodos , Biología de Sistemas/estadística & datos numéricos
14.
Nucleic Acids Res ; 43(7): 3442-53, 2015 Apr 20.
Artículo en Inglés | MEDLINE | ID: mdl-25779052

RESUMEN

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.


Asunto(s)
Neumonía por Mycoplasma/genética , Regiones Promotoras Genéticas , Genes Bacterianos , Modelos Teóricos
15.
Nucleic Acids Res ; 43(Database issue): D618-23, 2015 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-25378328

RESUMEN

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.


Asunto(s)
Bases de Datos Genéticas , Mycoplasma pneumoniae/genética , Mycoplasma pneumoniae/metabolismo , Biología de Sistemas , Genoma Bacteriano , Internet , Metaboloma , Proteoma , Transcriptoma
16.
Proteomics ; 16(4): 554-63, 2016 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-26702875

RESUMEN

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). All MS data have been deposited in the ProteomeXchange with identifier PXD002779 (http://proteomecentral.proteomexchange.org/dataset/PXD002779).


Asunto(s)
Proteínas Bacterianas/análisis , Proteínas Bacterianas/genética , Mycoplasma pneumoniae/química , Mycoplasma pneumoniae/genética , Bases de Datos de Proteínas , Genoma Bacteriano , Humanos , Mycoplasma pneumoniae/crecimiento & desarrollo , Neumonía por Mycoplasma/microbiología , Procesamiento Proteico-Postraduccional , Proteoma/análisis , Proteoma/genética , Proteómica , Espectrometría de Masas en Tándem , Transcriptoma
17.
Mol Syst Biol ; 11(1): 780, 2015 Jan 21.
Artículo en Inglés | MEDLINE | ID: mdl-25609650

RESUMEN

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.


Asunto(s)
ADN Bacteriano/genética , Genoma Bacteriano , Mycoplasma pneumoniae/genética , Sistemas de Lectura Abierta , ARN no Traducido/genética , Genes Esenciales , Conformación Proteica , Análisis de Secuencia de ADN , Transcripción Genética
18.
PLoS Genet ; 9(1): e1003191, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23300489

RESUMEN

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 N(6)-methyladenine (6 mA) and N(4)-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'-GAN(7)TAY-3'/3'-CTN(7)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.


Asunto(s)
Metilación de ADN/genética , Mycoplasma genitalium , Mycoplasma pneumoniae , Motivos de Nucleótidos/genética , Regulación de la Expresión Génica Arqueal , Genoma Bacteriano , Humanos , Metiltransferasas/genética , Mycoplasma genitalium/genética , Mycoplasma genitalium/metabolismo , Mycoplasma pneumoniae/genética , Mycoplasma pneumoniae/metabolismo
19.
BMC Genomics ; 15: 633, 2014 Jul 29.
Artículo en Inglés | MEDLINE | ID: mdl-25070459

RESUMEN

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. RESULTS: 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.


Asunto(s)
Artefactos , Bacterias/genética , Perfilación de la Expresión Génica/métodos , ARN Bacteriano/genética , Análisis de Secuencia de ARN/métodos , Estadística como Asunto/métodos , Secuencia de Bases , Isoformas de ARN/genética , Programas Informáticos
20.
Mol Syst Biol ; 9: 653, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23549481

RESUMEN

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.


Asunto(s)
Metabolismo Energético/genética , Regulación Bacteriana de la Expresión Génica , Genoma Bacteriano , Mycoplasma pneumoniae/genética , Mycoplasma pneumoniae/metabolismo , Simulación por Computador , Redes y Vías Metabólicas/genética , Modelos Biológicos , Mutación
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