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1.
Front Genet ; 15: 1346707, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38404664

RESUMEN

Mesoplasma florum is an emerging model organism for systems and synthetic biology due to its small genome (∼800 kb) and fast growth rate. While M. florum was isolated and first described almost 40 years ago, many important aspects of its biology have long remained uncharacterized due to technological limitations, the absence of dedicated molecular tools, and since this bacterial species has not been associated with any disease. However, the publication of the first M. florum genome in 2004 paved the way for a new era of research fueled by the rise of systems and synthetic biology. Some of the most important studies included the characterization and heterologous use of M. florum regulatory elements, the development of the first replicable plasmids, comparative genomics and transposon mutagenesis, whole-genome cloning in yeast, genome transplantation, in-depth characterization of the M. florum cell, as well as the development of a high-quality genome-scale metabolic model. The acquired data, knowledge, and tools will greatly facilitate future genome engineering efforts in M. florum, which could next be exploited to rationally design and create synthetic cells to advance fundamental knowledge or for specific applications.

2.
Microbiol Resour Announc ; 12(11): e0066523, 2023 Nov 16.
Artículo en Inglés | MEDLINE | ID: mdl-37843363

RESUMEN

We report the complete genome sequence and annotation of Escherichia coli DGF-298, a genome-reduced E. coli strain with interesting properties for systems and synthetic biology. DGF-298 has a single circular chromosome of 2,991,126 bp and 2,831 genes, including 2,691 coding sequences, with a mean G + C content of ~51%.

3.
Mol Syst Biol ; 17(10): e10574, 2021 10.
Artículo en Inglés | MEDLINE | ID: mdl-34612591

RESUMEN

Bacterial infections are commonly treated with antimicrobials, but the rise of multi-drug resistance and the presence of biofilms can compromise treatment efficacy. Recently, new approaches using live bacteria or engineered microorganisms have gained attention in the fight against several diseases. In their recent work, Lluch-Senar and colleagues (Garrido et al, 2021) genetically modified the lung pathogen Mycoplasma pneumoniae to attenuate its virulence and secrete antibiofilm and bactericidal enzymes. Their strategy successfully altered a Staphylococcus aureus biofilm on catheters implanted in mice, providing an additional demonstration of the potential of genetically engineered microorganisms as therapeutic agents.


Asunto(s)
Mycoplasma pneumoniae , Staphylococcus aureus , Animales , Antibacterianos/farmacología , Biopelículas , Ratones , Pruebas de Sensibilidad Microbiana , Mycoplasma pneumoniae/genética , Staphylococcus aureus/genética
4.
Mol Syst Biol ; 17(7): e10099, 2021 07.
Artículo en Inglés | MEDLINE | ID: mdl-34288418

RESUMEN

Mesoplasma florum, a fast-growing near-minimal organism, is a compelling model to explore rational genome designs. Using sequence and structural homology, the set of metabolic functions its genome encodes was identified, allowing the reconstruction of a metabolic network representing ˜ 30% of its protein-coding genes. Growth medium simplification enabled substrate uptake and product secretion rate quantification which, along with experimental biomass composition, were integrated as species-specific constraints to produce the functional iJL208 genome-scale model (GEM) of metabolism. Genome-wide expression and essentiality datasets as well as growth data on various carbohydrates were used to validate and refine iJL208. Discrepancies between model predictions and observations were mechanistically explained using protein structures and network analysis. iJL208 was also used to propose an in silico reduced genome. Comparing this prediction to the minimal cell JCVI-syn3.0 and its parent JCVI-syn1.0 revealed key features of a minimal gene set. iJL208 is a stepping-stone toward model-driven whole-genome engineering.


Asunto(s)
Genoma , Redes y Vías Metabólicas , Genoma/genética , Genómica , Redes y Vías Metabólicas/genética , Modelos Biológicos
5.
Mol Syst Biol ; 16(12): e9844, 2020 12.
Artículo en Inglés | MEDLINE | ID: mdl-33331123

RESUMEN

The near-minimal bacterium Mesoplasma florum is an interesting model for synthetic genomics and systems biology due to its small genome (~ 800 kb), fast growth rate, and lack of pathogenic potential. However, fundamental aspects of its biology remain largely unexplored. Here, we report a broad yet remarkably detailed characterization of M. florum by combining a wide variety of experimental approaches. We investigated several physical and physiological parameters of this bacterium, including cell size, growth kinetics, and biomass composition of the cell. We also performed the first genome-wide analysis of its transcriptome and proteome, notably revealing a conserved promoter motif, the organization of transcription units, and the transcription and protein expression levels of all protein-coding sequences. We converted gene transcription and expression levels into absolute molecular abundances using biomass quantification results, generating an unprecedented view of the M. florum cellular composition and functions. These characterization efforts provide a strong experimental foundation for the development of a genome-scale model for M. florum and will guide future genome engineering endeavors in this simple organism.


Asunto(s)
Entomoplasmataceae/fisiología , Secuencia de Bases , Biomasa , Entomoplasmataceae/genética , Entomoplasmataceae/crecimiento & desarrollo , Entomoplasmataceae/ultraestructura , Regulación Bacteriana de la Expresión Génica , Genoma Bacteriano , Espacio Intracelular/metabolismo , Cinética , Sustancias Macromoleculares/metabolismo , Ácidos Nucleicos/metabolismo , Sistemas de Lectura Abierta/genética , Regiones Promotoras Genéticas/genética , Ribosomas/metabolismo , Temperatura , Sitio de Iniciación de la Transcripción , Transcripción Genética
6.
Nat Commun ; 9(1): 4364, 2018 10 19.
Artículo en Inglés | MEDLINE | ID: mdl-30341288

RESUMEN

Termination of RNA polymerase II (RNAPII) transcription is a fundamental step of gene expression that is critical for determining the borders between genes. In budding yeast, termination at protein-coding genes is initiated by the cleavage/polyadenylation machinery, whereas termination of most noncoding RNA (ncRNA) genes occurs via the Nrd1-Nab3-Sen1 (NNS) pathway. Here, we find that NNS-like transcription termination is not conserved in fission yeast. Rather, genome-wide analyses show global recruitment of mRNA 3' end processing factors at the end of ncRNA genes, including snoRNAs and snRNAs, and that this recruitment coincides with high levels of Ser2 and Tyr1 phosphorylation on the RNAPII C-terminal domain. We also find that termination of mRNA and ncRNA transcription requires the conserved Ysh1/CPSF-73 and Dhp1/XRN2 nucleases, supporting widespread cleavage-dependent transcription termination in fission yeast. Our findings thus reveal that a common mode of transcription termination can produce functionally and structurally distinct types of polyadenylated and non-polyadenylated RNAs.


Asunto(s)
ARN/genética , Schizosaccharomyces/genética , Terminación de la Transcripción Genética/fisiología , ADN Helicasas/genética , ADN Helicasas/metabolismo , ADN Helicasas/fisiología , ARN Helicasas/genética , ARN Helicasas/metabolismo , ARN Helicasas/fisiología , ARN Polimerasa II/metabolismo , ARN Polimerasa II/fisiología , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/fisiología , Especificidad de la Especie
8.
mSystems ; 3(3)2018.
Artículo en Inglés | MEDLINE | ID: mdl-29657968

RESUMEN

The creation and comparison of minimal genomes will help better define the most fundamental mechanisms supporting life. Mesoplasma florum is a near-minimal, fast-growing, nonpathogenic bacterium potentially amenable to genome reduction efforts. In a comparative genomic study of 13 M. florum strains, including 11 newly sequenced genomes, we have identified the core genome and open pangenome of this species. Our results show that all of the strains have approximately 80% of their gene content in common. Of the remaining 20%, 17% of the genes were found in multiple strains and 3% were unique to any given strain. On the basis of random transposon mutagenesis, we also estimated that ~290 out of 720 genes are essential for M. florum L1 in rich medium. We next evaluated different genome reduction scenarios for M. florum L1 by using gene conservation and essentiality data, as well as comparisons with the first working approximation of a minimal organism, Mycoplasma mycoides JCVI-syn3.0. Our results suggest that 409 of the 473 M. mycoides JCVI-syn3.0 genes have orthologs in M. florum L1. Conversely, 57 putatively essential M. florum L1 genes have no homolog in M. mycoides JCVI-syn3.0. This suggests differences in minimal genome compositions, even for these evolutionarily closely related bacteria. IMPORTANCE The last years have witnessed the development of whole-genome cloning and transplantation methods and the complete synthesis of entire chromosomes. Recently, the first minimal cell, Mycoplasma mycoides JCVI-syn3.0, was created. Despite these milestone achievements, several questions remain to be answered. For example, is the composition of minimal genomes virtually identical in phylogenetically related species? On the basis of comparative genomics and transposon mutagenesis, we investigated this question by using an alternative model, Mesoplasma florum, that is also amenable to genome reduction efforts. Our results suggest that the creation of additional minimal genomes could help reveal different gene compositions and strategies that can support life, even within closely related species.

9.
ACS Synth Biol ; 7(1): 209-217, 2018 01 19.
Artículo en Inglés | MEDLINE | ID: mdl-28893065

RESUMEN

Cloning and transplantation of bacterial genomes is a powerful method for the creation of engineered microorganisms. However, much remains to be understood about the molecular mechanisms and limitations of this approach. We report the whole-genome cloning of Mesoplasma florum in Saccharomyces cerevisiae, and use this model to investigate the impact of a bacterial chromosome in yeast cells. Our results indicate that the cloned M. florum genome is subjected to weak transcriptional activity, and causes no significant impact on yeast growth. We also report that the M. florum genome can be transplanted into Mycoplasma capricolum without any negative impact from the putative restriction enzyme encoding gene mfl307. Using whole-genome sequencing, we observed that a small number of mutations appeared in all M. florum transplants. Mutations also arose, albeit at a lower frequency, when the M. capricolum genome was transplanted into M. capricolum recipient cells. These observations suggest that genome transplantation is mutagenic, and that this phenomenon is magnified by the use of genome donor and recipient cell belonging to different species. No difference in efficiency was detected after three successive rounds of genome transplantation, suggesting that the observed mutations were not selected during the procedure. Taken together, our results provide a more accurate picture of the events taking place during bacterial genome cloning and transplantation.


Asunto(s)
Clonación Molecular , Entomoplasmataceae/genética , Genoma Bacteriano , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas Bacterianas/genética , Desoxirribonucleasas de Localización Especificada Tipo II/genética , Perfilación de la Expresión Génica , Técnicas de Transferencia de Gen , Secuenciación de Nucleótidos de Alto Rendimiento , Hidroliasas/genética , Mutación , Plásmidos/genética , Plásmidos/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Análisis de Secuencia de ADN
10.
J Exp Bot ; 68(9): 2333-2344, 2017 04 01.
Artículo en Inglés | MEDLINE | ID: mdl-28369573

RESUMEN

An important branch of plant immunity involves the recognition of pathogens by nucleotide-binding, leucine-rich repeat (NB-LRR) proteins. However, signaling events downstream of NB-LRR activation are poorly understood. We have analysed the Arabidopsis translatome using ribosome affinity purification and RNA sequencing. Our results show that the translational status of hundreds of transcripts is differentially affected upon activation of the NB-LRR protein RPM1, showing an overall pattern of a switch away from growth-related activities to defense. Among these is the central translational regulator and growth promoter, Target of Rapamycin (TOR) kinase. Suppression of TOR expression leads to increased resistance to pathogens while overexpression of TOR results in increased susceptibility, indicating an important role for translational control in the switch from growth to defense. Furthermore, we show that several additional genes whose mRNAs are translationally regulated, including BIG, CCT2, and CIPK5, are required for both NB-LRR-mediated and basal plant innate immunity, identifying novel actors in plant defense.


Asunto(s)
Arabidopsis/genética , Arabidopsis/inmunología , Regulación de la Expresión Génica de las Plantas , Inmunidad de la Planta , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Transducción de Señal
11.
Appl Environ Microbiol ; 83(7)2017 04 01.
Artículo en Inglés | MEDLINE | ID: mdl-28115382

RESUMEN

The near-minimal bacterium Mesoplasma florum constitutes an attractive model for systems biology and for the development of a simplified cell chassis in synthetic biology. However, the lack of genetic engineering tools for this microorganism has limited our capacity to understand its basic biology and modify its genome. To address this issue, we have evaluated the susceptibility of M. florum to common antibiotics and developed the first generation of artificial plasmids able to replicate in this bacterium. Selected regions of the predicted M. florum chromosomal origin of replication (oriC) were used to create different plasmid versions that were tested for their transformation frequency and stability. Using polyethylene glycol-mediated transformation, we observed that plasmids harboring both rpmH-dnaA and dnaA-dnaN intergenic regions, interspaced or not with a copy of the dnaA gene, resulted in a frequency of ∼4.1 × 10-6 transformants per viable cell and were stably maintained throughout multiple generations. In contrast, plasmids containing only one M. florumoriC intergenic region or the heterologous oriC region of Mycoplasma capricolum, Mycoplasma mycoides, or Spiroplasma citri failed to produce any detectable transformants. We also developed alternative transformation procedures based on electroporation and conjugation from Escherichia coli, reaching frequencies up to 7.87 × 10-6 and 8.44 × 10-7 transformants per viable cell, respectively. Finally, we demonstrated the functionality of antibiotic resistance genes active against tetracycline, puromycin, and spectinomycin/streptomycin in M. florum Taken together, these valuable genetic tools will facilitate efforts toward building an M. florum-based near-minimal cellular chassis for synthetic biology.IMPORTANCEMesoplasma florum constitutes an attractive model for systems biology and for the development of a simplified cell chassis in synthetic biology. M. florum is closely related to the mycoides cluster of mycoplasmas, which has become a model for whole-genome cloning, genome transplantation, and genome minimization. However, M. florum shows higher growth rates than other Mollicutes, has no known pathogenic potential, and possesses a significantly smaller genome that positions this species among some of the simplest free-living organisms. So far, the lack of genetic engineering tools has limited our capacity to understand the basic biology of M. florum in order to modify its genome. To address this issue, we have evaluated the susceptibility of M. florum to common antibiotics and developed the first artificial plasmids and transformation methods for this bacterium. This represents a strong basis for ongoing genome engineering efforts using this near-minimal microorganism.


Asunto(s)
Entomoplasmataceae/genética , Plásmidos/genética , Origen de Réplica , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Replicación del ADN , ADN Bacteriano/genética , ADN Intergénico , Proteínas de Unión al ADN/genética , Farmacorresistencia Bacteriana Múltiple , Entomoplasmataceae/efectos de los fármacos , Escherichia coli/genética , Vectores Genéticos , Mycoplasma/genética , Recombinación Genética , Biología Sintética , Transformación Bacteriana
12.
Nucleic Acids Res ; 44(17): 8501-11, 2016 09 30.
Artículo en Inglés | MEDLINE | ID: mdl-27488189

RESUMEN

Genome transplantation (GT) allows the installation of purified chromosomes into recipient cells, causing the resulting organisms to adopt the genotype and the phenotype conferred by the donor cells. This key process remains a bottleneck in synthetic biology, especially for genome engineering strategies of intractable and economically important microbial species. So far, this process has only been reported using two closely related bacteria, Mycoplasma mycoides subsp. capri (Mmc) and Mycoplasma capricolum subsp. capricolum (Mcap), and the main factors driving the compatibility between a donor genome and a recipient cell are poorly understood. Here, we investigated the impact of the evolutionary distance between donor and recipient species on the efficiency of GT. Using Mcap as the recipient cell, we successfully transplanted the genome of six bacteria belonging to the Spiroplasma phylogenetic group but including species of two distinct genera. Our results demonstrate that GT efficiency is inversely correlated with the phylogenetic distance between donor and recipient bacteria but also suggest that other species-specific barriers to GT exist. This work constitutes an important step toward understanding the cellular factors governing the GT process in order to better define and eventually extend the existing genome compatibility limit.


Asunto(s)
Genoma Bacteriano , Mycoplasma capricolum/genética , Mycoplasma mycoides/genética , Filogenia , Transformación Genética , Clonación Molecular , Replicación del ADN/genética , ADN Bacteriano/genética , Marcadores Genéticos , Genotipo , Mutagénesis Insercional/genética , Fenotipo , Plásmidos/metabolismo , Reproducibilidad de los Resultados , Saccharomyces cerevisiae/genética
13.
Neural Dev ; 11(1): 13, 2016 07 28.
Artículo en Inglés | MEDLINE | ID: mdl-27468987

RESUMEN

BACKGROUND: Recent genetic studies in model organisms, such as Drosophila, C. elegans and mice, have highlighted a critical role for dual leucine zipper kinase (DLK) in neural development and axonal responses to injury. However, exactly how DLK fulfills these functions remains to be determined. Using RNA-seq profiling, we evaluated the global changes in gene expression that are caused by shRNA-mediated knockdown of endogenous DLK in differentiated Neuro-2a neuroblastoma cells. RESULTS: Our analysis led to the identification of numerous up- and down-regulated genes, among which several were found to be associated with system development and axon guidance according to gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses, respectively. Because of their importance in axonal growth, pruning and regeneration during development and adult life, we then examined by quantitative RT-PCR the mRNA expression levels of the identified axon guidance genes in DLK-depleted cells. Consistent with the RNA-seq data, our results confirmed that loss of DLK altered expression of the genes encoding neuropilin 1 (Nrp1), plexin A4 (Plxna4), Eph receptor A7 (Epha7), Rho family GTPase 1 (Rnd1) and semaphorin 6B (Sema6b). Interestingly, this regulation of Nrp1 and Plxna4 mRNA expression by DLK in Neuro-2a cells was also reflected at the protein level, implicating DLK in the modulation of the function of these axon guidance molecules. CONCLUSIONS: Collectively, these results provide the first evidence that axon guidance genes are downstream targets of the DLK signaling pathway, which through their regulation probably modulates neuronal cell development, structure and function.


Asunto(s)
Orientación del Axón/genética , Regulación de la Expresión Génica , Quinasas Quinasa Quinasa PAM/metabolismo , Neuronas/metabolismo , Animales , Línea Celular Tumoral , Células HEK293 , Humanos , Ratones , Neuritas/fisiología , Interferencia de ARN , Análisis de Secuencia de ARN , Transducción de Señal
14.
Mob Genet Elements ; 5(3): 1-5, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26442183

RESUMEN

Conjugative plasmids of the A/C incompatibility group (IncA/C) have become substantial players in the dissemination of multidrug resistance. These large conjugative plasmids are characterized by their broad host-range, extended spectrum of antimicrobials resistance, and prevalence in enteric bacteria recovered from both environmental and clinical settings. Until recently, relatively little was known about the basic biology of IncA/C plasmids, mostly because of the hindrance of multidrug resistance for molecular biology experiments. To circumvent this issue, we previously developed pVCR94ΔX, a convenient prototype that codes for a reduced set of antibiotic resistances. Using pVCR94ΔX, we then characterized the regulatory pathway governing IncA/C plasmid dissemination. We found that the expression of roughly 2 thirds of the genes encoded by this plasmid, including large operons involved in the conjugation process, depends on an FlhCD-like master activator called AcaCD. Beyond the mobility of IncA/C plasmids, AcaCD was also shown to play a key role in the mobilization of different classes of genomic islands (GIs) identified in various pathogenic bacteria. By doing so, IncA/C plasmids can have a considerable impact on bacterial genomes plasticity and evolution.

15.
Methods Mol Biol ; 1334: 143-59, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26404148

RESUMEN

Transcription start sites are commonly used to locate promoter elements in bacterial genomes. TSS were previously studied one gene at a time, often through 5'-rapid amplification of cDNA ends (5'-RACE). This technique has now been adapted for high-throughput sequencing and can be used to precisely identify TSS in a genome-wide fashion for practically any bacterium, which greatly contributes to our understanding of gene regulatory networks in microorganisms.


Asunto(s)
ADN Complementario/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Sitio de Iniciación de la Transcripción , Transcripción Genética , ADN Complementario/química , Redes Reguladoras de Genes/genética , Genoma Bacteriano , Regiones Promotoras Genéticas
16.
Methods Mol Biol ; 1334: 173-93, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26404150

RESUMEN

DNA-binding proteins play a crucial role in all living organisms by interacting with various DNA sequences across the genome. While several methods have been used to study the interaction between DNA and proteins in vitro, chromatin immunoprecipitation followed by sequencing (ChIP-seq) has become the standard technique for identifying the genome-wide location of DNA-binding proteins in vivo. However, the resolution of standard ChIP-seq methodology is limited by the DNA fragmentation process and presence of contaminating DNA. A significant improvement of the ChIP-seq technique results from the addition of an exonuclease treatment during the immunoprecipitation step (ChIP-exo) that lowers background noise and more importantly increases the identification of binding sites to a level near to single-base resolution by effectively footprinting DNA-bound proteins. By doing so, ChIP-exo offers new opportunities for a better characterization of the complex and fascinating architecture that resides in DNA-proteins interactions and provides new insights for the comprehension of important molecular mechanisms.


Asunto(s)
Inmunoprecipitación de Cromatina/métodos , Proteínas de Unión al ADN/química , Exonucleasas/química , Sitios de Unión , Fragmentación del ADN , Proteínas de Unión al ADN/genética , Exonucleasas/genética , Genoma , Análisis de Secuencia de ADN , Factores de Transcripción/química , Factores de Transcripción/genética
17.
PLoS One ; 10(7): e0133384, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26197065

RESUMEN

BACKGROUND: Continuous culture devices can be used for various purposes such as establishing reproducible growth conditions or maintaining cell populations under a constant environment for long periods. However, commercially available instruments are expensive, were not designed to handle small volumes in the milliliter range, and can lack the flexibility required for the diverse experimental needs found in several laboratories. METHODOLOGY/PRINCIPAL FINDINGS: We developed a versatile continuous culture system and provide detailed instructions as well as a graphical user interface software for potential users to assemble and operate their own instrument. Three culture chambers can be controlled simultaneously with the proposed configuration, and all components are readily available from various sources. We demonstrate that our continuous culture device can be used under different modes, and can easily be programmed to behave either as a turbidostat or chemostat. Addition of fresh medium to the culture vessel can be controlled by a real-time feedback loop or simply calibrated to deliver a defined volume. Furthermore, the selected light-emitting diode and photodetector enable the use of phenol red as a pH indicator, which can be used to indirectly monitor the bulk metabolic activity of a cell population rather than the turbidity. CONCLUSIONS/SIGNIFICANCE: This affordable and customizable system will constitute a useful tool in many areas of biology such as microbial ecology as well as systems and synthetic biology.


Asunto(s)
División Celular/fisiología , Técnicas Microbiológicas/instrumentación , Técnicas Microbiológicas/métodos , Modelos Teóricos , Entomoplasmataceae/citología , Entomoplasmataceae/crecimiento & desarrollo , Diseño de Equipo , Escherichia coli/citología , Escherichia coli/crecimiento & desarrollo , Concentración de Iones de Hidrógeno , Técnicas Microbiológicas/economía , Reproducibilidad de los Resultados , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/crecimiento & desarrollo
18.
Nucleic Acids Res ; 43(4): 2045-56, 2015 Feb 27.
Artículo en Inglés | MEDLINE | ID: mdl-25662215

RESUMEN

Integrative and conjugative elements (ICEs) of the SXT/R391 family have been recognized as key drivers of antibiotic resistance dissemination in the seventh-pandemic lineage of Vibrio cholerae. SXT/R391 ICEs propagate by conjugation and integrate site-specifically into the chromosome of a wide range of environmental and clinical Gammaproteobacteria. SXT/R391 ICEs bear setC and setD, two conserved genes coding for a transcriptional activator complex that is essential for activation of conjugative transfer. We used chromatin immunoprecipitation coupled with exonuclease digestion (ChIP-exo) and RNA sequencing (RNA-seq) to characterize the SetCD regulon of three representative members of the SXT/R391 family. We also identified the DNA sequences bound by SetCD in MGIVflInd1, a mobilizable genomic island phylogenetically unrelated to SXT/R391 ICEs that hijacks the conjugative machinery of these ICEs to drive its own transfer. SetCD was found to bind a 19-bp sequence that is consistently located near the promoter -35 element of SetCD-activated genes, a position typical of class II transcriptional activators. Furthermore, we refined our understanding of the regulation of excision from and integration into the chromosome for SXT/R391 ICEs and demonstrated that de novo expression of SetCD is crucial to allow integration of the incoming ICE DNA into a naive host following conjugative transfer.


Asunto(s)
Proteínas Bacterianas/metabolismo , Conjugación Genética , ADN Bacteriano/metabolismo , Regulación Bacteriana de la Expresión Génica , Regulón , Transactivadores/metabolismo , Proteínas Bacterianas/genética , ADN Bacteriano/química , Islas Genómicas , Mutación , Motivos de Nucleótidos , Regiones Operadoras Genéticas , Regiones Promotoras Genéticas , Transactivadores/genética , Activación Transcripcional
19.
PLoS Genet ; 10(10): e1004714, 2014 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-25340549

RESUMEN

Dissemination of antibiotic resistance genes occurs mostly by conjugation, which mediates DNA transfer between cells in direct contact. Conjugative plasmids of the IncA/C incompatibility group have become a substantial threat due to their broad host-range, the extended spectrum of antimicrobial resistance they confer, their prevalence in enteric bacteria and their very efficient spread by conjugation. However, their biology remains largely unexplored. Using the IncA/C conjugative plasmid pVCR94ΔX as a prototype, we have investigated the regulatory circuitry that governs IncA/C plasmids dissemination and found that the transcriptional activator complex AcaCD is essential for the expression of plasmid transfer genes. Using chromatin immunoprecipitation coupled with exonuclease digestion (ChIP-exo) and RNA sequencing (RNA-seq) approaches, we have identified the sequences recognized by AcaCD and characterized the AcaCD regulon. Data mining using the DNA motif recognized by AcaCD revealed potential AcaCD-binding sites upstream of genes involved in the intracellular mobility functions (recombination directionality factor and mobilization genes) in two widespread classes of genomic islands (GIs) phylogenetically unrelated to IncA/C plasmids. The first class, SGI1, confers and propagates multidrug resistance in Salmonella enterica and Proteus mirabilis, whereas MGIVmi1 in Vibrio mimicus belongs to a previously uncharacterized class of GIs. We have demonstrated that through expression of AcaCD, IncA/C plasmids specifically trigger the excision and mobilization of the GIs at high frequencies. This study provides new evidence of the considerable impact of IncA/C plasmids on bacterial genome plasticity through their own mobility and the mobilization of genomic islands.


Asunto(s)
Farmacorresistencia Bacteriana Múltiple/genética , Islas Genómicas/genética , Plásmidos/genética , Escherichia coli/genética , Genoma Bacteriano , Secuenciación de Nucleótidos de Alto Rendimiento , Proteus mirabilis/genética , Salmonella enterica/genética
20.
Genome Announc ; 2(5)2014 Oct 16.
Artículo en Inglés | MEDLINE | ID: mdl-25323716

RESUMEN

Escherichia coli BW25113 is the parent strain of the Keio collection comprising nearly 4,000 single-gene deletion mutants. We report the complete 4,631,469-bp genome sequence of this strain and the key variations from the type strain E. coli MG1655.

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