RESUMO
IncC conjugative plasmids and the multiple variants of Salmonella Genomic Island 1 (SGI1) are two functionally interacting families of mobile genetic elements commonly associated with multidrug resistance in the Gammaproteobacteria. SGI1 and its siblings are specifically mobilised in trans by IncC conjugative plasmids. Conjugative transfer of IncC plasmids is activated by the plasmid-encoded master activator AcaCD. SGI1 carries five AcaCD-responsive promoters that drive the expression of genes involved in its excision, replication, and mobilisation. SGI1 encodes an AcaCD homologue, the transcriptional activator complex SgaCD (also known as FlhDCSGI1) that seems to recognise and activate the same SGI1 promoters. Here, we investigated the relevance of SgaCD in SGI1's lifecycle. Mating assays revealed the requirement for SgaCD and its IncC-encoded counterpart AcaCD in the mobilisation of SGI1. An integrative approach combining ChIP-exo, Cappable-seq, and RNA-seq confirmed that SgaCD activates each of the 18 AcaCD-responsive promoters driving the expression of the plasmid transfer functions. A comprehensive analysis of the activity of the complete set of AcaCD-responsive promoters of SGI1 and the helper IncC plasmid was performed through reporter assays. qPCR and flow cytometry assays revealed that SgaCD is essential to elicit the excision and replication of SGI1 and destabilise the helper IncC plasmid.
Assuntos
Conjugação Genética/genética , Ilhas Genômicas/genética , Plasmídeos/genética , Salmonella/genética , Ativação Transcricional , Proteínas de Bactérias/genética , Replicação do DNA/genética , Farmacorresistência Bacteriana Múltipla/genética , Gammaproteobacteria/genética , Perfilação da Expressão Gênica/métodos , Regulação Bacteriana da Expressão Gênica , Modelos Genéticos , Regiões Promotoras Genéticas/genética , RNA-Seq/métodosRESUMO
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.
Assuntos
Mycoplasma pneumoniae , Staphylococcus aureus , Animais , Antibacterianos/farmacologia , Biofilmes , Camundongos , Testes de Sensibilidade Microbiana , Mycoplasma pneumoniae/genética , Staphylococcus aureus/genéticaRESUMO
Antibiotic resistance threatens our ability to treat infectious diseases, spurring interest in alternative antimicrobial technologies. The use of bacterial conjugation to deliver CRISPR-cas systems programmed to precisely eliminate antibiotic-resistant bacteria represents a promising approach but requires high in situ DNA transfer rates. We have optimized the transfer efficiency of conjugative plasmid TP114 using accelerated laboratory evolution. We hence generated a potent conjugative delivery vehicle for CRISPR-cas9 that can eliminate > 99.9% of targeted antibiotic-resistant Escherichia coli in the mouse gut microbiota using a single dose. We then applied this system to a Citrobacter rodentium infection model, achieving full clearance within four consecutive days of treatment.
Assuntos
Microbiota , Probióticos , Animais , Sistemas CRISPR-Cas/genética , Conjugação Genética , Edição de Genes , CamundongosRESUMO
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 Ë 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.
Assuntos
Genoma , Redes e Vias Metabólicas , Genoma/genética , Genômica , Redes e Vias Metabólicas/genética , Modelos BiológicosRESUMO
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.
Assuntos
Entomoplasmataceae/fisiologia , Sequência de Bases , Biomassa , Entomoplasmataceae/genética , Entomoplasmataceae/crescimento & desenvolvimento , Entomoplasmataceae/ultraestrutura , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Espaço Intracelular/metabolismo , Cinética , Substâncias Macromoleculares/metabolismo , Ácidos Nucleicos/metabolismo , Fases de Leitura Aberta/genética , Regiões Promotoras Genéticas/genética , Ribossomos/metabolismo , Temperatura , Sítio de Iniciação de Transcrição , Transcrição GênicaRESUMO
Staphylococcus aureus is one of the main pathogens leading to both clinical and subclinical bovine mastitis in dairy cattle. Prediction of disease evolution based on the characteristics of Staph. aureus isolates that cause intramammary infections and understanding the host-pathogen interactions may improve management of mastitis in dairy herds. For this study, several strains were selected from each of the 6 major Canadian spa types associated with mastitis (t267, t359, t529, t605, t2445, and t13401). Adherence to host cells and intracellular persistence of these strains were studied using a bovine mammary gland epithelial cell line (MAC-T). Additionally, relative virulence and host response (cytokines production) were also studied in vivo using a mouse model of mastitis. Whole-genome sequencing was performed on all strains and associations between clonal complex, sequence type, and presence of certain virulence factors were also investigated. Results show that spa type t2445 was correlated with persistence in MAC-T cells. Strains from spa t359 and t529 showed better ability to colonize mouse mammary glands. The exception was strain sa3154 (spa t529), which showed less colonization of glands compared with other t359 and t529 strains but possessed the highest number of superantigen genes including tst. All strains possessed hemolysins, but spa types t529 and t2445 showed the largest diameter of ß-hemolysis on blood agar plates. Although several spa types possessed 2 or 3 serine-aspartate rich proteins (Sdr) believed to be involved in many pathogenic processes, most t529 strains expressed only an allelic variant of sdrE. The spa types t605 (positive for the biofilm associated protein gene; bap+) and t13401 (bap-), that produced the largest amounts of biofilm in vitro, were the least virulent in vivo. Finally, strains from spa type t529 (ST151) elicited a cytokine expression profile (TNF-α, IL-1ß and IL-12) that suggests a potential for severe inflammation. This study suggests that determination of the spa type may help predict the severity of the disease and the ability of the immune system to eliminate intramammary infections caused by Staph. aureus.
Assuntos
Doenças dos Bovinos , Mastite Bovina , Mastite , Infecções Estafilocócicas , Animais , Canadá , Bovinos , Feminino , Mastite/veterinária , Leite , Infecções Estafilocócicas/veterinária , Staphylococcus aureus/genética , VirulênciaRESUMO
Genome-scale metabolic models (GEMs) are mathematically structured knowledge bases of metabolism that provide phenotypic predictions from genomic information. GEM-guided predictions of growth phenotypes rely on the accurate definition of a biomass objective function (BOF) that is designed to include key cellular biomass components such as the major macromolecules (DNA, RNA, proteins), lipids, coenzymes, inorganic ions and species-specific components. Despite its importance, no standardized computational platform is currently available to generate species-specific biomass objective functions in a data-driven, unbiased fashion. To fill this gap in the metabolic modeling software ecosystem, we implemented BOFdat, a Python package for the definition of a Biomass Objective Function from experimental data. BOFdat has a modular implementation that divides the BOF definition process into three independent modules defined here as steps: 1) the coefficients for major macromolecules are calculated, 2) coenzymes and inorganic ions are identified and their stoichiometric coefficients estimated, 3) the remaining species-specific metabolic biomass precursors are algorithmically extracted in an unbiased way from experimental data. We used BOFdat to reconstruct the BOF of the Escherichia coli model iML1515, a gold standard in the field. The BOF generated by BOFdat resulted in the most concordant biomass composition, growth rate, and gene essentiality prediction accuracy when compared to other methods. Installation instructions for BOFdat are available in the documentation and the source code is available on GitHub (https://github.com/jclachance/BOFdat).
Assuntos
Biomassa , Genômica/métodos , Redes e Vias Metabólicas , Modelos Biológicos , Software , Escherichia coli/genética , Escherichia coli/metabolismo , Genoma BacterianoRESUMO
There is an important need to develop new therapeutic tools to modulate the gene content of microbiomes. A potential strategy for microbiome engineering relies on the delivery of genetic payloads by conjugative plasmids. Yet, the introduction of large DNA molecules in conjugative plasmids can be challenging. Here, we describe the Double Recombinase Operated Insertion of DNA (DROID), an efficient method to assemble large DNA molecules without introducing antibiotic resistance genes or other unwanted sequences in the final construct. We exemplify this method by demonstrating that the Bxb1 integrase and FLP recombinase can be used successively to stably insert a relatively large DNA cargo consisting of a CRISPR-Cas9 system in a conjugative plasmid. We further show that the resulting CRISPR-Cas9 mobilization system was able to cure a multi-copy antibiotic resistance plasmid in a target bacterium. In addition to its utility for DNA payload integration in conjugative plasmids, the DROID method could readily be adapted to a multitude of other applications that require the manipulation of large DNA molecules.
Assuntos
Elementos de DNA Transponíveis , Mutagênese Insercional , Recombinases/metabolismo , Sistemas CRISPR-Cas , Conjugação GenéticaRESUMO
Methicillin-resistant Staphylococcus aureus (MRSA) is a leading cause of deadly hospital-acquired infections. The discovery of anti-Staphylococcus antibiotics and new classes of drugs not susceptible to the mechanisms of resistance shared among bacteria is imperative. We recently showed that tomatidine (TO), a steroidal alkaloid from solanaceous plants, possesses potent antibacterial activity against S. aureus small-colony variants (SCVs), the notoriously persistent form of this bacterium that has been associated with recurrence of infections. Here, using genomic analysis of in vitro-generated TO-resistant S. aureus strains to identify mutations in genes involved in resistance, we identified the bacterial ATP synthase as the cellular target. Sequence alignments were performed to highlight the modified sequences, and the structural consequences of the mutations were evaluated in structural models. Overexpression of the atpE gene in S. aureus SCVs or introducing the mutation found in the atpE gene of one of the high-level TO-resistant S. aureus mutants into the Bacillus subtilis atpE gene provided resistance to TO and further validated the identity of the cellular target. FC04-100, a TO derivative which also possesses activity against non-SCV strains, prevents high-level resistance development in prototypic strains and limits the level of resistance observed in SCVs. An ATP synthesis assay allowed the observation of a correlation between antibiotic potency and ATP synthase inhibition. The selectivity index (inhibition of ATP production by mitochondria versus that of bacterial ATP synthase) is estimated to be >105-fold for FC04-100.
Assuntos
Antibacterianos/farmacologia , ATPases Mitocondriais Próton-Translocadoras/química , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/enzimologia , Tomatina/análogos & derivados , Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/metabolismo , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/metabolismo , Testes de Sensibilidade Microbiana , ATPases Mitocondriais Próton-Translocadoras/genética , Mutação , Tomatina/farmacologiaRESUMO
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.
Assuntos
Genoma Bacteriano , Mycoplasma capricolum/genética , Mycoplasma mycoides/genética , Filogenia , Transformação Genética , Clonagem Molecular , Replicação do DNA/genética , DNA Bacteriano/genética , Marcadores Genéticos , Genótipo , Mutagênese Insercional/genética , Fenótipo , Plasmídeos/metabolismo , Reprodutibilidade dos Testes , Saccharomyces cerevisiae/genéticaRESUMO
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 â¼4.1 × 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.
Assuntos
Entomoplasmataceae/genética , Plasmídeos/genética , Origem de Replicação , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Replicação do DNA , DNA Bacteriano/genética , DNA Intergênico , Proteínas de Ligação a DNA/genética , Farmacorresistência Bacteriana Múltipla , Entomoplasmataceae/efeitos dos fármacos , Escherichia coli/genética , Vetores Genéticos , Mycoplasma/genética , Recombinação Genética , Biologia Sintética , Transformação BacterianaRESUMO
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.
Assuntos
Arabidopsis/genética , Arabidopsis/imunologia , Regulação da Expressão Gênica de Plantas , Imunidade Vegetal , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Transdução de SinaisRESUMO
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.
Assuntos
Proteínas de Bactérias/metabolismo , Conjugação Genética , DNA Bacteriano/metabolismo , Regulação Bacteriana da Expressão Gênica , Regulon , Transativadores/metabolismo , Proteínas de Bactérias/genética , DNA Bacteriano/química , Ilhas Genômicas , Mutação , Motivos de Nucleotídeos , Regiões Operadoras Genéticas , Regiões Promotoras Genéticas , Transativadores/genética , Ativação TranscricionalRESUMO
Gene regulatory programs in different cell types are largely defined through cell-specific enhancers activity. The histone variant H2A.Z has been shown to play important roles in transcription mainly by controlling proximal promoters, but its effect on enhancer functions remains unclear. Here, we demonstrate by genome-wide approaches that H2A.Z is present at a subset of active enhancers bound by the estrogen receptor alpha (ERα). We also determine that H2A.Z does not influence the local nucleosome positioning around ERα enhancers using ChIP sequencing at nucleosomal resolution and unsupervised pattern discovery. We further highlight that H2A.Z-enriched enhancers are associated with chromatin accessibility, H3K122ac enrichment and hypomethylated DNA. Moreover, upon estrogen stimulation, the enhancers occupied by H2A.Z produce enhancer RNAs (eRNAs), and recruit RNA polymerase II as well as RAD21, a member of the cohesin complex involved in chromatin interactions between enhancers and promoters. Importantly, their recruitment and eRNAs production are abolished by H2A.Z depletion, thereby revealing a novel functional link between H2A.Z occupancy and enhancer activity. Taken together, our findings suggest that H2A.Z acts as an important player for enhancer functions by establishing and maintaining a chromatin environment required for RNA polymerase II recruitment, eRNAs transcription and enhancer-promoters interactions, all essential attributes of enhancer activity.
Assuntos
Elementos Facilitadores Genéticos , Histonas/metabolismo , Ativação Transcricional , Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Metilação de DNA , Estradiol/farmacologia , Receptor alfa de Estrogênio/metabolismo , Genômica , Código das Histonas , Células MCF-7 , Nucleossomos/metabolismo , RNA Polimerase II/metabolismo , CoesinasRESUMO
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.
Assuntos
Farmacorresistência Bacteriana Múltipla/genética , Ilhas Genômicas/genética , Plasmídeos/genética , Escherichia coli/genética , Genoma Bacteriano , Sequenciamento de Nucleotídeos em Larga Escala , Proteus mirabilis/genética , Salmonella enterica/genéticaRESUMO
Tigecycline is an antibiotic of last resort for infections with carbapenem-resistant Acinetobacter baumannii. Plasmids harboring variants of the tetracycline destructase gene tetX promote rising tigecycline resistance rates. We report the earliest observation of tet(X3) in a clinical strain predating tigecycline's commercialization, suggesting selective pressures other than tigecycline contributed to its emergence. IMPORTANCE: We present the earliest observation of a tet(X3)-positive bacterial strain, predating by many years the earliest reports of this gene so far. This finding is significant as tigecycline is an antibiotic of last resort for carbapenem-resistant Acinetobacter baumannii (CRAB), which the World Health Organization ranks as one of its top three critical priority pathogens, and tet(X3) variants have become the most prevalent genes responsible for enabling CRAB to become tigecycline resistant. Moreover, the tet(X3)-positive strain we report is the first and only to be found that predates the commercialization of tigecycline, an antibiotic that was thought to have contributed to the emergence of this resistance gene. Understanding the factors contributing to the origin and spread of novel antibiotic resistance genes is crucial to addressing the major global public health issue, which is antimicrobial resistance.
Assuntos
Antibacterianos , Tetraciclina , Tigeciclina/farmacologia , Testes de Sensibilidade Microbiana , Antibacterianos/farmacologia , Tetraciclina/farmacologia , Plasmídeos , CarbapenêmicosRESUMO
Mesoplasma florum is an emerging model organism for systems and synthetic biology due to its small genome (â¼800 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.
RESUMO
Root-associated microorganisms play an important role in plant health, such as plant growth-promoting rhizobacteria (PGPR) from the Bacillus and Pseudomonas genera. Although bacterial consortia including these two genera would represent a promising avenue to efficient biofertilizer formulation, we observed that Bacillus subtilis root colonization is decreased by the presence of Pseudomonas fluorescens and Pseudomonas protegens. To determine if B. subtilis can adapt to the inhibitory effect of Pseudomonas on roots, we conducted adaptative laboratory evolution experiments with B. subtilis in mono-association or co-cultured with P. fluorescens on tomato plant roots. Evolved isolates with various colony morphology and stronger colonization capacity of both tomato plant and Arabidopsis thaliana roots emerged rapidly from the two evolution experiments. Certain evolved isolates also had better fitness on the root in the presence of other Pseudomonas species. In all independent lineages, whole-genome resequencing revealed non-synonymous mutations in genes ywcC or sinR encoding regulators involved in repressing biofilm development, suggesting their involvement in enhanced root colonization. These findings provide insights into the molecular mechanisms underlying B. subtilis adaptation to root colonization and highlight the potential of directed evolution to enhance the beneficial traits of PGPR.IMPORTANCEIn this study, we aimed to enhance the abilities of the plant-beneficial bacterium Bacillus subtilis to colonize plant roots in the presence of competing Pseudomonas bacteria. To achieve this, we conducted adaptive laboratory experiments, allowing Bacillus to evolve in a defined environment. We successfully obtained strains of Bacillus that were more effective at colonizing plant roots than the ancestor strain. To identify the genetic changes driving this improvement, we sequenced the genomes of these evolved strains. Interestingly, mutations that facilitated the formation of robust biofilms on roots were predominant. Many of these evolved Bacillus isolates also displayed the remarkable ability to outcompete Pseudomonas species. Our research sheds light on the mutational paths selected in Bacillus subtilis to thrive in root environments and offers exciting prospects for improving beneficial traits in plant growth-promoting microorganisms. Ultimately, this could pave the way for the development of more effective biofertilizers and sustainable agricultural practices.
Assuntos
Arabidopsis , Bacillus , Pseudomonas fluorescens , Bacillus subtilis/genética , Biofilmes , Arabidopsis/genéticaRESUMO
Mobilizable genomic islands (MGIs) are small genomic islands of less than 35 kbp containing an integrase gene and a sequence that resembles the origin of transfer (oriT) of an integrating conjugative element (ICE). MGIs have been shown to site-specifically integrate and excise from the chromosome of bacterial hosts and hijack the conjugative machinery of a coresident ICE to disseminate. To date, MGIs have been described in three strains belonging to three different Vibrio species. In this study, we report the discovery of 11 additional putative MGIs found in various species of Vibrio, Alteromonas, Pseudoalteromonas, and Methylophaga. We designed an MGI capture system that allowed us to relocate chromosomal MGIs onto a low-copy-number plasmid and facilitate their isolation and sequencing. Comparative genomics and phylogenetic analyses of these mobile genetic elements revealed their mosaic structure and their evolution through recombination and acquisition of exogenous DNA. MGIs were found to belong to a larger family of genomic islands (GIs) sharing a similar integrase gene and often integrated into the same integration site yet exhibiting a different mechanism of regulation of excision and mobilization. We found that MGIs can excise only when an ICE of the SXT/R391 family is coresident in the same cell, while GIs still excise regardless.