RESUMO
Mycoplasmas are atypical bacteria with small genomes that necessitate colonization of their respective animal or plant hosts as obligate parasites, whether as pathogens, or commensals. Some can grow axenically in specialized complex media yet show only host-cell-dependent growth in cell culture, where they can survive chronically and often through interactions involving surface colonization or internalization. To develop a mycoplasma-based system to identify genes mediating such interactions, we exploited genetically tractable strains of the goat pathogen Mycoplasma mycoides (Mmc) with synthetic designer genomes representing the complete natural organism (minus virulence factors; JCVI-syn1.0) or its reduced counterpart (JCVI-syn3B) containing only those genes supporting axenic growth. By measuring growth of surviving organisms, physical association with cultured human cells (HEK-293T, HeLa), and induction of phagocytosis by human myeloid cells (dHL-60), we determined that JCVI-syn1.0 contained a set of eight genes (MMSYN1-0179 to MMSYN1-0186, dispensable for axenic growth) conferring survival, attachment, and phagocytosis phenotypes. JCVI-syn3B lacked these phenotypes, but insertion of these genes restored cell attachment and phagocytosis, although not survival. These results indicate that JCVI-syn3B may be a powerful living platform to analyze the role of specific gene sets, from any organism, on the interaction with diverse mammalian cells in culture.
Assuntos
Mycoplasma mycoides , Mycoplasma , Animais , Humanos , Mycoplasma/genética , Mycoplasma mycoides/genética , Células HeLa , MamíferosRESUMO
Vaccination is the most cost-effective tool to control contagious bovine pleuropneumonia. The vaccines currently used in Africa are derived from a live strain called T1, which was attenuated by passage in embryonated eggs and broth culture. The number of passages is directly correlated to the degree of attenuation of the vaccinal strains and inversely correlated to their immunogenicity in cattle. Current quality control protocols applied to vaccine batches allow the assessment of identity, purity, and titers, but cannot assess the level of genetic drift form the parental vaccine strains. Deep sequencing was used to assess the genetic drift generated over controlled in vitro passages of the parental strain, as well as on commercial vaccine batches. Signatures of cloning procedures were detected in some batches, which imply a deviation from the standard production protocol. Deep sequencing is proposed as a new tool for the identity and stability control of T1 vaccines.
Assuntos
Doenças dos Bovinos , Mycoplasma mycoides , Pleuropneumonia Contagiosa , Pleuropneumonia , Animais , Bovinos , Vacinas Bacterianas/genética , África , Vacinas Atenuadas/genética , Controle de Qualidade , Sequenciamento de Nucleotídeos em Larga Escala , Pleuropneumonia Contagiosa/prevenção & controle , Mycoplasma mycoides/genéticaRESUMO
tRNA superwobbling, used by certain bacteria and organelles, is an intriguing decoding concept in which a single tRNA isoacceptor is used to decode all synonymous codons of a four-fold degenerate codon box. While Escherichia coli relies on three tRNAGly isoacceptors to decode the four glycine codons (GGN), Mycoplasma mycoides requires only a single tRNAGly. Both organisms express tRNAGly with the anticodon UCC, which are remarkably similar in sequence but different in their decoding ability. By systematically introducing mutations and altering the number and type of tRNA modifications using chemically synthesized tRNAs, we elucidated the contribution of individual nucleotides and chemical groups to decoding by the E. coli and M. mycoides tRNAGly. The tRNA sequence was identified as the key factor for superwobbling, revealing the T-arm sequence as a novel pivotal element. In addition, the presence of tRNA modifications, although not essential for providing superwobbling, was shown to delicately fine-tune and balance the decoding of synonymous codons. This emphasizes that the tRNA sequence and its modifications together form an intricate system of high complexity that is indispensable for accurate and efficient decoding.
Assuntos
Escherichia coli , Mycoplasma mycoides , RNA Bacteriano , RNA de Transferência de Glicina , Anticódon/genética , Sequência de Bases , Códon/genética , Escherichia coli/genética , Glicina/genética , RNA de Transferência/genética , RNA de Transferência de Glicina/genética , Mycoplasma mycoides/genética , Mycoplasma mycoides/metabolismo , RNA Bacteriano/genéticaRESUMO
Mycoplasma feriruminatoris is a fast-growing Mycoplasma species isolated from wild Caprinae and first described in 2013. M. feriruminatoris isolates have been associated with arthritis, kerato conjunctivitis, pneumonia and septicemia, but were also recovered from apparently healthy animals. To better understand what defines this species, we performed a genomic survey on 14 strains collected from free-ranging or zoo-housed animals between 1987 and 2017, mostly in Europe. The average chromosome size of the M. feriruminatoris strains was 1,040±0,024 kbp, with 24â% G+C and 852±31 CDS. The core genome and pan-genome of the M. feriruminatoris species contained 628 and 1312 protein families, respectively. The M. feriruminatoris strains displayed a relatively closed pan-genome, with many features and putative virulence factors shared with species from the M. mycoides cluster, including the MIB-MIP Ig cleavage system, a repertoire of DUF285 surface proteins and a complete biosynthetic pathway for galactan. M. feriruminatoris genomes were found to be mostly syntenic, although repertoires of mobile genetic elements, including Mycoplasma Integrative and Conjugative Elements, insertion sequences, and a single plasmid varied. Phylogenetic- and gene content analyses confirmed that M. feriruminatoris was closer to the M. mycoides cluster than to the ruminant species M. yeatsii and M. putrefaciens. Ancestral genome reconstruction showed that the emergence of the M. feriruminatoris species was associated with the gain of 17 gene families, some of which encode defence enzymes and surface proteins, and the loss of 25 others, some of which are involved in sugar transport and metabolism. This comparative study suggests that the M. mycoides cluster could be extended to include M. feriruminatoris. We also find evidence that the specific organization and structure of the DnaA boxes around the oriC of M. feriruminatoris may contribute to drive the remarkable fast growth of this minimal bacterium.
Assuntos
Mycoplasma mycoides , Mycoplasma , Animais , Genoma Bacteriano , Filogenia , Mycoplasma mycoides/genética , Mycoplasma mycoides/metabolismo , Mycoplasma/genética , Ruminantes/microbiologia , Genômica , Proteínas de Membrana/genéticaRESUMO
Possessing only essential genes, a minimal cell can reveal mechanisms and processes that are critical for the persistence and stability of life1,2. Here we report on how an engineered minimal cell3,4 contends with the forces of evolution compared with the Mycoplasma mycoides non-minimal cell from which it was synthetically derived. Mutation rates were the highest among all reported bacteria, but were not affected by genome minimization. Genome streamlining was costly, leading to a decrease in fitness of greater than 50%, but this deficit was regained during 2,000 generations of evolution. Despite selection acting on distinct genetic targets, increases in the maximum growth rate of the synthetic cells were comparable. Moreover, when performance was assessed by relative fitness, the minimal cell evolved 39% faster than the non-minimal cell. The only apparent constraint involved the evolution of cell size. The size of the non-minimal cell increased by 80%, whereas the minimal cell remained the same. This pattern reflected epistatic effects of mutations in ftsZ, which encodes a tubulin-homologue protein that regulates cell division and morphology5,6. Our findings demonstrate that natural selection can rapidly increase the fitness of one of the simplest autonomously growing organisms. Understanding how species with small genomes overcome evolutionary challenges provides critical insights into the persistence of host-associated endosymbionts, the stability of streamlined chassis for biotechnology and the targeted refinement of synthetically engineered cells2,7-9.
Assuntos
Evolução Molecular , Genes Essenciais , Genoma Bacteriano , Mycoplasma mycoides , Biologia Sintética , Biotecnologia/métodos , Biotecnologia/tendências , Divisão Celular , Genoma Bacteriano/genética , Mutação , Mycoplasma mycoides/citologia , Mycoplasma mycoides/genética , Mycoplasma mycoides/crescimento & desenvolvimento , Biologia Sintética/métodos , Tamanho Celular , Epistasia Genética , Seleção Genética , Aptidão Genética , Simbiose , Tubulina (Proteína)/químicaRESUMO
Bacterial pathogen-host interactions are a complex process starting with adherence and colonization followed by a variety of interactions such as invasion or cytotoxicity on one hand and pathogen recognition, secretion of proinflammatory/antibacterial substances and enhancing the barrier function of epithelial layers on the other hand. Therefore, a variety of in vitro, ex vivo and in vivo models have been established to investigate these interactions. Some in vitro models are composed of different cell types and extracellular matrices such as tissue explants or precision cut lung slices. These complex in vitro models mimic the in vivo situation more realistically, however, they often require new and more sophisticated methods for quantification of experimental results. Here we describe a multiplex qPCR-based method to quantify the number of bacteria of Mycoplasma (M.) mycoides interacting with their hosts in an absolute manner as well as normalized to the number of host cells. We choose the adenylate kinase (adk) gene from the pathogen and the Carcinoembryonic antigen-related cell adhesion molecule 18 (CEACAM18) gene from the host to determine cell numbers by a TaqMan-based assay system. Absolute copy numbers of the genes are calculated according to a standard containing a defined number of plasmids containing the sequence which is amplified by the qPCR. The new multiplex qPCR therefore allows the quantification of M. mycoides interacting with host cells in suspension, monolayer, 3D cell culture systems as well as in host tissues.
Assuntos
Doenças dos Bovinos , Mycoplasma mycoides , Mycoplasma , Animais , Bovinos , Mycoplasma mycoides/genética , Mycoplasma mycoides/metabolismo , Mycoplasma/genética , Pulmão/microbiologia , Técnicas de Cultura de Células , Doenças dos Bovinos/microbiologiaRESUMO
The new field of synthetic biology aims at the creation of artificially designed organisms. A major breakthrough in the field was the generation of the artificial synthetic organism Mycoplasma mycoides JCVI-syn3A. This bacterium possesses only 452 protein-coding genes, the smallest number for any organism that is viable independent of a host cell. However, about one third of the proteins have no known function indicating major gaps in our understanding of simple living cells. To facilitate the investigation of the components of this minimal bacterium, we have generated the database SynWiki (http://synwiki.uni-goettingen.de/). SynWiki is based on a relational database and gives access to published information about the genes and proteins of M. mycoides JCVI-syn3A. To gain a better understanding of the functions of the genes and proteins of the artificial bacteria, protein-protein interactions that may provide clues for the protein functions are included in an interactive manner. SynWiki is an important tool for the synthetic biology community that will support the comprehensive understanding of a minimal cell as well as the functional annotation of so far uncharacterized proteins.
Assuntos
Proteínas de Bactérias , Bases de Dados Genéticas , Genoma Bacteriano , Anotação de Sequência Molecular , Mycoplasma mycoides , Software , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Mycoplasma mycoides/química , Mycoplasma mycoides/genética , Mycoplasma mycoides/metabolismo , Biologia SintéticaRESUMO
Mycoplasma mycoides subsp. capri (Mmc) typically causes pneumonia, mastitis, arthritis, keratitis and septicaemia in goats. Mortality associated with Mmc in goat flocks is lower compared to Mycoplasma capricolum subsp. capripneumoniae-associated respiratory infections. Case fatality rates associated with Mmc ranged from 9.8 to 26.8% among several states in India. Molecular epidemiology approaches aimed at genotyping help to identify the diversity of isolates involved in a disease. Ten clinical pathogenic Mmc isolates were analysed by multilocus sequence typing (MLST) for studying genotypic relationships with 50 isolates available from public databases. The MLST analysis indicates high genetic diversity among Mmc isolates. From a total number of 60 isolates, 43 six sequence types (STs) were recognized comprising of six STs from India and 37 STs from other geographical regions. MLST profiles of isolates revealed none of the STs observed in Indian isolates were shared with global isolates. Some of the STs representing Indian isolates (four STs) were clustered into a novel clonal complex 1 (CC1). Maintenance of genetically related STs forming CCs among the goat population in India for longer periods indicates disease causing potentiality of these isolates. Based on various recombination analysis, weak clonal relationship among Mmc isolates were identified. The present study has enlightened further steps in disease investigations and to design future control measures by employing prevalent genotypes as vaccine candidates against Mmc infections.
Assuntos
Doenças das Cabras/microbiologia , Tipagem de Sequências Multilocus , Infecções por Mycoplasma/veterinária , Mycoplasma/classificação , Mycoplasma/genética , Animais , Feminino , Variação Genética , Genótipo , Doenças das Cabras/epidemiologia , Doenças das Cabras/mortalidade , Cabras , Índia/epidemiologia , Epidemiologia Molecular , Infecções por Mycoplasma/epidemiologia , Infecções por Mycoplasma/microbiologia , Infecções por Mycoplasma/mortalidade , Mycoplasma mycoides/genética , Mycoplasma mycoides/isolamento & purificaçãoRESUMO
Mycoplasma species are responsible for several economically significant livestock diseases for which there is a need for new and improved vaccines. Most of the existing mycoplasma vaccines are attenuated strains that have been empirically obtained by serial passages or by chemical mutagenesis. The recent development of synthetic biology approaches has opened the way for the engineering of live mycoplasma vaccines. Using these tools, the essential GTPase-encoding gene obg was modified directly on the Mycoplasma mycoides subsp. capri genome cloned in yeast, reproducing mutations suspected to induce a temperature-sensitive (TS+) phenotype. After transplantation of modified genomes into a recipient cell, the phenotype of the resulting M. mycoides subsp. capri mutants was characterized. Single-point obg mutations did not result in a strong TS+ phenotype in M. mycoides subsp. capri, but a clone presenting three obg mutations was shown to grow with difficulty at temperatures of ≥40°C. This particular mutant was then tested in a caprine septicemia model of M. mycoides subsp. capri infection. Five out of eight goats infected with the parental strain had to be euthanized, in contrast to one out of eight goats infected with the obg mutant, demonstrating an attenuation of virulence in the mutant. Moreover, the strain isolated from the euthanized animal in the group infected with the obg mutant was shown to carry a reversion in the obg gene associated with the loss of the TS+ phenotype. This study demonstrates the feasibility of building attenuated strains of mycoplasma that could contribute to the design of novel vaccines with improved safety.IMPORTANCE Animal diseases due to mycoplasmas are a major cause of morbidity and mortality associated with economic losses for farmers all over the world. Currently used mycoplasma vaccines exhibit several drawbacks, including low efficacy, short time of protection, adverse reactions, and difficulty in differentiating infected from vaccinated animals. Therefore, there is a need for improved vaccines to control animal mycoplasmoses. Here, we used genome engineering tools derived from synthetic biology approaches to produce targeted mutations in the essential GTPase-encoding obg gene of Mycoplasma mycoides subsp. capri Some of the resulting mutants exhibited a marked temperature-sensitive phenotype. The virulence of one of the obg mutants was evaluated in a caprine septicemia model and found to be strongly reduced. Although the obg mutant reverted to a virulent phenotype in one infected animal, we believe that these results contribute to a strategy that should help in building new vaccines against animal mycoplasmoses.
Assuntos
DNA Bacteriano/genética , GTP Fosfo-Hidrolases/genética , Mycoplasma mycoides/genética , Biologia Sintética/métodos , Animais , Proteínas de Bactérias/genética , Genoma Bacteriano , Cabras , Mutação , Infecções por Mycoplasma/sangue , Infecções por Mycoplasma/microbiologia , Mycoplasma mycoides/patogenicidade , Fenótipo , VirulênciaRESUMO
Orthologs document the evolution of genes and metabolic capacities encoded in extant and ancient genomes. However, the similarity between orthologs decays with time, and ultimately it becomes insufficient to infer common ancestry. This leaves ancient gene set reconstructions incomplete and distorted to an unknown extent. Here we introduce the "evolutionary traceability" as a measure that quantifies, for each protein, the evolutionary distance beyond which the sensitivity of the ortholog search becomes limiting. Using yeast, we show that genes that were thought to date back to the last universal common ancestor are of high traceability. Their functions mostly involve catalysis, ion transport, and ribonucleoprotein complex assembly. In turn, the fraction of yeast genes whose traceability is not sufficient to infer their presence in last universal common ancestor is enriched for regulatory functions. Computing the traceabilities of genes that have been experimentally characterized as being essential for a self-replicating cell reveals that many of the genes that lack orthologs outside bacteria have low traceability. This leaves open whether their orthologs in the eukaryotic and archaeal domains have been overlooked. Looking at the example of REC8, a protein essential for chromosome cohesion, we demonstrate how a traceability-informed adjustment of the search sensitivity identifies hitherto missed orthologs in the fast-evolving microsporidia. Taken together, the evolutionary traceability helps to differentiate between true absence and nondetection of orthologs, and thus improves our understanding about the evolutionary conservation of functional protein networks. "protTrace," a software tool for computing evolutionary traceability, is freely available at https://github.com/BIONF/protTrace.git; last accessed February 10, 2019.
Assuntos
Evolução Molecular , Modelos Genéticos , Proteínas/genética , Software , Simulação por Computador , Microsporídios/genética , Mycoplasma mycoides/genética , LevedurasRESUMO
JCVI-syn3A, a robust minimal cell with a 543 kbp genome and 493 genes, provides a versatile platform to study the basics of life. Using the vast amount of experimental information available on its precursor, Mycoplasma mycoides capri, we assembled a near-complete metabolic network with 98% of enzymatic reactions supported by annotation or experiment. The model agrees well with genome-scale in vivo transposon mutagenesis experiments, showing a Matthews correlation coefficient of 0.59. The genes in the reconstruction have a high in vivo essentiality or quasi-essentiality of 92% (68% essential), compared to 79% in silico essentiality. This coherent model of the minimal metabolism in JCVI-syn3A at the same time also points toward specific open questions regarding the minimal genome of JCVI-syn3A, which still contains many genes of generic or completely unclear function. In particular, the model, its comparison to in vivo essentiality and proteomics data yield specific hypotheses on gene functions and metabolic capabilities; and provide suggestions for several further gene removals. In this way, the model and its accompanying data guide future investigations of the minimal cell. Finally, the identification of 30 essential genes with unclear function will motivate the search for new biological mechanisms beyond metabolism.
One way that researchers can test whether they understand a biological system is to see if they can accurately recreate it as a computer model. The more they learn about living things, the more the researchers can improve their models and the closer the models become to simulating the original. In this approach, it is best to start by trying to model a simple system. Biologists have previously succeeded in creating 'minimal bacterial cells'. These synthetic cells contain fewer genes than almost all other living things and they are believed to be among the simplest possible forms of life that can grow on their own. The minimal cells can produce all the chemicals that they need to survive in other words, they have a metabolism. Accurately recreating one of these cells in a computer is a key first step towards simulating a complete living system. Breuer et al. have developed a computer model to simulate the network of the biochemical reactions going on inside a minimal cell with just 493 genes. By altering the parameters of their model and comparing the results to experimental data, Breuer et al. explored the accuracy of their model. Overall, the model reproduces experimental results, but it is not yet perfect. The differences between the model and the experiments suggest new questions and tests that could advance our understanding of biology. In particular, Breuer et al. identified 30 genes that are essential for life in these cells but that currently have no known purpose. Continuing to develop and expand models like these to reproduce more complex living systems provides a tool to test current knowledge of biology. These models may become so advanced that they could predict how living things will respond to changing situations. This would allow scientists to test ideas sooner and make much faster progress in understanding life on Earth. Ultimately, these models could one day help to accelerate medical and industrial processes to save lives and enhance productivity.
Assuntos
Genes Essenciais , Genoma Bacteriano , Mycoplasma mycoides/genética , Mycoplasma mycoides/metabolismo , Trifosfato de Adenosina/química , Simulação por Computador , Elementos de DNA Transponíveis , Escherichia coli , Ácido Fólico/metabolismo , Cinética , Substâncias Macromoleculares , Mutagênese , ProteômicaRESUMO
We previously discovered that intact bacterial chromosomes can be directly transferred to a yeast host cell where they can propagate as centromeric plasmids by fusing bacterial cells with S accharomyces cerevisiae spheroplasts. Inside the host any desired number of genetic changes can be introduced into the yeast centromeric plasmid to produce designer genomes that can be brought to life using a genome transplantation protocol. Earlier research demonstrated that the removal of restriction-systems from donor bacteria, such as Mycoplasma mycoides, Mycoplasma capricolum, or Haemophilus influenzae increased successful genome transfers. These findings suggested that other genetic factors might also impact the bacteria-to-yeast genome transfer process. In this study, we demonstrated that the removal of a particular genetic factor, the glycerol uptake facilitator protein gene glpF from M. mycoides, significantly increased direct genome transfer by up to 21-fold. Additionally, we showed that intact bacterial cells were endocytosed by yeast spheroplasts producing organelle-like structures within these yeast cells. These might lead to the possibility of creating novel synthetic organelles.
Assuntos
Genoma Bacteriano/genética , Mycoplasma mycoides/genética , Genoma Fúngico/genética , Glicerol/metabolismo , Haemophilus influenzae/genética , Mycoplasma capricolum/genética , Esferoplastos/citologia , Esferoplastos/metabolismoRESUMO
Capsular polysaccharides have been confirmed to be an important virulence trait in many gram-positive and gram-negative bacteria. Similarly, they are proposed to be virulence traits in minimal Mycoplasma that cause disease in humans and animals. In the current study, goats were infected with the caprine pathogen Mycoplasma mycoides subsp. capri or an engineered mutant lacking the capsular polysaccharide, galactofuranose. Goats infected with the mutant strain showed only transient fever. In contrast, 5 of 8 goats infected with the parental strain reached end-point criteria after infection. These findings confirm that galactofuranose is a virulence factor in M. mycoides.
Assuntos
Doenças das Cabras/microbiologia , Infecções por Mycoplasma/veterinária , Mycoplasma mycoides/metabolismo , Mycoplasma mycoides/patogenicidade , Polissacarídeos Bacterianos/genética , Animais , Doenças das Cabras/metabolismo , Cabras , Masculino , Mutação , Infecções por Mycoplasma/metabolismo , Infecções por Mycoplasma/microbiologia , Mycoplasma mycoides/química , Mycoplasma mycoides/genética , Polissacarídeos Bacterianos/metabolismoAssuntos
Biologia Celular/tendências , Engenharia Celular/tendências , Vida , Biologia Sintética/tendências , Complexos de ATP Sintetase/metabolismo , Trifosfato de Adenosina/biossíntese , Trifosfato de Adenosina/metabolismo , Fenômenos Biofísicos , Reatores Biológicos , Biologia Celular/ética , Divisão Celular/genética , Engenharia Celular/ética , Cloroplastos/metabolismo , Escherichia coli/citologia , Escherichia coli/enzimologia , Exobiologia , Genes Essenciais/genética , Humanos , Dispositivos Lab-On-A-Chip , Lipossomos , Viabilidade Microbiana/genética , Mitocôndrias/metabolismo , Mycoplasma capricolum/citologia , Mycoplasma capricolum/genética , Mycoplasma mycoides/citologia , Mycoplasma mycoides/genética , Força Próton-Motriz , Biologia Sintética/éticaRESUMO
OBJECTIVE: Regarded as one of the most expensive production diseases of dairy sheep and goats, contagious agalactia (CA) is caused by any of four agents: Mycoplasma agalactiae, M. mycoides subspecies capri (Mmc), M. capricolum subspecies capricolum (Mcc) and M. putrefaciens. Although CA is worldwide in distribution, it has not been reported in Australia, even though studies between the 1950s and 1980s isolated each agent from sheep or goats without any clinical signs associated with it. The aim of this study was to examine sheep and goats in Victoria, Australia, for the presence of CA-associated mycoplasmas and to investigate the evolutionary relationships of these isolates by comparing their genetic differences with their counterparts from other parts of the world. METHODS: A 3-year epidemiological survey of small ruminant populations in Victoria, Australia, was conducted for the presence of CA-associated mycoplasmas and the isolates obtained were genotyped by multilocus sequence typing (MLST). RESULTS: Mmc was the only CA-associated agent isolated from the 1358 samples analysed in the study, but was not associated with CA on the property where it was found. MLST analyses of Mmc strains revealed a distinct clustering of Australian isolates into a novel clade, with the closest relatives being strains from Europe. The distinct clustering is consistent with the absence of clinical disease in Australia. CONCLUSION: The isolation of Mmc indicates that this subspecies persists in Australian small ruminant populations. However, full genome sequencing and in vitro animal experimentation are needed to unequivocally demonstrate the avirulence of Australian strains.
Assuntos
Doenças das Cabras/epidemiologia , Infecções por Mycoplasma/veterinária , Mycoplasma mycoides/isolamento & purificação , Doenças dos Ovinos/epidemiologia , Animais , Doenças das Cabras/microbiologia , Cabras , Epidemiologia Molecular , Tipagem de Sequências Multilocus , Infecções por Mycoplasma/microbiologia , Mycoplasma mycoides/classificação , Mycoplasma mycoides/genética , Ovinos , Doenças dos Ovinos/microbiologia , Inquéritos e Questionários , Vitória/epidemiologiaAssuntos
Engenharia Celular , Engenharia Genética , Genoma/genética , Biologia Sintética/tendências , Animais , Cromatina/genética , Cromatina/metabolismo , Cromossomos Artificiais de Levedura/genética , DNA/biossíntese , DNA/genética , DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Redes Reguladoras de Genes/genética , Humanos , Mycoplasma mycoides/genética , Saccharomyces cerevisiae/genética , Salmonella typhimurium/genéticaRESUMO
Centromeres are essential for cell division and growth in all eukaryotes, and knowledge of their sequence and structure guides the development of artificial chromosomes for functional cellular biology studies. Centromeric proteins are conserved among eukaryotes; however, centromeric DNA sequences are highly variable. We combined forward and reverse genetic approaches with chromatin immunoprecipitation to identify centromeres of the model diatom Phaeodactylum tricornutum We observed 25 unique centromere sequences typically occurring once per chromosome, a finding that helps to resolve nuclear genome organization and indicates monocentric regional centromeres. Diatom centromere sequences contain low-GC content regions but lack repeats or other conserved sequence features. Native and foreign sequences with similar GC content to P. tricornutum centromeres can maintain episomes and recruit the diatom centromeric histone protein CENH3, suggesting nonnative sequences can also function as diatom centromeres. Thus, simple sequence requirements may enable DNA from foreign sources to persist in the nucleus as extrachromosomal episomes, revealing a potential mechanism for organellar and foreign DNA acquisition.
Assuntos
Centrômero/genética , DNA/metabolismo , Diatomáceas/genética , Plasmídeos/genética , Núcleo Celular/genética , Centrômero/metabolismo , Proteína Centromérica A/genética , Proteína Centromérica A/metabolismo , Imunoprecipitação da Cromatina/métodos , Cromossomos , DNA/genética , Mycoplasma mycoides/genéticaRESUMO
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
Bacteria are indispensable for the study of fundamental molecular biology processes due to their relatively simple gene and genome architecture. The ability to engineer bacterial chromosomes is quintessential for understanding gene functions. Here we demonstrate the engineering of the small-ribosomal subunit (16S) RNA of Mycoplasma mycoides, by combining the CRISPR/Cas9 system and the yeast recombination machinery. We cloned the entire genome of M. mycoides in yeast and used constitutively expressed Cas9 together with in vitro transcribed guide-RNAs to introduce engineered 16S rRNA genes. By testing the function of the engineered 16S rRNA genes through genome transplantation, we observed surprising resilience of this gene to addition of genetic elements or helix substitutions with phylogenetically-distant bacteria. While this system could be further used to study the function of the 16S rRNA, one could envision the "simple" M. mycoides genome being used in this setting to study other genetic structures and functions to answer fundamental questions of life.
Assuntos
Engenharia Genética/métodos , Mycoplasma mycoides/genética , RNA Ribossômico 16S/genética , Sistemas CRISPR-Cas , Clonagem Molecular , Genoma Bacteriano , Filogenia , RNA Bacteriano/genética , Saccharomyces cerevisiae/genéticaRESUMO
Mycoplasma mycoides subsp. capri (Mmc) is one of the main causative agents of caprine contagious agalactia. Besides, the absence of accurate control methods eases its dispersion between different herds within endemic areas of this disease. In this context, there is a need to implement molecular typing schemes which offer valuable information useful to establish control measures and enables the surveillance of this pathogen. The aim of this study was to assess the genetic variability of different strains of Mmc from a contagious agalactia endemic area through multilocus sequence typing (MLST). For this purpose, five house-keeping genes (fusA, glpQ, gyrB, lepA, rpoB) from 39 field isolates were analysed. These isolates were obtained from different geographic areas of Spain, between the years 2004 and 2015. The results obtained in this study suggest that the selected MLST scheme could be a useful technique to monitor the genetic variability of Mmc in endemic areas. Despite the significant differences found between the assessed field isolates, they could be classified according to their geographical origin. Moreover, it was also possible to detect genetic differences between Mmc strains coming from the same herd at the same sampling time, which may need to be taken into consideration when designing or arranging prophylactic strategies.