RESUMO
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
To unravel the underlying principles of membrane adaptation in small systems like bacterial cells, robust approaches to characterize membrane fluidity are needed. Currently available relevant methods require advanced instrumentation and are not suitable for high-throughput settings needed to elucidate the biochemical pathways involved in adaptation. We developed a fast, robust, and financially accessible quantitative method to measure the microviscosity of lipid membranes in bulk suspension using a commercially available plate reader. Our approach, which is suitable for high-throughput screening, is based on the simultaneous measurements of absorbance and fluorescence emission of a viscosity-sensitive fluorescent dye, 9-(2,2-dicyanovinyl)julolidine (DCVJ), incorporated into a lipid membrane. We validated our method using artificial membranes with various lipid compositions over a range of temperatures and observed values that were in good agreement with previously published results. Using our approach, we were able to detect a lipid phase transition in the ruminant pathogen Mycoplasma mycoides.
Assuntos
Corantes Fluorescentes/química , Ensaios de Triagem em Larga Escala , Lipídeos de Membrana/química , Mycoplasma mycoides/química , Tamanho da Partícula , ViscosidadeRESUMO
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/metabolismoRESUMO
Mycoplasma mycoides subsp. capri (Mmc) and subsp. mycoides (Mmm) are important ruminant pathogens worldwide causing diseases such as pleuropneumonia, mastitis and septicaemia. They express galactofuranose residues on their surface, but their role in pathogenesis has not yet been determined. The M. mycoides genomes contain up to several copies of the glf gene, which encodes an enzyme catalysing the last step in the synthesis of galactofuranose. We generated a deletion of the glf gene in a strain of Mmc using genome transplantation and tandem repeat endonuclease coupled cleavage (TREC) with yeast as an intermediary host for the genome editing. As expected, the resulting YCp1.1-Δglf strain did not produce the galactofuranose-containing glycans as shown by immunoblots and immuno-electronmicroscopy employing a galactofuranose specific monoclonal antibody. The mutant lacking galactofuranose exhibited a decreased growth rate and a significantly enhanced adhesion to small ruminant cells. The mutant was also 'leaking' as revealed by a ß-galactosidase-based assay employing a membrane impermeable substrate. These findings indicate that galactofuranose-containing polysaccharides conceal adhesins and are important for membrane integrity. Unexpectedly, the mutant strain showed increased serum resistance.
Assuntos
Adesinas Bacterianas/análise , Atividade Bactericida do Sangue , Membrana Celular/química , Membrana Celular/fisiologia , Dissacarídeos/análise , Mycoplasma mycoides/química , Mycoplasma mycoides/fisiologia , Animais , Aderência Bacteriana , Células Cultivadas , Deleção de Genes , Marcação de Genes , Immunoblotting , Microscopia Imunoeletrônica , Mycoplasma mycoides/genética , OvinosRESUMO
Contagious bovine pleuropneumonia (CBPP), caused by Mycoplasma mycoides subsp. mycoides (Mmm) is a severe cattle disease, present in many countries in sub-Saharan Africa. The development of improved diagnostic tests and vaccines for CBPP control remains a research priority. Polyacrylamide gel electrophoresis and mass spectrometry were used to characterize the Triton X-114 soluble proteome of nine Mmm strains isolated from Europe or Africa. Of a total of 250 proteins detected, 67 were present in all strains investigated. Of these, 44 were predicted to be lipoproteins or cytoplasmic membrane-associated proteins and are thus likely to be members of the core in vitro surface membrane-associated proteome of Mmm. Moreover, the presence of all identified proteins in other ruminant Mycoplasma pathogens were investigated. Two proteins of the core proteome were identified only in other cattle pathogens of the genus Mycoplasma pointing towards a role in host-pathogen interactions. The data generated will facilitate the identification and prioritization of candidate Mycoplasma antigens for improved control measures, as it is likely that surface-exposed membrane proteins will include those that are involved in host-pathogen interactions.
Assuntos
Proteínas da Membrana Bacteriana Externa/química , Doenças dos Bovinos/microbiologia , Mycoplasma mycoides/química , Mycoplasma mycoides/genética , Pleuropneumonia Contagiosa/microbiologia , Proteoma/genética , África , Animais , Bovinos , Cromatografia Líquida , Europa (Continente) , Mycoplasma/genética , Mycoplasma/metabolismo , Mycoplasma mycoides/isolamento & purificação , Pleuropneumonia Contagiosa/diagnóstico , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
A genome synthesized entirely from scratch has been used to replace the native genome of a living cell, thus creating a new cell. This achievement marks a new frontier in synthetic biology to design and create genomes for organisms with few genetic tools and for applications in areas of energy, health care and the environment.
Assuntos
Genes Sintéticos , Genoma Bacteriano , Mycoplasma mycoides/genética , Escherichia coli/química , Escherichia coli/genética , Engenharia Genética , Mycoplasma mycoides/químicaRESUMO
Analysis, using the polymerase chain reaction (PCR), restriction enzyme endonuclease analysis (REA), protein profile patterns, random amplification of polymorphic DNA (RAPD) fingerprinting, 16S rRNA gene sequencing and antisera growth inhibition tests, of 22 strains of Mycoplasma mycoides subsp. mycoides Large Colony type (MmmLC) and eight strains of M. mycoides subsp. capri (Mmc) are presented, along with a summary of comparative data from the literature for over 100 strains, all of which supports the reclassification of the MmmLC and Mmc strains into the single subspecies, M. mycoides subspecies capri.
Assuntos
Mycoplasma mycoides/classificação , Proteínas de Bactérias/análise , Impressões Digitais de DNA , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Mycoplasma mycoides/química , Mycoplasma mycoides/genética , RNA Ribossômico 16S/genética , Técnica de Amplificação ao Acaso de DNA Polimórfico , Análise de Sequência de DNARESUMO
As a step toward propagation of synthetic genomes, we completely replaced the genome of a bacterial cell with one from another species by transplanting a whole genome as naked DNA. Intact genomic DNA from Mycoplasma mycoides large colony (LC), virtually free of protein, was transplanted into Mycoplasma capricolum cells by polyethylene glycol-mediated transformation. Cells selected for tetracycline resistance, carried by the M. mycoides LC chromosome, contain the complete donor genome and are free of detectable recipient genomic sequences. These cells that result from genome transplantation are phenotypically identical to the M. mycoides LC donor strain as judged by several criteria.
Assuntos
DNA Bacteriano/genética , Genoma Bacteriano , Mycoplasma mycoides/genética , Mycoplasma/genética , Transformação Bacteriana , Acetato Quinase/química , Acetato Quinase/genética , Sequência de Aminoácidos , DNA Bacteriano/isolamento & purificação , Genótipo , Dados de Sequência Molecular , Mycoplasma/química , Mycoplasma mycoides/química , Fenótipo , Polietilenoglicóis , Proteoma/análise , Recombinação Genética , Análise de Sequência de DNARESUMO
The prokaryotic signal recognition particle (SRP) pathway comprises two proteins, Ffh and FtsY, homologous to the SRP54 and SRalpha proteins in the more complex eukaryotic system. All four proteins are part of a unique subfamily of GTPases. Four truncated versions of the 412 amino-acid FtsY receptor protein from Mycoplasma mycoides have been cloned, expressed in Escherichia coli and purified. Purified proteins from all constructs and the full-length FtsY protein were subjected to crystallization trials. Crystals were obtained for the construct which comprised residues 98-412 corresponding to the conserved NG-domain (residues 194-497 in E. coli). A native data set at 1.9 A resolution has been collected at 100 K using synchrotron radiation. The crystals belong to the space group P2(1)2(1)2, with unit-cell parameters a = 68.7, b = 101.1, c = 42.5 A and one molecule in the asymmetric unit.
Assuntos
Proteínas de Bactérias/química , Mycoplasma mycoides/química , Receptores Citoplasmáticos e Nucleares/química , Proteínas de Bactérias/genética , Sequência de Bases , Cristalização , Cristalografia por Raios X , Primers do DNA/genética , Escherichia coli/genética , Mycoplasma mycoides/genética , Receptores Citoplasmáticos e Nucleares/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Partícula de Reconhecimento de Sinal/química , Partícula de Reconhecimento de Sinal/genéticaRESUMO
Nucleotide sequence analysis of the 16S-23S rRNA intergenic spacer regions of six type or reference strains belonging to the Mycoplasma mycoides cluster and of Mycoplasma putrefaciens suggested the presence of two subclusters. One subcluster comprised M. mycoides subsp. mycoides small colony (SC) type, M. mycoides subsp. mycoides large colony (LC) type and M. mycoides subsp. capri, whereas the second subcluster comprised Mycoplasma capricolum subsp. capricolum, M. capricolum subsp. capripneumoniae and Mycoplasma sp. bovine group 7. The type strains from M. mycoides subsp. mycoides SC and M. mycoides subsp. capri had identical spacer sequences. The existence of two subclusters was supported by predicted secondary structures of the analysed region. The nucleotide variations in the loop domains of the secondary structures might be a useful genetic marker to distinguish between the two subclusters. The secondary structure differences delineated the differences between the two subclusters more clearly than the nucleotide sequence alignments, which only showed a small number of differences, and some of these were common to both clusters. The data also provided evidence in favour of a reclassification of Mycoplasma sp. bovine group 7 as another subspecies of M. capricolum.
Assuntos
DNA Bacteriano/genética , DNA Ribossômico/genética , Mycoplasma mycoides/classificação , Mycoplasma/classificação , Filogenia , Animais , Sequência de Bases , Bovinos , DNA Bacteriano/química , DNA Ribossômico/química , Cabras , Dados de Sequência Molecular , Mycoplasma/genética , Mycoplasma mycoides/química , Mycoplasma mycoides/genética , Conformação de Ácido Nucleico , Pleuropneumonia Contagiosa/microbiologia , RNA Ribossômico 16S/genética , RNA Ribossômico 23S/genética , Análise de Sequência de DNARESUMO
The Mycoplasma mycoides cluster is made of six species that are closely related both genetically and phenotypically. Two are of particular importance, M. mycoides subsp. mycoides SC causing contagious bovine pleuropneumonia and M. capricolum subsp. capripneumoniae causing contagious caprine pleuropneumonia. The sequences of a putative membrane protein gene and partial flanking open reading frames have been obtained from various strains in this cluster, including all reference strains. Sequence analysis showed this locus is present and fully conserved in all strains of M. mycoides subsp. mycoides SC isolated from geographically most distant places worldwide. In M. capricolum subsp. capripneumoniae polymorphism in this locus has been found at seven positions and revealed that they can be used as epidemiological markers. Conserved regions were used to define a primer pair that enables the amplification by PCR of two fragments 302 and 1298bp long, respectively. The 302bp long fragment contains an intergenic sequence that can be used for phylogenetic studies or for identification purposes. Parsimony analysis on an alignment of 49 DNA sequences show a subdivision of the M. mycoides cluster into two subgroups that is in accordance with results obtained by phenotypic methods. Two lineages exist within the capricolum subgroup, one of them clustering strains identified as M. capricolum subsp. capricolum, M. capricolum subsp. capricolum and M. sp Bovine Group 7. However M. capricolum subsp. capripneumoniae strains can readily be identified by three specific nucleotide positions or by sequencing the 1298bp long fragment. There is no clear subdivision within the mycoides subgroup, supporting the idea that M. mycoides subsp. mycoides LC and M. mycoides subsp. capri should not be separated into two subspecies. Mycoplasma mycoides subsp. mycoides SC strains can easily be distinguished as they bear an insertion sequence 15bp downstream from the stop codon of the membrane protein gene.
Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Doenças dos Bovinos/microbiologia , Doenças das Cabras/microbiologia , Mycoplasma mycoides/genética , Filogenia , Pleuropneumonia Contagiosa/microbiologia , Animais , Proteínas da Membrana Bacteriana Externa/química , Sequência de Bases , Bovinos , Clonagem Molecular , Análise por Conglomerados , Primers do DNA/química , DNA Bacteriano/química , Biblioteca Gênica , Cabras , Dados de Sequência Molecular , Mycoplasma mycoides/química , Mycoplasma mycoides/classificação , Reação em Cadeia da Polimerase , Alinhamento de Sequência/veterinária , Análise de Sequência de DNARESUMO
The lppA gene, encoding the lipoprotein named LppA[Mcaca] was characterised in Mycoplasma capricolum subsp. capricolum. It encodes a lipoprotein with an apparent molecular mass of 57 kDa as determined by SDS-PAGE. Using antibodies directed against recombinant LppA[Mcaca], we showed the expression of this lipoprotein in all M. capricolum subsp. capricolum by immunoblot analysis. The serum did not cross-react with other members of the Mycoplasma mycoides cluster, hence showing that LppA[Mcaca] was antigenically specific to M. capricolum subsp. capricolum. The lppA gene was conserved within the subspecies and was used for the development of a specific PCR assay for the identification of M. capricolum subsp. capricolum. The taxonomically related Mycoplasma capricolum subsp. capripneumoniae (F38) was found to contain an lppA-pseudo-gene. It showed high similarity to functional lppA genes of other mycoplasmas in the M. mycoides cluster. However, it contained interrupted open reading frames. Moreover, the nucleotide sequence of the lppA pseudo-genes in different strains of M. capricolum subsp. capripneumoniae were quite variable. Interestingly, the lppA pseudo-gene had a size similar to that of the functional lppA genes of other mycoplasmas of the M. mycoides cluster and occupied the same genomic location as the latter ones in the vicinity of the mtlD genes. This study showed that all members of the M. mycoides cluster contain each a species-, subspecies- respectively type- specific lppA gene analogue which encodes a lipoprotein that has structural and functional relationship to the surface lipoprotein LppA [MmymySC], previously named P72, of M. mycoides subsp mycoides SC, with the exception of M. capricolum subsp. capripneumoniae which seems not to express an LppA analogue.
Assuntos
Proteínas de Bactérias/genética , Doenças das Cabras/microbiologia , Lipoproteínas/genética , Infecções por Mycoplasma/veterinária , Mycoplasma mycoides/genética , Animais , Anticorpos Antibacterianos/biossíntese , Proteínas de Bactérias/química , Sequência de Bases , Western Blotting/veterinária , Mapeamento Cromossômico , Primers do DNA/química , DNA Bacteriano/química , Biblioteca Gênica , Cabras , Lipoproteínas/química , Camundongos , Dados de Sequência Molecular , Infecções por Mycoplasma/microbiologia , Mycoplasma mycoides/química , Mycoplasma mycoides/classificação , Hibridização de Ácido Nucleico , Reação em Cadeia da Polimerase/veterinária , Proteínas Recombinantes/genética , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
The genes encoding the 62-kDa lipoproteins from the Mycoplasma mycoides subsp. mycoides large-colony type (LC) strain Y-goat and the M. mycoides subsp. capri strain PG3 were cloned and analyzed by sequencing. These two lipoproteins have been named LppA[MmymyLC] and LppA[Mmyca], and their corresponding genes have been named lppA[MmymyLC] and lppA[Mmyca], respectively. The nucleotide and deduced amino acid sequences of these two lipoproteins showed a very high degree of similarity between these two mycoplasmas. Given the sequence data, LppA seems to fulfill the same structural functions as the previously described major lipoproteins P72 of M. mycoides subsp. mycoides small-colony type and P67 of the Mycoplasma species bovine group 7. Based on lppA gene sequences of M. mycoides subsp. mycoides LC and M. mycoides subsp. capri type strains, a specific PCR assay was developed so that it amplified this gene in all field strains of the two species analyzed in this study but not in the other members of the M. mycoides cluster. Analysis of the PCR-amplified lppA genes with frequently cutting restriction enzymes showed a certain degree of genetic variability which, however, did not cluster the two subspecies. This PCR therefore allows a rapid identification of M. mycoides subsp. mycoides LC and M. mycoides subsp. capri but does not distinguish between these two closely related subspecies. LppA was expressed in Escherichia coli K-12 and used for the production of polyclonal mouse antiserum. Antibodies against recombinant LppA[MmymyLC] reacted with a 62-kDa protein in all M. mycoides subsp. mycoides LC and M. mycoides subsp. capri type strains and field strains tested but not with the other members of the M. mycoides cluster, thus showing the antigenic specificity of LppA and further supporting the concept that a close relationship exists between these two mycoplasmas.
Assuntos
Lipoproteínas/genética , Lipoproteínas/imunologia , Mycoplasma mycoides/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Southern Blotting , Clonagem Molecular , Primers do DNA , DNA Bacteriano/análise , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Dados de Sequência Molecular , Mycoplasma mycoides/química , Mycoplasma mycoides/imunologia , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
The course of immune reactions of the manifold antigens of Mycoplasma mycoides subsp. mycoides small colony type (SC) was analysed in serum and bronchial lavage of cattle experimentally infected with the African strain Afadé and the European strain L2 using Western-blots and complement fixation. Western-blot analysis of total antigens of both strains with sera from animals infected with the homologous and heterologous strain revealed the common dominant immunogenic antigens with the molecular masses of 110, 95, 85, 80, 72, 62, 48 and 39 kDa. The sequential sampling of the blood and bronchial lavages before and after contact infections allowed us to identify the antigens of 85, 80, 72, 48 and 39 kDa as particularly early immunogens. The IgA Western blots of the bronchial lavages showed distinct, early and persistent reactions to the 110, 85, 80, 72, 48 and 45 kDa proteins. These proteins were the predominant lipoproteins as determined by [14C]palmitic acid labelling. Only relatively weak reactions of the bronchial lavages were detected with IgG. In general immune responses were significantly stronger in the animals infected with the African strain Afadé, which gave positive results two weeks after contact infection. In contrast, the animals infected with the European strain L2 induced much lower reactions with a delay of three months after contact infection. In one animal strain L2 caused no sero-conversion and no infection. The results indicate a difference in virulence between the African strain Afadé and the European strain L2.
Assuntos
Anticorpos Antibacterianos/biossíntese , Brônquios/imunologia , Doenças dos Bovinos/imunologia , Imunoglobulinas/biossíntese , Mycoplasma mycoides/imunologia , Pleuropneumonia Contagiosa/imunologia , África , Animais , Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/análise , Antígenos de Bactérias/química , Antígenos de Bactérias/imunologia , Western Blotting/veterinária , Lavagem Broncoalveolar/veterinária , Bovinos , Europa (Continente) , Feminino , Imunoglobulina G/biossíntese , Imunoglobulina G/sangue , Imunoglobulina M/biossíntese , Imunoglobulina M/sangue , Imunoglobulinas/sangue , Lipoproteínas/análise , Mycoplasma mycoides/químicaAssuntos
Doenças dos Bovinos/microbiologia , Mycoplasma mycoides/isolamento & purificação , Pleuropneumonia Contagiosa/microbiologia , Sêmen/microbiologia , Animais , Bovinos , Masculino , Técnicas Microbiológicas/veterinária , Mycoplasma mycoides/química , Mycoplasma mycoides/classificação , Pênis/microbiologia , Irrigação Terapêutica/veterináriaRESUMO
In all the strains of M. gallisepticum investigated, a protein with apparent molecular weight 40 kDa was revealed by immunoblotting with polyclonal anti-calf brain tubulin antibodies and monoclonal anti-chicken alpha-tubulin antibodies. In other 8 investigated Mycoplasma species no positive reactions with the same antibodies were found. The M. Gallisepticum cells were examined under electron microscope on fine serial sections and on some sections going at different angles to the long cell axis. Undermembrane system of tubules was revealed and the intracellular pattern of the tubular structures were reconstructed. The immunoelectron microscopic data suggest that tubulin-like protein may be included into the structures.