RESUMO
Marine cyanobacteria of the genus Prochlorococcus represent numerically dominant photoautotrophs residing throughout the euphotic zones in the open oceans and are major contributors to the global carbon cycle. Prochlorococcus has remained a genetically intractable bacterium due to slow growth rates and low transformation efficiencies using standard techniques. Our recent successes in cloning and genetically engineering the AT-rich, 1.1 Mb Mycoplasma mycoides genome in yeast encouraged us to explore similar methods with Prochlorococcus. Prochlorococcus MED4 has an AT-rich genome, with a GC content of 30.8%, similar to that of Saccharomyces cerevisiae (38%), and contains abundant yeast replication origin consensus sites (ACS) evenly distributed around its 1.66 Mb genome. Unlike Mycoplasma cells, which use the UGA codon for tryptophane, Prochlorococcus uses the standard genetic code. Despite this, we observed no toxic effects of several partial and 15 whole Prochlorococcus MED4 genome clones in S. cerevisiae. Sequencing of a Prochlorococcus genome purified from yeast identified 14 single base pair missense mutations, one frameshift, one single base substitution to a stop codon and one dinucleotide transversion compared to the donor genomic DNA. We thus provide evidence of transformation, replication and maintenance of this 1.66 Mb intact bacterial genome in S. cerevisiae.
Assuntos
Genoma Bacteriano , Prochlorococcus/genética , Clonagem Molecular , Genes Bacterianos , Mutação , Origem de Replicação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Análise de Sequência de DNARESUMO
Most microbes have not been cultured, and many of those that are cultivatable are difficult, dangerous or expensive to propagate or are genetically intractable. Routine cloning of large genome fractions or whole genomes from these organisms would significantly enhance their discovery and genetic and functional characterization. Here we report the cloning of whole bacterial genomes in the yeast Saccharomyces cerevisiae as single-DNA molecules. We cloned the genomes of Mycoplasma genitalium (0.6 Mb), M. pneumoniae (0.8 Mb) and M. mycoides subspecies capri (1.1 Mb) as yeast circular centromeric plasmids. These genomes appear to be stably maintained in a host that has efficient, well-established methods for DNA manipulation.
Assuntos
Clonagem Molecular/métodos , Genoma Bacteriano , Mycoplasma/genética , Saccharomyces cerevisiae/genética , Sequência de Bases , Diploide , Vetores Genéticos/química , Dados de Sequência Molecular , Mycoplasma genitalium/genética , Mycoplasma mycoides/genética , Mycoplasma pneumoniae/genética , Recombinação GenéticaRESUMO
We previously reported assembly and cloning of the synthetic Mycoplasma genitalium JCVI-1.0 genome in the yeast Saccharomyces cerevisiae by recombination of six overlapping DNA fragments to produce a 592-kb circle. Here we extend this approach by demonstrating assembly of the synthetic genome from 25 overlapping fragments in a single step. The use of yeast recombination greatly simplifies the assembly of large DNA molecules from both synthetic and natural fragments.
Assuntos
DNA/biossíntese , Genoma Bacteriano/genética , Mycoplasma genitalium/genética , Oligodesoxirribonucleotídeos/genética , Leveduras/genética , Clonagem Molecular/métodos , Oligodesoxirribonucleotídeos/metabolismo , Recombinação GenéticaRESUMO
Most gene knockouts in mycoplasmas are achieved through labor-intensive transposon mutagenesis. Here, we describe a method for making targeted deletions in Mycoplasma pneumoniae by use of homologous recombination. In this method, M. pneumoniae is transformed with a plasmid carrying an antibiotic resistance marker flanked by 1-kb regions surrounding the target gene. Following selection for the antibiotic resistance, colonies are screened for double crossovers which indicate complete deletion of the target open reading frame.
Assuntos
Técnicas de Inativação de Genes/métodos , Marcação de Genes/métodos , Genética Microbiana/métodos , Mycoplasma pneumoniae/genética , Antibacterianos/farmacologia , DNA Bacteriano/química , DNA Bacteriano/genética , Farmacorresistência Bacteriana , Vetores Genéticos , Dados de Sequência Molecular , Plasmídeos , Recombinação Genética , Seleção Genética , Análise de Sequência de DNARESUMO
Many bacterial and archaeal genomes are of a similar size to molecules that have been cloned in the yeast Saccharomyces cerevisiae and thus might be clonable as single, circular episomes in this host. Yeast offers a variety of efficient tools for the manipulation and study of cloned DNA. One strategy to clone a genome in yeast is to cotransform yeast spheroplasts with the genome of interest and a linear yeast vector whose termini are homologous to a spot in the genome. Clones are selected on auxotrophic medium and then screened for completeness and size; they may also be sequenced.
Assuntos
Clonagem Molecular/métodos , Genoma Bacteriano/genética , Saccharomyces cerevisiae/genética , Técnicas de Cultura , DNA/genética , DNA/isolamento & purificação , DNA/metabolismo , Enzimas de Restrição do DNA/metabolismo , Eletroforese em Gel de Ágar , Engenharia Genética , Vetores Genéticos/genética , Reação em Cadeia da Polimerase , Saccharomyces cerevisiae/citologia , Esferoplastos/genética , Biologia Sintética , Transformação GenéticaRESUMO
We report the design, synthesis, and assembly of the 1.08-mega-base pair Mycoplasma mycoides JCVI-syn1.0 genome starting from digitized genome sequence information and its transplantation into a M. capricolum recipient cell to create new M. mycoides cells that are controlled only by the synthetic chromosome. The only DNA in the cells is the designed synthetic DNA sequence, including "watermark" sequences and other designed gene deletions and polymorphisms, and mutations acquired during the building process. The new cells have expected phenotypic properties and are capable of continuous self-replication.
Assuntos
Bioengenharia , Engenharia Genética , Genoma Bacteriano , Mycoplasma capricolum/genética , Mycoplasma mycoides/genética , Proteínas de Bactérias/análise , Sequência de Bases , Clonagem Molecular , DNA Bacteriano/síntese química , DNA Bacteriano/genética , Escherichia coli/genética , Deleção de Genes , Genes Bacterianos , Dados de Sequência Molecular , Mycoplasma mycoides/crescimento & desenvolvimento , Mycoplasma mycoides/fisiologia , Mycoplasma mycoides/ultraestrutura , Fenótipo , Plasmídeos , Reação em Cadeia da Polimerase , Polimorfismo Genético , Saccharomyces cerevisiae/genética , Transformação BacterianaRESUMO
We recently reported the chemical synthesis, assembly, and cloning of a bacterial genome in yeast. To produce a synthetic cell, the genome must be transferred from yeast to a receptive cytoplasm. Here we describe methods to accomplish this. We cloned a Mycoplasma mycoides genome as a yeast centromeric plasmid and then transplanted it into Mycoplasma capricolum to produce a viable M. mycoides cell. While in yeast, the genome was altered by using yeast genetic systems and then transplanted to produce a new strain of M. mycoides. These methods allow the construction of strains that could not be produced with genetic tools available for this bacterium.
Assuntos
Clonagem Molecular , Técnicas de Transferência de Genes , Engenharia Genética , Genoma Bacteriano , Mycoplasma capricolum/genética , Mycoplasma mycoides/genética , Saccharomyces cerevisiae/genética , Centrômero , Metilação de DNA , Enzimas de Restrição do DNA/genética , Enzimas de Restrição do DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo III/genética , Mycoplasma mycoides/crescimento & desenvolvimento , Mycoplasma mycoides/isolamento & purificação , Plasmídeos , Análise de Sequência de DNA , Deleção de Sequência , Transformação BacterianaRESUMO
We have synthesized a 582,970-base pair Mycoplasma genitalium genome. This synthetic genome, named M. genitalium JCVI-1.0, contains all the genes of wild-type M. genitalium G37 except MG408, which was disrupted by an antibiotic marker to block pathogenicity and to allow for selection. To identify the genome as synthetic, we inserted "watermarks" at intergenic sites known to tolerate transposon insertions. Overlapping "cassettes" of 5 to 7 kilobases (kb), assembled from chemically synthesized oligonucleotides, were joined by in vitro recombination to produce intermediate assemblies of approximately 24 kb, 72 kb ("1/8 genome"), and 144 kb ("1/4 genome"), which were all cloned as bacterial artificial chromosomes in Escherichia coli. Most of these intermediate clones were sequenced, and clones of all four 1/4 genomes with the correct sequence were identified. The complete synthetic genome was assembled by transformation-associated recombination cloning in the yeast Saccharomyces cerevisiae, then isolated and sequenced. A clone with the correct sequence was identified. The methods described here will be generally useful for constructing large DNA molecules from chemically synthesized pieces and also from combinations of natural and synthetic DNA segments.
Assuntos
Clonagem Molecular , DNA Bacteriano/síntese química , Genoma Bacteriano , Genômica/métodos , Mycoplasma genitalium/genética , Sequência de Bases , Cromossomos Artificiais Bacterianos , Cromossomos Artificiais de Levedura , DNA Recombinante , Escherichia coli/genética , Vetores Genéticos , Oligodesoxirribonucleotídeos/síntese química , Plasmídeos , Recombinação Genética , Saccharomyces cerevisiae/genética , Análise de Sequência de DNA , Transformação GenéticaRESUMO
Several experimental approaches were used to construct a detailed transcriptional profile of the phylogenetically conserved ftsZ cell division gene cluster in both Mycoplasma genitalium and its closest relative, Mycoplasma pneumoniae. We determined initiation and termination points for the cluster, as well as an absolute steady-state RNA level for each gene. Transcription of this cluster in both these organisms was shown to be highly strand specific. While the four genes in this cluster are cotranscribed, their transcription unit also includes two genes of close proximity yet disparate function. A transcription initiation point immediately upstream of these two genes was detected in M. genitalium but not M. pneumoniae. In M. pneumoniae, transcription of the six genes terminates at a poly(U)-tailed hairpin. In M. genitalium, this transcription terminates at two closely spaced points by an unknown mechanism. Real-time reverse transcription-PCR analysis of this cluster in M. pneumoniae shows that mRNA levels for all six genes vary at most fivefold and form a gradient of decreasing quantity with increasing distance from the promoter at the beginning of the cluster. mRNA from coding regions was approximately 20- to 100-fold more abundant than that from intergenic regions. We estimated the most abundant mRNA we detected at 0.6 copy per cell. We conclude that groups of functionally related genes in M. genitalium and M. pneumoniae are often preceded by promoters but rarely followed by terminators. This causes functionally unrelated genes to be commonly cotranscribed in these organisms.