Cloning whole bacterial genomes in yeast

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

Transcriptional Analysis of the Conserved ftsZ Gene Cluster in Mycoplasma genitalium and Mycoplasma pneumoniae

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

Targeted Chromosomal Knockouts in Mycoplasma pneumoniae▿ †

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

Creation of a Bacterial Cell Controlled by a Chemically Synthesized Genome

We report the design, synthesis and assembly of the 1.08-Mbp Mycoplasma mycoides JCVI-syn1.0 genome starting from digitized genome sequence information and its transplantation into a Mycoplasma capricolum recipient cell to create new Mycoplasma 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. In 1977, Sanger and colleagues determined the complete genetic code of phage φX174 (1), the first DNA genome to be completely sequenced. Eighteen years later, in 1995, our tea