Sequence analysis of a complete 1.66 Mb Prochlorococcus marinus MED4 genome cloned in yeast

• Published in: Nucleic acids research 2012-11, Vol.40 (20), p.10375-10383
• Main Authors: Tagwerker, Christian, Dupont, Christopher L, Karas, Bogumil J, Ma, Li, Chuang, Ray-Yuan, Benders, Gwynedd A, Ramon, Adi, Novotny, Mark, Montague, Michael G, Venepally, Pratap, Brami, Daniel, Schwartz, Ariel, Andrews-Pfannkoch, Cynthia, Gibson, Daniel G, Glass, John I, Smith, Hamilton O, Venter, J Craig, Hutchison, 3rd, Clyde A
• Format: Article
• Language: English
• Subjects: Base pairs; Carbon cycle; Cloning, Molecular; Codons; Cyanobacteria; DNA; DNA sequencing; Euphotic zone; Genes, Bacterial; Genetic code; Genome, Bacterial; Genomes; genomics; Growth rate; Guanylate cyclase; Missense mutation; Molecular Biology; Mutation; Mycoplasma; Mycoplasma mycoides; Nucleotide sequence; Oceans; Prochlorococcus; Prochlorococcus - genetics; Prochlorococcus marinus; Replication; Replication Origin; Replication origins; Saccharomyces cerevisiae; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - growth & development; Sequence Analysis, DNA; Stop codon; Transformation; Transversion
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gks823

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.