title: Sequence analysis of a complete 1.66 Mb Prochlorococcus marinus MED4 genome cloned in yeast.
abstract: 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.
authors.affiliation: Department of Synthetic Biology and Bioenergy, J. Craig Venter Institute, 10355 Science Center Drive, San Diego, CA 92121, USA. ctagwerker@jcvi.org
authors.forename: Christian
authors.initials: C
authors.lastname: Tagwerker
authors.forename: Christopher L
authors.initials: CL
authors.lastname: Dupont
authors.forename: Bogumil J
authors.initials: BJ
authors.lastname: Karas
authors.forename: Li
authors.initials: L
authors.lastname: Ma
authors.forename: Ray-Yuan
authors.initials: RY
authors.lastname: Chuang
authors.forename: Gwynedd A
authors.initials: GA
authors.lastname: Benders
authors.forename: Adi
authors.initials: A
authors.lastname: Ramon
authors.forename: Mark
authors.initials: M
authors.lastname: Novotny
authors.forename: Michael G
authors.initials: MG
authors.lastname: Montague
authors.forename: Pratap
authors.initials: P
authors.lastname: Venepally
authors.forename: Daniel
authors.initials: D
authors.lastname: Brami
authors.forename: Ariel
authors.initials: A
authors.lastname: Schwartz
authors.forename: Cynthia
authors.initials: C
authors.lastname: Andrews-Pfannkoch
authors.forename: Daniel G
authors.initials: DG
authors.lastname: Gibson
authors.forename: John I
authors.initials: JI
authors.lastname: Glass
authors.forename: Hamilton O
authors.initials: HO
authors.lastname: Smith
authors.forename: J Craig
authors.initials: JC
authors.lastname: Venter
authors.forename: Clyde A
authors.initials: CA
authors.lastname: Hutchison
mesh_terms: D003001:Cloning, Molecular; D005798:Genes, Bacterial; D016680:Genome, Bacterial; D009154:Mutation; D048929:Prochlorococcus / Q000235:genetics*; D018741:Replication Origin; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development; D017422:Sequence Analysis, DNA