J. Gen. Plant Pathol. zyxwvutsr 68 : 225-236 (2002) zyxwvut Minimal Set of Metabolic Pathways Suggested from the Genome of Onion Yellows Phytoplasmat Kenro OSHIMA1,4, Shin-ichi MIYATA'>4, Toshimi SAWAYANAGI', Shigeyuki KAKIZAWAl, Hisashi NISHIGAWA2,Hee-Young JUNG2, Ken-ichiro FURUKI', Masaki YANAZAKV, Shiho SUZUKI', Wei WEl2, Tsutomu KUBOYAMA3, Masashi UGAKP2 and Shigetou NAMBA'72* zyxw ABSTRACT The phytoplasmas are a group of plant pathogenic bacteria that cause devastating damage to over zyx 700 plant species worldwide. They can propagate intracellularly in both insect and plant hosts. Despite their economic importance and unique biological features, phytoplasmas remain the most poorly characterized plant pathogens, primarily because efforts at zyxwvutsrq in vitro culture, gene delivery, and mutagenesis have been unsuccessful. To shed light on these microorgan- isms, we constructed a DNA library for the onion yellows (OY) phytoplasma, Candidatus Phytoplasma sp. 16s-group I, AY-subgroup (onion), and obtained a draft sequence of phage clones that cover the whole chromosome, which is estimated to be 1 Mbp at most, and completely sequenced and annotated a significant portion of the chromosome (760 kbp). We also cloned and sequenced all three of its extrachromosomal (EC) DNAs (7, 6 and 4 kbp). The EC DNAs are thought to replicate via a rolling-circle replication mechanism with their own unique replicases. The chromosome encodes genes for basic housekeeping functions, such as DNA replication, transcription and translation, but none for amino acid or fatty acid biosynthesis, the TCA cycle, or oxidative phosphorylation. These genes seem to have been replaced by homolog in the host nuclear genome, since phytoplasmas can import many biological substances from host cells, as is the case with parasitic mycoplasmas. Surprisingly, the phytoplasma genome encodes even fewer genes for metabolic functions than that of mycoplasmas, which are known to possess minimal gene sets; genes for the pentose phosphate cycle, conserved in the genomes of all other reported bacteria, were not found in the OY phytoplasma genome. Phytoplasmas appear to possess the most minimal set of metabolic pathways identified in an organism to date; this minimalism may be related to the fact that phytoplasmas inhabit the nutrient-rich environment of the phloem. More than half of the unknown proteins are predicted to localize in the cell membrane, suggesting that they participate in interactions between the intracellular phytoplasma and the host cell surface or cytoplasm. Although the OY phytoplas- ma does not possess the typical genes related to pathogenicity found in other phytopathogenic bacteria, some of these unknown genes may be related to pathogenicity via their unique metabolic profile, such as actively importing hos! metabolites and affecting normal cellular functiqns. This work is the first genome analysis of a plant pathogen that inhabits plant phloem cells. (Received June 3, 2002 ; Accepted June 27, 2002) Key words : phytoplasma, genome, sequence, metabolic pathway. Detailed information about the OY phytoplasma genome sequencing project and annotated data processed by COG program provided by NCBI is available on our Web site (http: //user.ecc.u-tokyo.ac.jp/-knamba/phytoplasma/). Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, 202 Bioscience Bldg., 5-1-5, Kashiwanoha, Kashiwa 277-8562, Japan Department of Agricultural and Environmental Biology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan Department of Agricultural Sciences, School of Agriculture, Ibaraki University, 3-21-1, Chuo, Ami-machi, Inashiki-gun, Ibaraki 300-0393, Japan Both authors contributed equally to this work. * Corresponding author (E-mail : snamba@ims.u-tokyo.ac.jp)