Targeted Mutagenesis of ORF326, frxC and ORF469 of a Cyanobacterium, Synechocysitis PCC6803, Homologous to Liverwort Chloroplast Genes

ORF326, frxC and ORF469 of a transfomable cynobacterium, Synechcystis PCC6803, have sequence similarity with ORF465 on the choroplast genome of a livewort, Marchantia polymorpha, respectively. To elucidate their functions,targeted mutagenesis was performed by transformation with clened DNA in which the ORF was disrupted by insertion of a kanamycin resistancen gene cassette.Streak-purifications of a single colony of each transformant were repeatde to segregate homozygous mutants for disrupted copies, because Synechocystis PCC6803 was reported to have approximately 10 chromosomal DNA copies. Southern blot analysis revealed that mutants for ORF326 had not only disrupted ORF326 copies but also wild type ORF326 copies. This suggests that ORF326 is indispensable for growth under the mixotrophic growth condition used. However, mutants for frxC and mutants for ORF469 had only mutated copies, indicating that they dispensable for growth. Growth and chlorophyll a content of an ORF469-disrupted mutant were compared and chlorophyll a content of an ORF469-disrupted mutant were compared to those of wild type under mixotrophic growth condition, but no significant difference was detected. This indicates that ORF469 is required for neither normal growth nor chlorophyll biosynthesis under thie condition.

ゼニゴケ葉緑体ORF316、frxCおよびORFと相同性を持つ、形質転換型ラン藻Synechocystis PCC6803株のORF326、frxCおよびORF469の機能についての情報を得るため、これらの欠損株の作製を行った。コード領域にカナマイシン耐性遺伝子カセットを挿入することにより不活性化された変異型のORF（オーブンリーディングフレーム）を持つプラスミドを用いてSynechocystis PCC6803株の形質転換を行い、カナマイシンを含む培地で選抜した。Synechocystis PCC6803株は約10コピーのクロモソームを持つが、サザンブロット解析の結果、ORF326については、変異型と野生型のORF326の双方を持つ株しか得られず、増殖に必要と推測された。一方frxCおよびORF469については、ともに全て変異型に置き換わった株が得られ、増殖には必要ないことが示された。さらにORF469欠損株を光照射下で培養し、増殖速度とクロロフィルa濃度を測定したが、いずれも野生株とほぼ同じであり、ORF469は、この条件下では、増殖やクロロフィル生合成に必要ないと推察された。