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Enterobacter属細菌による軟腐病制御のためのファージ単離と特性解析

グエン, コン, タイン NGUYEN, CONG, THANH 九州大学

2020.09.25

概要

軟腐病(soft rot)は野菜、果物、草花などの葉、茎、根、球根などに発生し、農業生産に深刻な被害を及ぼす世界共通の疾病である。本病の原因菌はsoft rot Pectobacteriaceae(SRP)と称され、Pectobacterium属とDickeya属細菌に大別されていた。近年、蒟蒻、唐辛子、ドラゴンフルーツの軟腐病からEnterobacter属細菌が単離された。Enterobacter属細菌はグラム陰性の通性嫌気性桿菌で、Enterobacter–Escherichiaクレードに属し、SRPとされるPectobacterium–Dickeyaクレードとは異なるEnterobacteriales目のグループに属するため、、同属菌がSRPであるか否かの議論が生じている。

 一方、軟腐病をはじめとする植物病原菌制御のため、東南アジア諸国では農薬や化学薬品の多施用が問題となっている。これを解決する一方策として細菌ウイルスであるバクテリオファージ(ファージ)を用いた生物防除(ファージセラピー)が提唱されている。ファージは細菌の寄生因子で、特異的な細菌を宿主として感染し、最終的には宿主を溶菌して娘ファージ粒子を放出する。このため、ファージセラピーは特異的な宿主菌のみを破壊し、環境負荷を最小化できる方策と考えられる。そこで本論文では、ベトナムの軟腐病発生地域より単離した細菌の分子系統解析を行い、その多様性を明らかにし、当該菌に感染するファージを単離してその性状、ゲノム・タンパク質構造を明らかにすることで、軟腐病制御を目的としたファージセラピーの基盤を構築する事を目的とした。まず、ベトナム各地の軟腐病発生域の野菜、果物より7株の細菌を単離した。単離株については、16SrDNA塩基配列から、Acinetobacter baumannii、Pantoea dispersa、およびEnterobacter属に分類した。しかし、Enterobacter属に分類した5株については、生理・生化学試験や分子系統解析によってもEnterobacter属基準株と明確な一致が認められなかった。そこで、EnterobacterM4-VN株についてMiseqを用いた全ゲノム解読を行なった。その結果、本株ゲノム(accession No. BLVN01000000)は4,754,309bp(G+C含量55.1%)から成り、65個のtRNA、7個のrRNA、1個のCRISPRの他、4,424個のORFが推定された。これらのORFのうち、病原性に関与する遺伝子としてTonBタンパク質、M16プロテアーゼをコードする遺伝子が検索できた。一方、M4-VN株ゲノムとEnterobacter属基準株とのin silico DNA-DNAハイブリダイゼーション値は42.5%であったことから、本株はEnterobacter属の新種であると推測した。

 次に、分離株および既存の植物病原菌に感染するファージの取得を試み、ベトナムバクニン省の土壌からプラークアッセイでファージEspM4VNを単離した。本ファージは、正二十面体(直径100nm)の頭部、収縮性の尾部(長さ100nm、幅18nm)から成り、尾部には特徴的なアンブレラ構造を有していた。これらの形態的特徴は、Ackermannviridae科のShigellaファージAg3やDickeyaファージJA15、XF4と類似していた。また、本ファージは分離株、既存株20株のうち、M4-VN株のみにクリアープラークを形成する狭い宿主域を示す溶菌性ファージであった。さらに、EspM4VNは10〜50℃、pH4~10で安定で、1.5%(v/v)までのエタノール、Tween20にも耐性を示した。一段増殖曲線から、EspM4VNの潜伏期は20分、上昇期は10分、プラトー期40分で平均放出数は122であった。

 本ファージEspM4VNのゲノムは160,766bpで219個のORFが推定され、これらの遺伝子配座はAg3ファージの他、SalmonellaファージSKML-39、DickeyaファージCoodle、PP35、JA15、Limestoneと類似していた。上記の結果に加えて、ESI-MS/MS分析により本ファージ構造タンパク質の解析を行い、4種の特異的tail spikeタンパク質を同定し、また、構造タンパク質やDNA polymerase、DNA ligaseなどの分子系統解析を行った。これらの結果から、新規のEnterobacter属M4-VN株に感染するファージEspM4VNをAckermannviridae科、Aglimvirinae亜科、Agtrevirus属に分類した。

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