{"created":"2023-05-15T13:38:20.884746+00:00","id":2274,"links":{},"metadata":{"_buckets":{"deposit":"14f56230-cad0-4359-a3c6-6c0f2d1df592"},"_deposit":{"created_by":12,"id":"2274","owners":[12],"pid":{"revision_id":0,"type":"depid","value":"2274"},"status":"published"},"_oai":{"id":"oai:repository.naro.go.jp:00002274","sets":["87:633:634:133:241"]},"author_link":["1321"],"item_10002_biblio_info_7":{"attribute_name":"書誌情報","attribute_value_mlt":[{"bibliographicIssueDates":{"bibliographicIssueDate":"2009-03-01","bibliographicIssueDateType":"Issued"},"bibliographicPageEnd":"70","bibliographicPageStart":"23","bibliographicVolumeNumber":"9","bibliographic_titles":[{"bibliographic_title":"畜産草地研究所研究報告"},{"bibliographic_title":"Bulletin of National Institute of Livestock and Grassland Science","bibliographic_titleLang":"en"}]}]},"item_10002_description_5":{"attribute_name":"抄録","attribute_value_mlt":[{"subitem_description":"Chapter 1. Manipulation for selective Plasmid Elimination from Lactococcus lactis  Strains of lactococcal bacteria are used as starters in the fermentation of dairy products. Such strains generally carry a number of plasmids, varying in size from approximately 2 kb to 80 kb. Some plasmids encode properties essential to the manufacture of dairy products such as lactose fermentation, proteolysis, diacetyl production, and phage resistance, and others encode nonessential or unknown properties. Plasmid elimination is a fundamental technique for investigating the diverse properties of encoding plasmids. It is currently performed by culturing with a mutagenic chemical such as acridine orange, culturing in unbuffered medium, exposing cells to elevated growth temperatures, regenerating bacterial protoplasts, or a composite of these methods. With these methods, plasmids cannot be chosen for elimination, and the simultaneous loss of more than one plasmid is frequent. In addition, the resulting variants that have lost co-existing essential plasmids are ineffective as starters. 　This study was designed selectively to eliminate a θ-plasmid from Lactococcus lactis strains by transforming synthetic competitors. A shuttle vector for Escherichia coli and L. lactis, pDB1, was constructed by ligating a partial replicon of pDR1-1B, which is a 7.3 kb θ-plasmid in L. lactis DRC1, with an erythromycin resistance gene into pBluescript II KS^+. This versatile vector was used to construct competitors to common lactococcal θ-plasmids. pDB1 contains the 5' half of the replication origin and the 3' region of repB of pDR1-1B, but lacks the 1.1-kb region normally found between these two segments. A set of primers, Pv3 and Pv4, was designed to amplify the 1.1-kb middle parts of the general θ-replicons of lactococcal plasmids. When the PCR products were cloned into the Nru I and Xho I sites of pDB1, synthetic replicons were constructed and replication activity was restored. A number of θ-plasmids in L. lactis ssp. lactis and cremoris were eliminated selectively by transforming the synthetic competitors. These competitors were easily eliminated by subculture for a short time in the absence of selection. The resulting variants contained no exogenous DNA and are suitable for food products, since part of the phenotype was altered without altering other plasmids indispensable for fermentation.  Chapter 2. Breeding of new Lactococcus lactis starters by plasmid elimination  Lactococcus lactis subsp. lactis biovar diacetilactis DRC1 carries more than 6 plasmids, including a 7.4 kb cryptic plasmid, which was designated as pDR1-1. pDR1-1 was found to significantly affect the specific growth rate of the host cells because of its limiting effect on growth. When pDR1-1 was eliminated by an unstable competitor to pDR1-1, as described in chapter 1, the resulting variant, L. lactis DRC1ΔpDR1-1, grew more efficiently than the DRC1 wild type. In addition, Lactococcus lactis subsp. lactis biovar diacetilactis N7 carried an 8.3-kb plasmid, which was expected to be the citrate permease plasmid (CitP-plasmid). When the 8.3-kb plasmid was eliminated, the variant, L. lactis N7ΔpCit, lost the ability to metabolize citrate and to produce the aromatic compound diacetyl from citrate. Diacetyl produces a buttery flavor in fermented dairy products, but this aroma is undesirable for yoghurt. Therefore, selective elimination of CitP-plasmid may serve to breed a variant preferable for yoghurt starter. Neither L. lactis DRC1ΔpDR1-1 nor L. lactis N7ΔpCit contained exogenous DNA, making both suitable for food products.  Chapter 3. Characterization of a cryptic plasmid that contributes to the stable maintenance of host genome in Lactococcus lactis 　Lactococcus lactis subsp. cremoris NIAI712 carries five different plasmids, including an 8.7-kb plasmid designated pAG6. pAG6 encodes a subunit of a type-I restriction and modification system (HsdS), as well as proteins involved in cadmium resistance (CadA and CadC). When we eliminated pAG6 by inserting a competitor into strain NIAI712, the resulting ΔpAG6 variants showed a slow-milk–coagulation phenotype, even though the cells retained their lactose fermentation and proteolysis activities. Pulsed-field gel electrophoresis followed by Southern hybridization analysis showed that chromosomal rearrangements as well as co-elimination of the 50-kb plasmid pAG3, which carried an oligopeptide transport system gene cluster (opp-cluster), occurred consistently in the genome of ΔpAG6 variants. These results suggest that the stable maintenance of pAG6 prevents destabilization of a co-existing plasmid and constant genome rearrangement of chromosome. In ΔpAG6 variants, transposases of IS982 and ISS1 were expressed at lower levels than in the parent NIAI712 strain. The expression of these transposases increased in an intermediate variant containing both pAG6 and competitor. Therefore, the frequency of chromosomal rearrangements and loss of pAG3 in association with the IS982 and ISS1 elements may increase during the process of pAG6 elimination. 　Out of the entire sequence of pAG6, we have focused on the function of HsdS as a factor that serves in stable maintenance of the host genome. HsdS is part of multi-functional complexes, i.e. Type-I R/M systems composed of three different subunits, HsdS, HsdM, and HsdR. This complex is active in an N-6 adenine-specific DNA methylase, a DNA dependent ATPase, a DNA translocase, and a restriction endonuclease. Since HsdS is responsible for the recognition of a specific DNA sequence, the restriction and methylation sites in the genomes would be altered by the elimination of HsdS/pAG6. We therefore expected that the restriction complex with HsdS/pAG6 would cleave pAG3 and part of the host chromosome, or that the gene transpositions regulated by IS982 or ISS1 would be promoted by aberrant transcription of the tnp genes following the methylation changes near the promoter regions.","subitem_description_type":"Abstract"},{"subitem_description":"乳製品製造に汎用されているLactococcus lactis の遺伝子構成は, 2 Mb 程度の小型の染色体遺伝子と, 複数のプラスミド遺伝子を細胞内に保有することが特徴的である. L. lactis のプラスミドは, ごく一部の例外を除いてθ - 複製型プラスミドであり, 乳発酵に必須な形質をコードする場合が多く, ラクトース資化, プロティナーゼ活性, クエン酸取込み, ファージ耐性, バクテリオシン生産, 粘性物質生産などの形質に関与する. L. lactis の内在プラスミドの種類や組合せは菌株ごとに異なり, 菌株特異的な表現型を決定する. L. lactis の分離原は乳製品, 生乳, 漬物, 生草など多岐にわたり, プラスミド構成を変えながら生育環境に適応していると考えられる. 中には細胞内に10 種類程度のプラスミドを保有する株もあり, 機能が特定されていないプラスミドも多く残されている. プラスミドの機能解析は, 通常まずプラスミド除去株を作出し, 変異株の表現形質と親株の表現形質を比較して研究の端緒とする. 従って, 除去するプラスミドを任意にコントロールすることができれば, プラスミド上の遺伝子機能や関係する表現形質を効率良く推定することができる. また, 必要不可欠なプラスミドを損なわずに, 1 種類のプラスミドを除去する方法は, 発酵産業に利用可能な実用菌株の改良にも利用できる. 第1章では, 宿主DNA にランダムに作用する変異剤処理などを行わず, 複数の内在プラスミドのうち1種類のプラスミドを選択的に除去し, 親株と発酵特性の異なる新菌株を作出する方法, 即ち, 任意のθプラスミドの複製単位をin vitro で再構成し, 不和合性プラスミド（競合プラスミド）によるθ - プラスミド選択的除去法を開発した. 本法は, 1）複製単位の再構成に共通して用いることのできるプラスミドベクター（pDB1）の作成, 2）任意のL. lactis θプラスミドの不和合性配列を増幅しうるPCR プライマーペア（VF3 - VF4）の設計, 3）in vitro での不和合性プラスミドの再構成と, L. lactis wild type プラスミドの除去操作, からなる. 　この方法で作出した変異株は, 細胞内に外来遺伝子を保有せず, また発酵に不都合な遺伝変異も起こっていないと考えられるため, 食品加工用のスターターに利用できる. そこで第2章では本法を用いたL. lactis プラスミド変異株の育種例2例について記述した. 1 例目としては, 前段でL. lactis DRC1 に内在し, 宿主の増殖速度を抑制するプラスミドの発見と, その解析について述べ, 続いてプラスミドの選択的除去法を用い, 当該プラスミドを除去することで親株より増殖速度の早いプラスミド変異株を作出したことを上げた. また2 例目としては, L. l. lactis bioval.diacetylactis N7 からクエン酸透過性プラスミドを選択的に除去し, クエン酸の代謝産物であるジアセチルの生成能を失わせたフレーバー変異株育種の試みについて記述し, 作出したプラスミド変異株の乳発酵スターターとしての能力について考察した. 　第3章には, プラスミドの選択的除去で見出された新しいプラスミド性因子の解析例「宿主遺伝子の安定化に働くプラスミドの発見」についてまとめた. 　L. lactis subsp. cremoris NIAI712 は, 乳発酵スターター乳酸菌のプロトタイプとして, 世界中で広く研究に用いられているL. lactis NCDO712 の派生株である. L. lactis NIAI712 は, 5 種類のプラスミドを有し, そのうち約9 kb のプラスミドpAG6 はコピー数も多く, 非常に安定である. それゆえ従来のプラスミド除去法では欠失されず, その機能は調べられていなかった. そこで開発したプラスミドの選択的除去方法を試みたところ, 効率よくpAG6 除去株（712Δ pAG6）が得られた. 712 Δ pAG6 の乳発酵能を調べたところ, 乳中での増殖能および乳酸生成能が親株よりも著しく劣っていた. 712 Δ pAG6 と親株からゲノム遺伝子を抽出し, その制限分解パターンを比較したところ, pAG6 の除去に伴って, 短期間のうちに遺伝子組み換えによる変異が起こることが明らかとなった. さらにこのゲノム変異によって, カゼイン分解物の取り込みに働く一連の遺伝子群opp-pepO が, 例外なく消失していることを突き止め, 発酵遅延の主原因であると結論した. 　pAG6 には, 宿主DNA のメチル化配列を決定する因子がコードされていた. 遺伝子プロモーター近傍のDNA のメチル化状態が, 遺伝子の転写活性に影響することは周知の事実である. 特に遺伝子の転移を仲介するトランスポゾンの転移酵素遺伝子tnp の転写減衰はよく知られている. それゆえpAG6 の除去操作中, すなわち, pAG6 と競合プラスミドが同一細胞中に共存する状態でtnp の転写活性が上昇するのではないかと予想した. そこで, pAG6 と 競合プラスミドが共存する変異株を作成し, tnp 転写活性を解析した. その結果, 競合プラスミドの共存によってpAG6 の複製が不安定になっている最中には, ある種のtnp の発現量が特異的に上昇することを明らかにした. L. lactis のプラスミドが, 共存する他のプラスミドやクロモゾームなど宿主のゲノム構造の安定化に働く現象は, 本研究で明らかにされた新規な知見である. 宿主は細胞内でpAG6 を安定に保持することで, ゲノム遺伝子のメチル化状態を正常に保ち, ゲノム内トランスポゾンなど可動性遺伝因子の転移活性を小さくし, ゲノム構造や菌株特異的なプラスミド構成を維持するのかもしれない. L. lactis において, DNA メチル化による転写制御の研究はごく少ない. 本研究で作出した変異株が, メチル化と菌株特異的な遺伝子発現との関連を解析するモデル菌株になるのではないかと期待している. ","subitem_description_type":"Abstract"}]},"item_10002_identifier_registration":{"attribute_name":"ID登録","attribute_value_mlt":[{"subitem_identifier_reg_text":"10.24514/00002175","subitem_identifier_reg_type":"JaLC"}]},"item_10002_publisher_8":{"attribute_name":"出版者","attribute_value_mlt":[{"subitem_publisher":"独立行政法人 農業・食品産業技術総合研究機構"}]},"item_10002_relation_14":{"attribute_name":"DOI","attribute_value_mlt":[{"subitem_relation_type":"isIdenticalTo","subitem_relation_type_id":{"subitem_relation_type_id_text":"10.24514/00002175","subitem_relation_type_select":"DOI"}}]},"item_10002_source_id_9":{"attribute_name":"ISSN","attribute_value_mlt":[{"subitem_source_identifier":"1347-0825","subitem_source_identifier_type":"ISSN"}]},"item_10002_version_type_20":{"attribute_name":"著者版フラグ","attribute_value_mlt":[{"subitem_version_resource":"http://purl.org/coar/version/c_970fb48d4fbd8a85","subitem_version_type":"VoR"}]},"item_creator":{"attribute_name":"著者","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"小林, 美穂"},{"creatorName":"コバヤシ, ミホ","creatorNameLang":"ja-Kana"},{"creatorName":"KOBAYASHI, Miho","creatorNameLang":"en"}],"nameIdentifiers":[{},{},{}]}]},"item_files":{"attribute_name":"ファイル情報","attribute_type":"file","attribute_value_mlt":[{"accessrole":"open_date","date":[{"dateType":"Available","dateValue":"2019-03-20"}],"displaytype":"detail","filename":"ilgs_report_No9p23-70p.pdf","filesize":[{"value":"6.1 MB"}],"format":"application/pdf","licensetype":"license_note","mimetype":"application/pdf","url":{"label":"ilgs_report_No9p23-70p.pdf","url":"https://repository.naro.go.jp/record/2274/files/ilgs_report_No9p23-70p.pdf"},"version_id":"dba9807a-f6a5-4f9c-9fe7-24c12719d45e"}]},"item_keyword":{"attribute_name":"キーワード","attribute_value_mlt":[{"subitem_subject":"乳酸菌","subitem_subject_scheme":"Other"},{"subitem_subject":"プラスミド","subitem_subject_scheme":"Other"},{"subitem_subject":"生育速度","subitem_subject_scheme":"Other"},{"subitem_subject":"乳発酵","subitem_subject_scheme":"Other"},{"subitem_subject":"Lactic acid bacteria","subitem_subject_language":"en","subitem_subject_scheme":"Other"},{"subitem_subject":"plasmid","subitem_subject_language":"en","subitem_subject_scheme":"Other"},{"subitem_subject":"growth rate","subitem_subject_language":"en","subitem_subject_scheme":"Other"},{"subitem_subject":"milk fermentation","subitem_subject_language":"en","subitem_subject_scheme":"Other"}]},"item_language":{"attribute_name":"言語","attribute_value_mlt":[{"subitem_language":"jpn"}]},"item_resource_type":{"attribute_name":"資源タイプ","attribute_value_mlt":[{"resourcetype":"departmental bulletin paper","resourceuri":"http://purl.org/coar/resource_type/c_6501"}]},"item_title":"乳業用乳酸菌Lactococcus lactis のプラスミド育種改良法の開発と乳発酵特性変異の解明に関する研究","item_titles":{"attribute_name":"タイトル","attribute_value_mlt":[{"subitem_title":"乳業用乳酸菌Lactococcus lactis のプラスミド育種改良法の開発と乳発酵特性変異の解明に関する研究"},{"subitem_title":"New methods for selective plasmid elimination from Lactococcus lactis and characterization of the genetic variability of variants derived from Lactococcal starter for milk fermentation","subitem_title_language":"en"}]},"item_type_id":"10002","owner":"12","path":["241"],"pubdate":{"attribute_name":"公開日","attribute_value":"2019-03-22"},"publish_date":"2019-03-22","publish_status":"0","recid":"2274","relation_version_is_last":true,"title":["乳業用乳酸菌Lactococcus lactis のプラスミド育種改良法の開発と乳発酵特性変異の解明に関する研究"],"weko_creator_id":"12","weko_shared_id":12},"updated":"2023-05-15T16:10:33.439966+00:00"}