Alanine Racemase Mutants of Burkholderia pseudomallei and Burkholderia mallei and Use of Alanine Racemase as a Non-Antibiotic-Based Selectable Marker

Burkholderia pseudomallei and Burkholderia mallei are category B select agents and must be studied under BSL3 containment in the United States. They are typically resistant to multiple antibiotics, and the antibiotics used to treat B. pseudomallei or B. mallei infections may not be used as selective agents with the corresponding Burkholderia species. Here, we investigated alanine racemase deficient mutants of B. pseudomallei and B. mallei for development of non-antibiotic-based genetic selection methods and for attenuation of virulence. The genome of B. pseudomallei K96243 has two annotated alanine racemase genes (bpsl2179 and bpss0711), and B. mallei ATCC 23344 has one (bma1575). Each of these genes encodes a functional enzyme that can complement the alanine racemase deficiency of Escherichia coli strain ALA1. Herein, we show that B. pseudomallei with in-frame deletions in both bpsl2179 and bpss0711, or B. mallei with an in-frame deletion in bma1575, requires exogenous d-alanine for growth. Introduction of bpsl2179 on a multicopy plasmid into alanine racemase deficient variants of either Burkholderia species eliminated the requirement for d-alanine. During log phase growth without d-alanine, the viable counts of alanine racemase deficient mutants of B. pseudomallei and B. mallei decreased within 2 hours by about 1000-fold and 10-fold, respectively, and no viable bacteria were present at 24 hours. We constructed several genetic tools with bpsl2179 as a selectable genetic marker, and we used them without any antibiotic selection to construct an in-frame ΔflgK mutant in the alanine racemase deficient variant of B. pseudomallei K96243. In murine peritoneal macrophages, wild type B. mallei ATCC 23344 was killed much more rapidly than wild type B. pseudomallei K96243. In addition, the alanine racemase deficient mutant of B. pseudomallei K96243 exhibited attenuation versus its isogenic parental strain with respect to growth and survival in murine peritoneal macrophages

Development and Practice of Introductory Teaching Materials for Science "Problem- Based Learning" on the Theme of "Removal of Roadside Utility Poles"

P(論文)平成29年～ 30年に改訂された学習指導要領においては，持続可能な開発のための教育（ESD）や主権者教育などが取り上げられている．本稿では，新・逆向き設計に基づき，SDGsに関連した政策の評価を「総合的な学習の時間」で生徒が取り組めるようにするため，各教科で行う探究活動の準備段階で用いるゲーミング教材について，特に理科の物理と生物の知識を活用する教材を開発した．教材は無電柱化の政策に関する評価を題材とし，学習した知識と関連付ける形で優先的に無電柱化に取り組む地域の優先度を検証する活動を通じて，探究活動の流れと探究における知識活用の方法論を学び今後取り組む探究活動や市民生活における理科の知識の活用の見通しを得ることを教材の目標とした．実践の結果，生徒は理科の知識の重要性を認識した一方，教材に関しては化学の知識活用の追加，時間配分の改善，定量的評価を用いた探究の導入の3 点の改善点があった．departmental bulletin pape