腸道出血性大腸桿菌（EHEC）感染會引起嚴重的症狀，也會發生危及生命的病例，尤其是在幼兒和老年人中。EHEC 的主要毒力因子是志賀毒素，該基因編碼在噬菌體基因組中，在 EHEC 染色體中溶原化。正常情況下，CI阻遏物嚴格抑制噬菌體和stx2AB基因編碼的志賀毒素產生。然而，噬菌體的產生是在某些壓力下誘導的，例如 DNA 損傷引發的 SOS 反應。由於stx2AB基因編碼在噬菌體基因組中，因此在產生噬菌體時也會產生毒素。最終，當噬菌體裂解細胞時，產生的志賀毒素從細菌細胞中釋放出來。眾所周知，RecBCD 路徑誘導 SOS 反應和噬菌體產生。然而，在 EHEC 中沒有很好地研究誘導毒素產生的其他機制。因此，我們使用雙報告菌株對參與 DNA 修復、應激反應、全體調節和群體感應的基因進行了篩選。為了構建篩選菌株（MG1655 933W Δstx2AB::gfp/ pSulA-mCherry），將來自EHEC EDL933的編碼毒素基因的噬菌體 (933W) 溶原到大腸桿菌K-12 MG1655染色體中，並將毒素基因 (stx2AB) 替換為gfp基因。在 SOS 報導基因 (pSulA-mCherry) 的質體中，mCherry 基因與受SOS反應調節的sulA 啟動子融合。用於篩選的具有突變的菌株在有或沒有誘導SOS反應的MMC的情況下培養，並測定OD600nm、mCherry和GFP表現量。OD600 nm用於觀測細菌生長和細胞裂解。mCherry 和 GFP 分別用於 SOS 反應和志賀毒素產生的報導。從篩選中，我們發現 recF、recO、recR 和 recJ 的突變體與野生型相比不裂解細胞並且顯示出較低的 GFP 表達，儘管突變體中的 SOS 反應與野生型沒有顯著差異。為了研究噬菌體的產生，用 MG1655 933W 中的 recF、recO、recR 和 recJ 單突變株進行噬菌斑形成單位 (pfu) 測定，與野生型相比，突變體顯示出較低的 pfu。還進行了西方墨點法以確定毒素的產生，結果表明，與野生型相比，recF、recO、recR 和 recJ 單突變體減少了志賀毒素的表達。結果與篩選中 recF、recO、recR 和 recJ 突變體中的低 GFP 表達一致。總之，這些結果表明 recF、recO、recR 和recJ 涉及了大腸桿菌中的噬菌體和志賀毒素的產生。藉由qPCR確定涉及噬菌體lytic cycle級聯中基因的mRNA表現量。結果表明，ΔrecF顯示出較低的晚期基因mRNA表現量，這意味著recF涉及了933W噬菌體產生的晚期基因表達。在這裡，我們證明 RecFORJ通過 N 抗終止作用涉及大腸桿菌中志賀毒素的產生，以激活933W噬菌體的晚期基因表達。

Infection of enterohemorrhagic Escherichia coli (EHEC) causes severe symptom, and life-threatening case also happens, especially in young children and old people. The major virulence factor in EHEC is Shiga toxin, and the gene is encoded in a bacteriophage genome, which is lysogenized in the EHEC chromosome. Under normal condition, CI repressor tightly represses production of the phage and the Shiga toxin encoded by stx2AB gene. However, the phage production is induced under some stress such as DNA damage via SOS response. Since stx2AB gene is encoded in the phage genome, the toxin is also produced when the phage is produced. Eventually, the produced Shiga toxin is released out from the bacterial cells, when the phage lyses the cells. It's well known that RecBCD pathway induces SOS response and the phage production. However, the other mechanism to induce the toxin production is not investigated well in EHEC. So, we performed a screening for genes involved in DNA repair, stress response, global regulator and quorum sensing using a double reporter strain. To construct the screening strain (MG1655 933W Δstx2AB::gfp/ pSulA-mCherry), the phage (933W) encoding toxin gene from EHEC EDL933 was lysogenized into E. coli K-12 MG1655 chromosome and the toxin gene (stx2AB) was replaced with gfp gene. In a plasmid for SOS reporter (pSulA-mCherry), mCherry gene is fused with sulA promoter, which is regulated by SOS response. The screening strain with a mutation for the screening was cultured with or without MMC which induces SOS response, and OD600nm, mCherry and GFP levels were determined. OD600nm is to see bacterial growth and cell lysis. mCherry and GFP are reporter for SOS response and Shiga toxin production, respectively. From the screening, we found that mutants for recF, recO, recR and recJ did not lyse the cells and showed lower GFP expression compared to the wild type, although SOS response in the mutants were not significantly different from wild type. To investigate the phage production, plaque forming unit (pfu) assay was performed with recF, recO, recR and recJ single mutation in MG1655 933W and the mutants showed significantly lower pfu compared to the wild type. Western blotting was also performed to determine the toxin production, and the result showed that recF, recO, recR and recJ single mutants decreased the expression of Shiga toxin compared to wild type. The result is consistent with low GFP expression in recF, recO, recR and recJ mutants in the screening. Taken together, these results suggest that recF, recO, recR, and recJ are involved in phage and Shiga toxin production in E. coli. The mRNA expression of genes involved in the phage lytic cycle cascade was determined by qPCR. The results showed that, ΔrecF showed a lower expression level of late genes for phage production, which means that recF is involved in the late gene expression of 933W phage production. Here, we demonstrate that RecFORJ are involved in Shiga toxin production in E. coli through N antitermination to activate late gene expression in 933W phage.

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