C. difficle p-cresol production

To identify the cellular localisation of the HpdBCA decarboxylase complex, a plasmid based HpdB-SNAP-tag translational fusion was constructed using a C. difficile compatible plasmid (pMTL84151) under control of the hpdBCA promoter. The hpdB coding sequence omitting the stop codon, was fused via a linker to a SNAP-tag (Table 1). Confirmation of HpdB linked to the SNAP-tag was carried out via western blot, and mass spectrometry, in which we identified six peptides, four with 100% identity and two with 99% identity unique to HpdB. Localisation of HpdB was visualised by confocal microscopy in 630Δerm and the p-cresol deficient mutant (hpdC::CT) (carrying the HpdB-SNAP-tag fusion (PhpdB-CDS-SNAP), in the presence and absence of p-HPA) to induce HpdBCA production.为确定HpdBCA脱羧酶复合物的细胞定位，本研究采用适配艰难梭菌（C. difficile）的质粒pMTL84151，在hpdBCA启动子的调控下，构建了基于质粒的HpdB-SNAP标签翻译融合表达载体。将去除终止密码子的hpdB编码序列通过肽接头与SNAP标签（SNAP-tag）相融合（详见表1）。通过蛋白质印迹（western blot）与质谱分析（mass spectrometry）对HpdB与SNAP标签的融合效果进行验证，共鉴定出6条HpdB特异性肽段，其中4条与HpdB序列完全匹配，2条与HpdB的匹配度达99%。随后，在添加或不添加对羟基苯乙酸（p-HPA，用以诱导HpdBCA复合物表达）的条件下，本研究借助共聚焦显微镜（confocal microscopy），对携带HpdB-SNAP标签融合质粒（PhpdB-CDS-SNAP）的630Δerm菌株及对甲酚缺陷突变株（hpdC::CT）中HpdB的亚细胞定位进行了可视化观测。