Duang-Nkern Jintana, Nontaleerak Benya, Thongphet Apasiri, Asano Krisana, Chujan Suthipong, Satayavivad Jutamaad, Sukchawalit Rojana, Mongkolsuk Skorn
Laboratory of Biotechnology, Chulabhorn Research Institute, Bangkok 10210, Thailand.
Program in Applied Biological Sciences, Chulabhorn Graduate Institute, Bangkok 10210, Thailand.
Gene. 2024 Nov 15;927:148754. doi: 10.1016/j.gene.2024.148754. Epub 2024 Jul 5.
Pseudomonas aeruginosa PA2196 is a TetR family transcriptional repressor. In this study, the deletion of the PA2196 gene caused increased expression of the downstream gene curA (PA2197), which encodes for a NADPH-dependent curcumin/dihydrocurcumin reductase. The PA2196 gene was then identified as curR, and a DNA footprinting assay showed that CurR directly bound to the curA promoter at an imperfect 15-bp inverted repeat, 5'-TAGTTGA-C-TGGTCTA-3'. A curA promoter-lacZ fusion assay and site-directed mutagenesis further demonstrated that the identified CurR binding site plays a crucial role in curA repression by CurR. curA transcription was inducible by sodium hypochlorite (NaOCl) and N-ethylmaleimide (NEM) but not by hydrogen peroxide, organic hydroperoxide, or curcumin. The oxidation and alkylation of CurR by NaOCl and NEM, respectively, resulted in the inactivation of its DNA-binding activity, which induced curA expression. Under the tested conditions, the deletion of either curR or curA did not affect the survival of P. aeruginosa under NaOCl stress in the absence or presence of curcumin.
铜绿假单胞菌PA2196是一种TetR家族转录阻遏物。在本研究中，PA2196基因的缺失导致下游基因curA（PA2197）的表达增加，curA编码一种依赖NADPH的姜黄素/二氢姜黄素还原酶。随后PA2196基因被鉴定为curR，DNA足迹分析表明CurR直接结合到curA启动子上一个不完美的15bp反向重复序列5'-TAGTTGA-C-TGGTCTA-3'处。curA启动子与lacZ的融合分析以及定点诱变进一步证明，所鉴定的CurR结合位点在CurR对curA的阻遏中起关键作用。curA转录可被次氯酸钠（NaOCl）和N-乙基马来酰亚胺（NEM）诱导，但不能被过氧化氢、有机氢过氧化物或姜黄素诱导。NaOCl和NEM分别对CurR进行氧化和烷基化，导致其DNA结合活性失活，从而诱导curA表达。在测试条件下，缺失curR或curA在有无姜黄素的情况下均不影响铜绿假单胞菌在NaOCl胁迫下的存活。
