Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada.
FEMS Microbiol Lett. 2010 Oct;311(1):27-35. doi: 10.1111/j.1574-6968.2010.02076.x. Epub 2010 Aug 16.
The complete genome sequence of the bovine pathogen Mannheimia haemolytica A1 was analyzed by blast searches for the presence of two-component regulatory system proteins. Five complete sets of putative two-component systems were identified, and the NarQ/P system was further investigated. in silico analysis of the NarQ and NarP proteins showed features that are typical of the sensor and response regulator proteins. A narP knock-out mutant was constructed. The narP mutant has lost its ability to respond to NaNO(3) in the media and fail to alter the expression of several proteins. One of the proteins that showed increased production in the parent strain in response to NaNO(3) was analyzed by matrix-assisted laser desorption ionization time-of-flight MS. Unexpectedly, the protein was identified to be FbpA, a periplasmic component of the iron transporter system. Sequence analysis of the promoter region of fbpA identified motifs typical for NarP-regulated genes. The expression of the leukotoxin gene was also altered in the narP mutant as shown by Western immunoblot analysis and reverse transcription-PCR.
通过对牛支原体 A1 的全基因组序列进行生物信息学 blast 搜索，分析了其两套蛋白的存在情况。鉴定出了 5 套完整的假定双组分调控系统，进一步对 NarQ/P 系统进行了研究。对 NarQ 和 NarP 蛋白的计算机分析显示，其具有典型的传感器和反应调节蛋白的特征。构建了 narP 敲除突变株。该突变株丧失了对培养基中 NaNO3 的响应能力，并且不能改变几种蛋白质的表达。通过基质辅助激光解吸电离飞行时间质谱分析，对亲本菌株中响应 NaNO3 表达增加的一种蛋白质进行了分析。出乎意料的是，该蛋白质被鉴定为 FbpA，即铁转运系统的周质成分。FbpA 基因启动子区域的序列分析鉴定出了 NarP 调控基因的典型基序。Western 免疫印迹分析和反转录-PCR 显示，白细胞毒素基因的表达在 narP 突变株中也发生了改变。
