Romero Alba, Ruiz Beatriz, Sohng Jae Kyung, Koirala Niranjan, Rodríguez-Sanoja Romina, Sánchez Sergio
Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, 04510, México, D. F., Mexico.
Appl Biochem Biotechnol. 2015 Mar;175(6):3207-17. doi: 10.1007/s12010-015-1493-6. Epub 2015 Jan 27.
In Streptomyces, carbon utilization is of significant importance for the expression of genes involved in morphological differentiation and antibiotic production. Glucose is mainly transported by GlcP, a membrane protein encoded by glcp. In Streptomyces coelicolor, this protein is encoded by sco5578. However, there is little information about the physiology of the GlcP promoter in Streptomyces. The aim of the present work was to clone and perform a functional analysis of the sp7066 promoter (ortholog of sco5578) from Streptomyces peucetius var. caesius. Hydrophobicity and cellular location analysis of the putative amino acid sequence of the cloned gene predicted SP7066 would be a membrane protein with a topology of six plus six transmembrane segments interrupted by a large cytoplasmic loop. In silico analysis of the upstream region of the sp7066 transcription initiation site predicted the sequences 5'-AGGAATAGT-3' and 5'-TTGACT-3' for regions -10 and -35 of sp7066 promoter. To reflect sp7066 expression, the promoter sequence was amplified, subcloned, and fused to the egfp reporter gene. Immunoblot analysis revealed that D-glucose and its analog 2-deoxyglucose were able to induce sp7066 expression. This effect was not modified by the presence of equimolar concentrations of D-galactose or N-acetylglucosamine. No expression of egfp was detected with the use of other carbon sources such as L-arabinose, D-fructose, and glycerol. Based on these analyses, we conclude that D-glucose is a preferred carbon source in S. peucetius var. caesius and that the sp7066 expression product, a putative non-PTS glucose permease, likely is a H+/symporter, localized to the membrane, and shows a strong specificity for D-glucose for inducing expression.
在链霉菌中，碳源利用对于参与形态分化和抗生素生产的基因表达具有重要意义。葡萄糖主要通过GlcP进行转运，GlcP是由glcp编码的一种膜蛋白。在天蓝色链霉菌中，该蛋白由sco5578编码。然而，关于链霉菌中GlcP启动子的生理学信息很少。本研究的目的是克隆来自卡氏链霉菌（Streptomyces peucetius var. caesius）的sp7066启动子（sco5578的直系同源物）并进行功能分析。对克隆基因的推定氨基酸序列进行疏水性和细胞定位分析预测，SP7066将是一种膜蛋白，其拓扑结构为六个跨膜段加上六个跨膜段，中间被一个大的细胞质环中断。对sp7066转录起始位点上游区域的计算机分析预测，sp7066启动子的-10区和-35区的序列分别为5'-AGGAATAGT-3'和5'-TTGACT-3'。为了反映sp7066的表达情况，对启动子序列进行了扩增、亚克隆，并与egfp报告基因融合。免疫印迹分析表明，D-葡萄糖及其类似物2-脱氧葡萄糖能够诱导sp7066的表达。等摩尔浓度的D-半乳糖或N-乙酰葡糖胺的存在不会改变这种效应。使用其他碳源如L-阿拉伯糖、D-果糖和甘油时，未检测到egfp的表达。基于这些分析，我们得出结论：D-葡萄糖是卡氏链霉菌中首选的碳源，并且sp7066的表达产物，一种推定的非磷酸转移酶系统（PTS）葡萄糖通透酶，可能是一种H+/同向转运体，定位于膜上，并且对D-葡萄糖诱导表达具有很强的特异性。
