Reid Sharon J, Rafudeen M Suhail, Leat Neil G
Microbiology (Reading). 1999 Jun;145 ( Pt 6):1461-1472. doi: 10.1099/13500872-145-6-1461.
The sucrose operon of Clostridium beijerinckii NCIMB 8052 comprises four genes, which encode a sucrose-specific enzyme IIBC(Scr) protein of the phosphotransferase system (ScrA), a transcriptional repressor (ScrR), a sucrose hydrolase (ScrB) and an ATP-dependent fructokinase (ScrK). The scrARBK operon was cloned in Escherichia coli in three stages. Initial isolation was achieved by screening a C. beijerinckii genomic library in E. coli for clones able to utilize sucrose, while the remainder of the operon was isolated by inverse PCR and by plasmid rescue of flanking regions from a scrB mutant constructed by targeted gene disruption. Substrate specificity assays confirmed that the sucrose hydrolase was a beta-fructofuranosidase, able to hydrolyse sucrose and raffinose but not inulin or levans, and that the scrK gene encoded an ATP/Mg2+-dependent fructokinase. Both enzyme activities were induced by sucrose in C. beijerinckii. Disruption of the scr operon of C. beijerinckii by targeted plasmid integration into either the scrR or the scrB gene resulted in strains unable to utilize sucrose, indicating that this was the only inducible sucrose catabolic pathway in this organism. RNA analysis confirmed that the genes of the scr operon were co-transcribed on a 5 kb mRNA transcript and that transcription was induced by sucrose, but not by glucose, fructose, maltose or xylose. Primer extension experiments identified the transcriptional start site as lying 44 bp upstream of the scrA ATG start codon, immediately adjacent to the imperfect pelindrome sequence proposed to be a repressor binding site. Disruption of the scrR gene resulted in constitutive transcription of the upstream scrA gene, suggesting that ScrR encodes a transcriptional repressor which acts at the scrA operator sequence. The scrR gene is therefore itself negatively autoregulated as part of the polycistronic scrARBK mRNA
拜氏梭菌（Clostridium beijerinckii）NCIMB 8052的蔗糖操纵子由四个基因组成，它们编码磷酸转移酶系统的蔗糖特异性酶IIBC（Scr）蛋白（ScrA）、转录阻遏物（ScrR）、蔗糖水解酶（ScrB）和ATP依赖性果糖激酶（ScrK）。scrARBK操纵子分三个阶段克隆到大肠杆菌中。最初的分离是通过在大肠杆菌中筛选拜氏梭菌基因组文库，寻找能够利用蔗糖的克隆来实现的，而操纵子的其余部分则通过反向PCR以及从通过靶向基因破坏构建的scrB突变体的侧翼区域进行质粒拯救来分离。底物特异性分析证实，蔗糖水解酶是一种β-呋喃果糖苷酶，能够水解蔗糖和棉子糖，但不能水解菊粉或果聚糖，并且scrK基因编码一种ATP/Mg2+依赖性果糖激酶。在拜氏梭菌中，这两种酶活性均由蔗糖诱导。通过将靶向质粒整合到scrR或scrB基因中破坏拜氏梭菌的scr操纵子，导致菌株无法利用蔗糖，这表明这是该生物体中唯一可诱导的蔗糖分解代谢途径。RNA分析证实，scr操纵子的基因在一个5 kb的mRNA转录本上共转录，并且转录由蔗糖诱导，但不由葡萄糖、果糖、麦芽糖或木糖诱导。引物延伸实验确定转录起始位点位于scrA ATG起始密码子上游44 bp处，紧邻被认为是阻遏物结合位点的不完全回文序列。scrR基因的破坏导致上游scrA基因的组成型转录，这表明ScrR编码一种转录阻遏物，其作用于scrA操纵序列。因此，scrR基因本身作为多顺反子scrARBK mRNA的一部分受到负向自我调节。
