Abratt V R, Mbewe M, Woods D R
Department of Microbiology, University of Cape Town, Rondebosch, South Africa.
Mol Gen Genet. 1998 May;258(4):363-72. doi: 10.1007/s004380050742.
Investigations of possible regulators of Bacteroides fragilis glutamine synthetase (GS) activity were done in Escherichia coli using a compatible dual-plasmid system. The B. fragilis glnA gene, together with upstream and downstream flanking regions, was cloned onto the low copy number plasmid pACYC184 and expressed in the E. coli glnA ntrB ntrC deletion strain, YMC11. GS activity was monitored following co-transformation with a B. fragilis genomic library carried on the compatible plasmid pEcoR251. A gene was cloned that caused a twofold increase in B. fragilis GS activity but did not affect the activity of the E. coli GS enzyme or the B. fragilis sucrase (ScrL). Deletion of the B. fragilis glnA downstream region decreased basal levels of GS activity, but did not affect the ability of the cloned gene to increase the B. fragilis GS activity. Reporter gene analysis, using the B. fragilis glnA promoter region fused to the promoterless Clostridium acetobutylicum endoglucanase gene, showed no increase in reporter gene activity. This demonstrated that the increase in GS activity was not regulated at the transcriptional level, and that the cloned gene product was not affecting the copy number of the plasmid in trans. Sequence data indicated that the cloned gene had good amino acid identity to a range of elongation factor P (EF-P) proteins, the highest being to that of a Synechocystis sp (48%), and the least to Mycobacterium genitalium (27%). Amino acid identity to the E. coli EF-P was intermediate (37%). A possible role for EF-P in enhancing translation of the B. fragilis glnA mRNA is proposed.
利用兼容双质粒系统在大肠杆菌中对脆弱拟杆菌谷氨酰胺合成酶（GS）活性的可能调节因子进行了研究。将脆弱拟杆菌glnA基因及其上下游侧翼区域克隆到低拷贝数质粒pACYC184上，并在大肠杆菌glnA ntrB ntrC缺失菌株YMC11中表达。与携带在兼容质粒pEcoR251上的脆弱拟杆菌基因组文库共转化后，监测GS活性。克隆到一个基因，它使脆弱拟杆菌GS活性提高了两倍，但不影响大肠杆菌GS酶或脆弱拟杆菌蔗糖酶（ScrL）的活性。缺失脆弱拟杆菌glnA下游区域会降低GS活性的基础水平，但不影响克隆基因提高脆弱拟杆菌GS活性的能力。使用与无启动子的丙酮丁醇梭菌内切葡聚糖酶基因融合的脆弱拟杆菌glnA启动子区域进行报告基因分析，结果显示报告基因活性没有增加。这表明GS活性的增加不是在转录水平上调节的，并且克隆的基因产物不会反式影响质粒的拷贝数。序列数据表明，克隆的基因与一系列延伸因子P（EF-P）蛋白具有良好的氨基酸同一性，与集胞藻属的同一性最高（48%），与生殖支原体的同一性最低（27%）。与大肠杆菌EF-P的氨基酸同一性处于中间水平（37%）。提出了EF-P在增强脆弱拟杆菌glnA mRNA翻译中的可能作用。
