L-Glutamine metabolism and its regulation in C. glutamicum. Corynebacterium glutamicum ATCC 13032

Corynebacterium glutamicum, a gram-positive soil bacterium used for the industrial production of amino acids such as L-glutamate and L-lysine, is able to use a number of different nitrogen sources, such as ammonium, urea, or creatinine. In this communication, we show that L-glutamine serves as an excellent nitrogen source for C. glutamicum and allows similar growth rates in glucose minimal medium as ammonium. A transcriptome comparison revealed a strong induction of the nitrogen starvation response when glutamine was used as nitrogen source. Subsequent growth experiments with a variety of mutants defective in nitrogen metabolism showed that glutamate synthase is crucial for glutamine utilization, while a putative glutaminase is dispensable under the experimental conditions used. The fact that the glutamate synthase encoding gltBD operon is under strict nitrogen control explains the necessity for induction of the nitrogen starvation response. The paradox situation that the nitrogen starvation response is induced although intracellular L-glutamine levels are high has implications on nitrogen sensing. In contrast to other gram-positive and gram-negative bacteria such as Bacillus subtilis, Salmonella typhimurium, and Klebsiella pneumoniae, a drop in glutamine concentration obviously does not serve as a nitrogen starvation signal in C. glutamicum. Overall design: Three biological replicates were performed. To analyse how L-glutamine influences global gene expression when used as sole nitrogen source instead of ammonium, DNA microarray analyses were performed. For this purpose RNA was isolated from exponentially growing cells cultivated in CgXII medium containing glucose as carbon source and either L-glutamine or ammonium sulphate as nitrogen source.

谷氨酸棒杆菌（Corynebacterium glutamicum）是一种革兰氏阳性土壤细菌，广泛用于L-谷氨酸（L-glutamate）、L-赖氨酸（L-lysine）等氨基酸的工业化生产。该菌可利用铵、尿素、肌酐等多种不同氮源。

本研究证实，L-谷氨酰胺（L-glutamine）可作为谷氨酸棒杆菌的优质氮源，在葡萄糖基本培养基中，其支持的生长速率与铵作为氮源时相当。转录组（transcriptome）比较分析显示，当以谷氨酰胺为唯一氮源时，谷氨酸棒杆菌会强烈诱导氮饥饿响应（nitrogen starvation response）。后续针对一系列氮代谢缺陷突变体的生长实验表明，谷氨酸合酶（glutamate synthase）是谷氨酰胺利用过程中的关键酶，而推定谷氨酰胺酶（putative glutaminase）在所采用的实验条件下并非必需。编码谷氨酸合酶的gltBD操纵子（gltBD operon）受严格的氮调控，这一机制解释了为何即便胞内L-谷氨酰胺水平较高，仍需诱导氮饥饿响应。尽管胞内L-谷氨酰胺浓度未出现下降，却仍触发了氮饥饿响应，这一矛盾现象对氮感知（nitrogen sensing）机制的研究具有重要意义。与枯草芽孢杆菌（Bacillus subtilis）、鼠伤寒沙门氏菌（Salmonella typhimurium）、肺炎克雷伯菌（Klebsiella pneumoniae）等其他革兰氏阳性、革兰氏阴性细菌不同，谷氨酰胺浓度下降显然并非谷氨酸棒杆菌的氮饥饿信号。

总体实验设计：本研究设置3个生物学重复。为探究以L-谷氨酰胺替代硫酸铵作为唯一氮源时，其对谷氨酸棒杆菌全球基因表达谱的影响，我们开展了DNA微阵列（DNA microarray）分析。具体实验流程为：从以葡萄糖为碳源、分别以L-谷氨酰胺或硫酸铵（ammonium sulphate）为氮源的CgXII培养基中培养的指数生长期细胞中提取总RNA。