Nolden L, Ngouoto-Nkili C E, Bendt A K, Krämer R, Burkovski A
Institut für Biochemie, Universität zu Köln, Zülpicher-Str. 47, D-50674 Köln, Germany.
Mol Microbiol. 2001 Dec;42(5):1281-95. doi: 10.1046/j.1365-2958.2001.02694.x.
A novel nitrogen control system regulating the transcription of genes expressed in response to nitrogen starvation in Corynebacterium glutamicum was identified by us recently. In this communication, we also show that the nitrogen regulation cascade in C. glutamicum functions by a new mechanism, although components highly similar to sensor and signal transmitter proteins of Escherichia coli are used, namely uridylyltransferase and a PII-type GlnK protein. The genes encoding these key components of the nitrogen regulation cascade, glnD and glnK, are organized in an operon together with amtB, which codes for an ammonium permease. Using a combination of site-directed mutagenesis, RNA hybridization experiments, reporter gene assays, transport measurements and non-denaturing gel electrophoresis followed by immunodetection, we showed that GlnK is essential for nitrogen control and that signal transduction is transmitted by uridylylation of this protein. As a consequence of the latter, a glnD deletion strain lacking uridylyltransferase is impaired in its response to nitrogen shortage. The glnD mutant revealed a decreased growth rate in the presence of limiting amounts of ammonium or urea; additionally, changes in its protein profile were observed, as shown by in vivo labelling and two-dimensional PAGE. In contrast to E. coli, expression of glnD is upregulated upon nitrogen limitation in C. glutamicum. This indicates that the glnD gene product is probably not the primary sensor of nitrogen status in C. glutamicum as shown for enterobacteria. In accordance with this hypothesis, we found a deregulated nitrogen control as a result of the overexpression of glnD. Furthermore, quantification of cytoplasmic amino acid pools excluded the possibility that a fall in glutamine concentration is perceived as the signal for nitrogen starvation by C. glutamicum, as is found in enterobacteria. Direct measurements of the intracellular ammonium pool indicated that the concentration of this compound might indicate the cellular nitrogen status. Deduced from glnK and glnD expression patterns and the genetic organization of these genes, this regulatory mechanism is also present in Corynebacterium diphtheriae, the causative agent of diphtheria.
我们最近在谷氨酸棒杆菌中鉴定出一种新型氮控制系统,该系统可调节响应氮饥饿而表达的基因的转录。在本通讯中,我们还表明,尽管谷氨酸棒杆菌中使用了与大肠杆菌的传感器和信号转导蛋白高度相似的成分,即尿苷酰转移酶和PII型GlnK蛋白,但其氮调节级联通过一种新机制发挥作用。编码氮调节级联这些关键成分的基因glnD和glnK与编码铵通透酶的amtB一起组成一个操纵子。通过定点诱变、RNA杂交实验、报告基因检测、转运测量以及非变性凝胶电泳后免疫检测相结合的方法,我们表明GlnK对于氮控制至关重要,并且信号转导通过该蛋白的尿苷酰化进行传递。后者的结果是,缺乏尿苷酰转移酶的glnD缺失菌株对氮短缺的反应受损。glnD突变体在存在限量铵或尿素的情况下生长速率降低;此外,如体内标记和二维聚丙烯酰胺凝胶电泳所示,观察到其蛋白质谱发生了变化。与大肠杆菌不同,谷氨酸棒杆菌中glnD的表达在氮限制时上调。这表明,如肠杆菌中所示,glnD基因产物可能不是谷氨酸棒杆菌中氮状态的主要传感器。根据这一假设,我们发现由于glnD的过表达导致氮控制失调。此外,对细胞质氨基酸库的定量排除了谷氨酸棒杆菌像肠杆菌那样将谷氨酰胺浓度下降视为氮饥饿信号的可能性。对细胞内铵库的直接测量表明,该化合物的浓度可能指示细胞的氮状态。从glnK和glnD的表达模式以及这些基因的遗传组织推断,这种调节机制也存在于白喉棒杆菌中,白喉棒杆菌是白喉的病原体。