Inactivation of gltB Abolishes Expression of the Assimilatory Nitrate Reductase Gene (nasB) in Pseudomonas putida KT2442
Inactivation of gltB Abolishes Expression of the Assimilatory Nitrate Reductase Gene (nasB) in Pseudomonas putida KT2442
复制标题
DOI：
10.1128/jb.182.12.3368-3376.2000
复制
发表时间：
2000-06
影响因子：
3.2
通讯作者：
L. Eberl;A. Ammendola;M. Rothballer;M. Givskov;C. Sternberg;M. Kilstrup;K. Schleifer;S. Molin
中科院分区：
文献类型：
--
作者：
L. Eberl;A. Ammendola;M. Rothballer;M. Givskov;C. Sternberg;M. Kilstrup;K. Schleifer;S. Molin
摘要：通过使用 mini-Tn5 转座子诱变，产生无启动子细菌荧光素酶 luxAB 与恶臭假单胞菌 KT2442 基因的随机转录融合。选择通过诱导生物发光来响应铵缺乏的插入突变体。对反应最强烈的突变体进行了基因分析，并证明其在恶臭假单胞菌 KT2442 的同化硝酸还原酶基因 (nasB) 内携带转座子。遗传证据以及 nasB 侧翼 DNA 区域的序列分析表明，硝酸盐同化所需的基因并未聚集在一起。我们分离了三个第二位点突变体，其中 nasB 表达的诱导在氮限制条件下完全消失。染色体连接点的核苷酸序列分析表明，在所有三个突变体中，二级转座子已插入恶臭假单胞菌 KT2442 编码谷氨酸合酶主要亚基的 gltB 基因的不同位点。 gltB突变体的详细生理特征表明，它们无法利用许多潜在的氮源，在表达氮饥饿蛋白的能力方面存在缺陷，在氮限制条件下表现出异常的细胞形态，并且在长期氮饥饿时期生存的能力受损。
ABSTRACT By using mini-Tn5 transposon mutagenesis, random transcriptional fusions of promoterless bacterial luciferase,luxAB, to genes of Pseudomonas putida KT2442 were generated. Insertion mutants that responded to ammonium deficiency by induction of bioluminescence were selected. The mutant that responded most strongly was genetically analyzed and is demonstrated to bear the transposon within the assimilatory nitrate reductase gene (nasB) of P. putida KT2442. Genetic evidence as well as sequence analyses of the DNA regions flanking nasBsuggest that the genes required for nitrate assimilation are not clustered. We isolated three second-site mutants in which induction ofnasB expression was completely abolished under nitrogen-limiting conditions. Nucleotide sequence analysis of the chromosomal junctions revealed that in all three mutants the secondary transposon had inserted at different sites in the gltB gene of P. putida KT2442 encoding the major subunit of the glutamate synthase. A detailed physiological characterization of thegltB mutants revealed that they are unable to utilize a number of potential nitrogen sources, are defective in the ability to express nitrogen starvation proteins, display an aberrant cell morphology under nitrogen-limiting conditions, and are impaired in the capacity to survive prolonged nitrogen starvation periods.