Maeda Shin-ichi, Sugita Chieko, Sugita Mamoru, Omata Tatsuo
Laboratory of Molecular Plant Physiology, Graduate School of Bioagricultural Sciences, Nagoya University, Furucho, Chikusaku, Nagoya, Aichi 464-8601, Japan.
J Biol Chem. 2006 Mar 3;281(9):5869-76. doi: 10.1074/jbc.M513196200. Epub 2006 Jan 3.
The Synechococcus elongatus mutant lacking the nrtABCD gene cluster (NA3) is defective in active nitrate transport and requires high nitrate concentrations (>30 mm) for sustained growth. Prolonged incubation of NA3 in medium containing 2 mm nitrate led to isolation of a pseudorevertant (NA3R) capable of transport of millimolar concentrations of nitrate, from which three mutants with improved affinity for nitrate were obtained. We identified three genes responsible for the latent transport activity for nitrate: ltnA, which encodes a response regulator with no effector domain; ltnB, which encodes a hybrid histidine kinase with two receiver domains; and ltnT, which encodes a sulfate permease-like protein with a putative cyclic nucleoside monophosphate (cNMP)-binding domain. Missense mutations of the high affinity derivatives of NA3R were found in ltnT, verifying that LtnT acts as the transporter. Overexpression of truncated LtnT lacking the cNMP-binding domain (but not full-length LtnT) conferred nitrate transport activity on NA3, suggesting that the cNMP-binding domain inhibits transport under normal conditions. A nonsense mutation in ltnB that resulted in elimination of the receiver domains of the encoded protein was responsible for expression of nitrate transport activity in NA3R. Expression of LtnB derivatives lacking the receiver domains also conferred low affinity nitrate transport activity on NA3. The phosphoryl group of the histidine kinase domain of LtnB was transferred to Asp(52) of LtnA in vitro. Overexpression of LtnA (but not LtnA(D52E)) led to manifestation of the latent nitrate transport activity in NA3, indicating involvement of phosphorylated LtnA in activation of the novel transporter.
缺少nrtABCD基因簇的聚球藻突变体（NA3）在活性硝酸盐转运方面存在缺陷，需要高浓度硝酸盐（>30 mM）才能持续生长。将NA3在含有2 mM硝酸盐的培养基中长时间培养，得到了一个能够转运毫摩尔浓度硝酸盐的假回复突变体（NA3R），从该突变体中获得了三个对硝酸盐亲和力提高的突变体。我们鉴定出了三个负责硝酸盐潜在转运活性的基因：ltnA，编码一个没有效应结构域的应答调节蛋白；ltnB，编码一个具有两个接收结构域的杂合组氨酸激酶；ltnT，编码一个具有推定环核苷酸单磷酸（cNMP）结合结构域的硫酸盐通透酶样蛋白。在ltnT中发现了NA3R高亲和力衍生物的错义突变，证实LtnT作为转运蛋白发挥作用。缺少cNMP结合结构域的截短型LtnT（而非全长LtnT）的过表达赋予了NA3硝酸盐转运活性，表明cNMP结合结构域在正常条件下抑制转运。ltnB中的一个无义突变导致编码蛋白的接收结构域缺失，这是NA3R中硝酸盐转运活性表达的原因。缺少接收结构域的LtnB衍生物的表达也赋予了NA3低亲和力硝酸盐转运活性。在体外，LtnB组氨酸激酶结构域的磷酸基团转移到了LtnA的Asp(52)上。LtnA（而非LtnA(D52E)）的过表达导致NA3中潜在的硝酸盐转运活性显现，表明磷酸化的LtnA参与了新型转运蛋白的激活。
