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J. Biol. Chem., Vol. 281, Issue 9, 5869-5876, March 3, 2006

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Latent Nitrate Transport Activity of a Novel Sulfate Permease-like Protein of the Cyanobacterium Synechococcus elongatus^*

Shin-ichi Maeda¹, Chieko Sugita, Mamoru Sugita, and Tatsuo Omata

From the Laboratory of Molecular Plant Physiology, Graduate School of Bioagricultural Sciences, and the Center for Gene Research, Nagoya University, Nagoya 464-8601, Japan

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 Asp⁵² 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.

Received for publication, December 12, 2005 , and in revised form, January 3, 2006.

^* This work was supported by Grant-in-aid for Scientific Research in Priority Areas 13206027 and in part by Grant-in-aid for Specially Promoted Research 13CE2005 and the 21st Century Center of Excellence Program of the Ministry of Education, Culture, Sports, Science, and Technology of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Laboratory of Molecular Plant Physiology, Graduate School of Bioagricultural Sciences, Nagoya University, Furocho, Chikusaku, Nagoya, Aichi 464-8601, Japan. Tel.: 81-52-789-4104; Fax: 81-52-789-4107; E-mail: maeda{at}agr.nagoya-u.ac.jp.

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