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J. Biol. Chem., Vol. 282, Issue 46, 33641-33648, November 16, 2007

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PII Is Important in Regulation of Nitrogen Metabolism but Not Required for Heterocyst Formation in the Cyanobacterium Anabaena sp. PCC 7120^*

From the State Key Laboratory of Protein and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871, China

PII is an important signal protein for regulation of nitrogen metabolism in bacteria and plants. We constructed a mutant of glnB, encoding PII, in a heterocystous cyanobacterium, Anabaena sp. PCC 7120, with a cre-loxP system. The mutant (MP2) grew more slowly than the wild type under all nitrogen regimens. It excreted a large amount of ammonium when grown on nitrate due to altered activities of glutamine synthetase and nitrate reductase. MP2 had a low nitrogenase activity but was able to form heterocysts under diazotrophic conditions, suggesting that PII is not required for heterocyst differentiation. Analysis of the PII with mass spectroscopy found tyrosine nitration at Tyr-51 under diazotrophic conditions while no phosphorylation at Ser-49 was detected. The strains 51F and 49A, which have PII with mutations of Y51F and S49A, respectively, were constructed to analyze the functions of the two key residues on the T-loop. Like MP2, they had low nitrogenase activity and grew slowly under diazotrophic conditions. 49A was also impaired in nitrate uptake and formed heterocysts in the presence of nitrate. The up-regulation of ntcA after nitrogen step-down, which was present in the wild type, was not observed in 51F and 49A. While our results showed that the Ser-49 residue is important to the function of PII in Anabaena sp. PCC 7120, evidence from the PII pattern of the wild type and 49A in non-denaturing gel electrophoresis suggested that Ser-49 is not modified. The possible physiological roles of tyrosine nitration of PII are discussed.

Received for publication, August 6, 2007 , and in revised form, September 17, 2007.

^* This study was supported by National Science Foundation of China Grants 30230040 and 30540017 and Ministry of Science and Technology of China Grant 01CB108903. 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S3 and Tables S1 and S2.

¹ To whom correspondence should be addressed. Tel.: 86-10-6275-6421; Fax: 86-10-6275-1526; E-mail: jzhao{at}pku.edu.cn.

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