A glutamine-amidotransferase-like protein modulates FixT anti-kinase activity in Sinorhizobium meliloti.

Hélène Bergès,Sébastien Guiral,Anne-Marie Garnerone,Claire Checroun,Pierre Boistard,Jacques Batut

doi:10.1186/1471-2180-1-6

Abstract

Nitrogen fixation gene expression in Sinorhizobium meliloti, the alfalfa symbiont, depends on a cascade of regulation that involves both positive and negative control. On top of the cascade, the two-component regulatory system FixLJ is activated under the microoxic conditions of the nodule. In addition, activity of the FixLJ system is inhibited by a specific anti-kinase protein, FixT. The physiological significance of this negative regulation by FixT was so far unknown. We have isolated by random Tn5 mutagenesis a S. meliloti mutant strain that escapes repression by FixT. Complementation test and DNA analysis revealed that inactivation of an asparagine synthetase-like gene was responsible for the phenotype of the mutant. This gene, that was named asnO, encodes a protein homologous to glutamine-dependent asparagine synthetases. The asnO gene did not appear to affect asparagine biosynthesis and may instead serve a regulatory function in S. meliloti. We provide evidence that asnO is active during symbiosis. Isolation of the asnO mutant argues for the existence of a physiological regulation associated with fixT and makes it unlikely that fixT serves a mere homeostatic function in S. meliloti. Our data suggest that asnO might control activity of the FixT protein, in a way that remains to be elucidated. A proposed role for asnO might be to couple nitrogen fixation gene expression in S. meliloti to the nitrogen needs of the cells.

Highlights

Nitrogen fixation gene expression in Sinorhizobium meliloti, the alfalfa symbiont, depends on a cascade of regulation that involves both positive and negative control
NifA mediates activation of nif genes involved in nitrogenase biosynthesis whereas FixK, a member of the Crp/Fnr family, activates expression of genes involved in the synthesis of a respihttp://www.biomedcentral.com/1471-2180/1/6 ratory oxidase complex [4, 5]. fixK is indirectly responsible for negative regulation of the cascade since it controls expression of a gene, fixT, that negatively affects expression of FixLJ dependent genes
Whether FixT serves a mere homeostatic function in S. meliloti or whether FixT allows integration of a physiological signal by the FixLJ system was so far unknown. We addressed this question by looking for S. meliloti mutants in which the FixT protein would not be active in repression

Summary

Introduction

Nitrogen fixation gene expression in Sinorhizobium meliloti, the alfalfa symbiont, depends on a cascade of regulation that involves both positive and negative control. Expression of nitrogen fixation genes is under both positive and negative control This regulation depends on a regulatory cascade, on top of which the twocomponent regulatory system FixLJ activates expression of nitrogen fixation genes in response to microoxic conditions [1], such as those that prevail inside the nodule [2]. We have shown recently that the FixT protein negatively affects the expression of nifA and fixK by inhibiting phosphorylation of the sensor hemoprotein kinase FixL and, by consequence, phosphorylation of FixJ [6]. Whether FixT serves a mere homeostatic function in S. meliloti (the level of FixT protein feed-back controlling activity of the FixLJ system) or whether FixT allows integration of a physiological signal by the FixLJ system was so far unknown. We addressed this question by looking for S. meliloti mutants in which the FixT protein would not be active in repression

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Results

Conclusion