Klessig DF etal. (2000) PNAS 97(16), 8849-8855 Seregelyes C et al. (2000) FEBS Lett. 482, 125-130


H. Berges, C. Checroun, S. Guiral, A.M. Garnerone, P. Boistard, J. Batut

Lab. de Bio. Mol. des Relations Plantes-Microorganismes, CNRS-INRA, Castanet, France

1. Introduction

S. meliloti forms N2-fixing nodules on the roots of alfalfa and closely related plants. Expression of nitrogen fixation genes is under both positive and negative control. This regulation depends on a regulatory cascade, on top of which the two-component regulatory system FixLJ activates expression of nitrogen fixation genes in response to microoxic conditions, such as those that prevail inside the nodule. Under microoxic conditions, the sensor histidine kinase FixL autophosphorylates and transfers its phosphate to the FixJ transcriptional regulator protein. Phosphorylated FixJ then activates transcription of two intermediate regulatory genes, nifA and fixK, that both encode transcriptional regulators. FixK is also indirectly responsible for negative regulation of the cascade since it controls expression of a gene, fixT, that negatively affects expression of FixLJ dependent genes (Foussard et al. 1997). 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 (Garnerone et al. 1999). 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.

2. Results and Discussion

We have isolated an S. meliloti mutant strain that phenotypically escapes the repressor activity exerted by FixT. The mutation lies in a gene named asnO. This gene encodes a protein homologous to glutamine-dependent asparagine synthetases. The asnO gene did not appear to affect asparagine biosynthesis and may serve a regulatory function in S. meliloti. We provide evidence that asnO is active during symbiosis and that its expression is induced in microoxic conditions (Berges et al. 2001).

The present work argues in favor of a physiological function associated with fixT. This finding brings support to the previous suggestion that FixT may allow integration of an additional signal by the FixLJ two-component regulatory system whose activity is primarily regulated by oxygen. We propose as a working model that the absence of AsnO may result in an imbalance in the pool of a metabolite (e.g. a substrate or a product of AsnO), that would affect the intrinsic repressing activity of FixT or, equally, the interaction between FixT and FixL. Because glutamine, a likely byproduct of nitrogen fixation in symbiotic rhizobia, is a predicted substrate of the AsnO protein, it is tempting to speculate that asnO and fixT may provide a link between the nitrogen status of bacteria -or of the plant cell - and nitrogen fixation activity and reducing power generation. Possibly, such a genetic device may connect the nitrogen needs of the plant to the nitrogen fixation activity of the microsymbiont.

3. References

Berges H et al. (2001) BMC Microbiol. 1, 6 Foussard M et al. (1997) Mol. Microbiol. 25, 27-37 Garnerone AM et al. (1999) J. Biol. Chem. 274, 32500-32506


S. Povolo, S. Casella

Dept Biotecnologie Agrarie, University of Padova, 35020 Legnaro, Padova, Italy

Most rhizobia accumulate poly-|3-hydroxybutyrate (PHB) during free-living growth but not all are able to store the polymer in symbiotic life. The role of PHB during nitrogen fixation in bacteroid is unclear. A PHB-synthase (phbC) mutant of Rhizobium etli has more nitrogenase activity and higher seed content (Cevallos et al. 1996). In contrast, bacteroids of Sinorhizobium meliloti do not accumulate PHB and nitrogen fixation is not affected by the lack of PHB-synthase (Povolo 1994). In S. meliloti a mutation in a gene involved in carbon flux regulation (aniA) reduces nitrogen fixation suggesting a complex interplay among different metabolic pathways, namely PHB, glycogen and nitrogenase activity (Povolo, Casella 2000). Recently a Tn5-mutant of Rhizobium tropici with enhanced symbiotic nitrogen fixation was isolated. The mutation mapped in a gene of glycogen synthesis (Marroqui 2001). The aim of this work was to isolate and characterize R. tropici mutants unable to accumulate PHB.

Mutants deficient in phbC were obtained by chromosomal integration of a kanamycin resistance cassette. DNA isolation and handling were performed as described (Sambrook et al. 1989). PHB was assayed by gas chromatography. Glycogen was extracted from cells and analyzed according to Rua et al. (1993). For microscopy, dry smears of formalin-treated cells or bacteroids were stained with 0.05 mM Nile red and examined by epifluorescence microscopy. Assay for symbiotic performance on Phaseolus vulgaris was carried out in Leonard jars using a nitrogen-free medium. Not inoculated controls were used. Protein concentration was determined by the method of Bradford.

R. tropici CIAT899 accumulated PHB during free-living growth but not much in the symbiotic state. The PHB-mutants of R. tropici (strains 900 and 901) showed nitrogen fixation activities similar to wild type (Table 1). Another effect of the phbC mutation was an increase of glycogen content (Table 1), confirming what was previously observed for R. etli. If PHB in R. tropici plays a role in storage/regeneration of reducing equivalents, thus interacting with the nitrogen-fixing apparatus during symbiotic life, glycogen can take the place of PHB when the phbC gene is knocked out.

Table 1. Poly-P-hydroxybutyrate, glycogen content and symbiotic traits of It tropici strains.


(mg protein)"1

Glycogen Hg(mg protein)"1

Nodule per plant number mg dw

C2H4 production mg nod"1 h"'



36.6± 1.1

86 55.3

466.8 ± 25



71.1 ± 1.3

73 41.7

428.2 ± 32



62.2 ± 2.2

81 32.1

Cevallos MA et al. (1996) J. Bacteriol. 178, 1646-1654 Marroqui S et al. (2001) J. Bacteriol. 183, 854-864 Povolo S, Casella S (2000) Arch. Microbiol. 174, 42-49 Povolo S et al. (1994) Can. J. Microbiol. 40, 823-829 Rua J et al. (1993) J. Gen. Microbiol. 139, 217-222

Sambrook J et al. (1989) Molecular Cloning: A Laboratory Manual. 2nd edn, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

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