Results and Discussion

We showed that addition of "AICAR" to the culture medium reduces the microaerobic level of expression offixN-lacZ, fixT-lacZ, fixK-lacZ and nifA-lacZ gene fusion by 10-, 3.7-, 7.5- and 1.6-fold respectively. "AICAR" has no effect when fixK is constitutively expressed. These results suggest that "AICAR" affects fix gene expression upstream of fixK and nifA. We speculate that the metabolite interferes with the activity of the two components FixLJ system either directly or indirectly via an unknown protein intermediate. This is consistent with the proposed role of AICAR in eukaryotic cells (Kaiser et al. 1996).

It is to point out that the repression by "AICAR" occurs in a strain mutated for a gene known as the ORF151, located downstream of fixK. ORF151 gene product is homologous to Nim proteins of Bacteroides fragilis, to an unkown protein from Methanobacterium thermoautotrophicum and to 4 homologous genes found in Mezorhizobium loti genome. Nim proteins are 5-nitroimidazole reductase (Carlier et al. 1997). We purified the ORF151 protein as an inteine fusion and further biochemical analysis reveals that it encodes a flavoprotein. These properties of the ORF151 suggest that, as Nim proteins do on 5-nitroimidazole drugs, it could have a reductase activity on AICAR.

In the symbiosis, an ORF151 mutant is Nod+ and fix+ and symbiotic activity of a fixN-lacZ fusion is only reduced by 2-fold in the ORF151 mutant. However when AICAR is endogenously accumulated (in a purH mutant), fixK is repressed by 10-fold in the nodule. We speculate that AICAR may accumulate under particular symbiotic conditions as a result of either bacteroids or plant cell metabolism and inhibit fixK and nifA expression. ORF151 would prevent AICAR effect and preserve nitrogen fixation and respiration.

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