Expression of the Sinorhizobium meliloti C4-dicarboxylate transport gene during symbiosis with the Medicago host plant

During symbiosis between Sinorhizobium meliloti and the Medicago host plant, the energy required to fix atmospheric nitrogen, is derived from the plant photosynthate. Current evidence indicates that C ₄ -dicarboxylates (dCA) are the major and probably only source of carbon provided to the bacteroids in sufficiently high amounts to support symbiotic N ₂ -fixation. The ability of the microsymbiont to take up dCA is essential for the establishment of an effective symbiosis. The dct A gene codes for a high affinity dCA permease (DctA). The regulatory dct BD genes code for a two-component regulatory system. Under free-living conditions, the regulatory dct BD genes are essential for activation of the dct A promoter. Bacteroids isolated from nodules induced by regulatory dct BD mutants take up dCA efficiently. This demonstrated that in the specific environment of the nodule, regulatory molecules other than DctBD, are involved in the expression of the dct A promoter.

The aim of this work was to study the regulation of the dct A gene during symbiosis of S.meliloti with the Medicago host plants and in particular to characterise this alternative mechanism of symbiotic activation (ASA). Using gene fusions of the dct A expression signals to the lac Z reporter gene, in combination with histochemical staining of plant tissue, the regulation of the S.melilotidct A gene was studied in situ during the symbiosis with the Medicago host plants. The major findings of this work are the following.

1) The ASA mechanism is only active at the late stages of the symbiosis.

First, it is established that in a wild-type background, the dct A gene is expressed during the early and late stages of symbiosis (A). In contrast in nodules induced by a regulatory dct B or dct D mutant, a distinct late symbiotic pattern of dct A expression was observed (B). This meant that the ASA is an exclusive late symbiotic mechanism. The temporal and spatial pattern of DctBD-independent dct A expression in nodules induced on the alfalfa host plant was always very similar to that of the nif H gene fusion. This late symbiotic manifestation of the ASA means that the early expression of dct A observed in wild-type nodules is necessarily DctBD dependent.

2) The ASA requires sequences downstream of the dct A promoter for activity.

We also found that certain gene fusions lacking the extreme N-terminal domain of the dct A coding region did not respond to the ASA. This was remarkable since these gene fusions appeared to be correctly regulated by the DctBD system under free-living conditions. In contrast, gene fusions containing the first 8 amino-acids of the dct A coding region were efficiently activated by the ASA (B). We concluded that some cis-acting nucleotide sequences, located immediately downstream of the dct A promoter in the beginning of the coding region, are required for ASA activity

3) The UAS sites of the dct A promoter are not essential for ASA activity.

During symbiosis dct A:: lac Z gene fusions lacking the upstream activator sites (UAS) were expressed in wild-type nodules (D) and at a similar level in a dct D mutant background (E). The pattern of temporal and spatial expression in a wild-type nodule (D) was similar to that observed for DctBD independent activation of other gene fusions containing the UAS (B). We concluded that the UAS, which are indispensable for the activation by the DctBD system, are not essential for the activation of the dct A promoter by the ASA.

4) The alternative symbiotic activator requires NifA.

We observed gene fusions lacking the UAS sequences are efficiently expressed in the wild-type and dct D mutant backgrounds (D;E), but were not activated in nodules formed by a nif A mutant strain (F).

Working with the Medicago truncatula host plant, we found that in nodules induced by a dct D mutant strain, the dct A gene was not expressed efficiently. We also observed that the nif A gene is expressed in these nodules at a level, which is strongly reduced in comparison to the level of nif A expression in nodules induced on M.sativa . We concluded that an efficient expression of the nif A gene is required for ASA activity.

5) The ASA is not essential for symbiotic N ₂ -fixation.

Monitoring dct A activity with the dct A:: lac Z gene fusions which are not activated by the ASA, demonstrated that the DctBD system alone is sufficient to express the dct A promoter during symbiosis. Additionally we observed that although the ASA does not function in nodules induced on the Medicago truncatula host plant, these nodules were efficient for symbiotic N ₂ -fixation. This indicates that activation of the dct A promoter by the ASA is not required for symbiotic N ₂ -fixation.