The Quest for the FixJ Regulon

In the regulatory cascade controlling nif and fix genes, FixJ is a global regulator, while nif A and fixK are specialized regulators of nif and fix genes, respectively. This regulatory network architecture suggested that FixJ might control yet other sets of genes in response to microaerobiosis in the nodule environment.

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Figure 3. Distribution of FixJ binding sites on the S. meliloti genome.

Figure 3. Distribution of FixJ binding sites on the S. meliloti genome.

In order to identify potential new FixJ targets in the genome of S. meliloti we developed an in vitro cyclic selection (SELEX) procedure using a fusion protein between GST and the DNA binding domain of FixJ. DNA fragments covering the entire S. meliloti genome were generated by random priming as described by Singer et al. (1997), protein-DNA complexes were adsorbed on glutathione-sepharose beads, bound DNA was eluted and amplified. Resulting DNA, enriched in FixJ binding fragments, was subjected to additional selection cycles until protein-DNA complexes could be detected by gel retardation analysis. Three selection rounds appeared to be sufficient. Selected fragments were cloned and sequenced, and fragments originating from the same region were clustered on the basis of sequence comparison. This analysis yielded 32 independent candidate FixJ targets. These candidate targets were further tested by gel retardation and DNase I foot-printing experiments in the presence of FixJ~P, which led to the identification of 22 FixJ~P binding sites on the S. meliloti genome. These include the known fixK and fixK' promoters (Waelkens et al. 1992) and 20 novel sites unequally distributed through the genome (Figure 3). pSymA contains 10 sites distributed throughout the replicon, while only 3 sites are located on pSymB. Similarly the chromosome contains very few FixJ~P binding sites, with the exception of a region which was previously recognized as resulting from horizontal transfer (Capela et al. 2001). Thus the fixJ gene and the majority of its targets appear to be carried by pSymA, which is consistent with the proposal that pSymA has been acquired relatively recently (Galibert et al. 2001), together with the fix./ regulon. In this scenario, the cluster of FixJ binding sites found on the chromosome would mostly derive from pSymA.

In agreement with this view, two of the new FixJ targets appear to result from a duplication of the fixK promoter, evident from the presence of a remnant truncated fixK ORF. This duplication of the fixK promoter confers /«./-dependent microaerobic induction upon the downstream gene, as evidenced by RT-PCR and reporter gene experiments. Similar promoter duplications, including a truncated nifH ORF, were previously observed for the nifH promoter and were found to confer nif'A regulation upon downstream genes (Better et al. 1983; Murphy et al. 1993). Such a 'promoter hijacking' may therefore be a more common phenomenon in the S. meliloti genome than originally thought, allowing for the recruitment of new genes into pre-existing regulatory networks.

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