Progr. de Genet. Mol. de Plásmidos Bact. Centro de Investigación sobre Fijación de Nitrógeno, UNAM. Av. Universidad s/n. C. P. 62210, Cuernavaca, Morelos, México
Recently, we reported the presence of two fix regions in Rhizobium etli CFN42 (Girard et al. 2000; Soberón et al. 1999). Gene fusion analysis coupled with mutation of each regulatory element was used to study the mechanisms employed in R. etli CFN42 for the regulation of nitrogen fixation genes. This circuit integrates the participation of genes located in two different plasmids and shows several novel characteristics when compared with the conventional systems as R. meliloti and B. japonicum. Among these, microaerobic expression of the fixN reiterations exhibits a differential dependence for FixL, where transcription offixNf was suppressed in the absence of FixL; expression of fixNd, however, is reduced only 50% in this mutant (Girard et al. 2000). Moreover, new data indicate the participation of fnrN homologs in the fixNd microaerobic expression (López et al. submitted).
To localize which sequences in the fixN regulatory regions are responsible for this differential expression, derivatives containing deletions of the upstream and 5' coding sequence were generated by PCR, and fused with a promoterless (3-glucuronidase gene. The expression patterns were analyzed under aerobic and microaerobic conditions. Plasmids containing transcriptional fusions were introduced into different R. etli CFN42 derivatives and cultured under aerobic and microaerobic conditions to study its transcriptional regulation.
Differential regulation of fixN reiterated genes is maintained. However, a very interesting behavior was observed for a new fixNd deleted derivative. Deletion of codons 17 to 95 increases the fixNd microaerobic expression and allows its aerobic expression. Surprisingly, the aerobic expression observed in derivatives harboring this deletion is still dependent on the same regulators operating under microaerobic conditions, FixKf, FnrNd and FnrNchr. These results suggest the existence of a regulatory region within the fixNd coding region that diminish its expression in both aerobiosis and microaerobiosis. To define the minimal region involved in this new transcriptional pattern for a cytochrome oxidase, new derivatives carrying small deletions will be generated.
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