azotoformans failed to complement the SP8 phenotype (Fig. 3a). When these plasmids were introduced into SP7 (ΔrpoN2::kan), we observed that only rpoN2 from R. azotoformans was able to restore the swimming defect of this strain (Fig. 3b). To further evaluate the ability of the different rpoN genes to complement the phenotype of the SP8 strain, we determined their capacity to restore the wild-type level of the transcriptional activity of the nifU promoter (nifUp) in the SP8 background. It has been previously shown that the activity of this promoter is mainly

dependent on RpoN1 and the bEBP NifA (Poggio et al., 2002, 2006). For this, a plasmid carrying a transcriptional fusion of the uidA gene (encoding the β-glucuronidase Androgen Receptor Antagonist enzyme) with nifUp was introduced into the SP8 derivative strains expressing the different rpoN genes. As expected, the rpoN genes that complemented the phenotype of the SP8 strain allowed a wild-type level of activity IWR-1 research buy of nifUp, while in the strains expressing the noncomplementing rpoNs, the activity of the nifUp was reduced

approximately 100 times (data not shown). This result suggests that the strains that showed a growth defect under diazotrophic growth conditions are unable to induce the genes required for nitrogen fixation. Together, these results suggest that rpoN1 and rpoN2 of R. azotoformans are also specialized to transcribe a particular set of genes, as occurs in R. sphaeroides. In addition, the fact that rpoN3 of R. azotoformans cannot express the σ54-dependent fli and nif genes of R. sphaeroides strengthens the notion Adenosine triphosphate that in these species rpoN3 may be specialized to transcribe a different subset of genes. With the exception of rpoN3 from R. azotoformans, all the other rpoN genes complemented either SP7 or SP8 strains, indicating that the tested rpoN1 and rpoN2 genes were being expressed in at least that condition. Nevertheless, it could be argued that the rpoN genes cloned in pRK415 could be

conditionally expressed, and R. azotoformans rpoN3 not expressed at all. To discard these possibilities, we cloned the rpoN gene from R. blasticus, and rpoN1 and rpoN3 from R. azotoformans in a construct that added a 6His-tag at the carboxy terminus of the protein. This tag allowed us to detect the resulting proteins by western blot. All the proteins were present when the cells were grown under aerobic or anaerobic N-limiting conditions (Fig. 4a and b), supporting our previous conclusions. Our results show that the orthologues of rpoN1Rs and rpoN2Rs can complement the mutants in these genes, suggesting that following duplication, a fast process of specialization occurred, after which each of the copies has maintained the characteristics that allow them to transcribe their particular set of genes. Given that rpoN1 from R.