Suppression analysis of positive control mutants of NifA reveals two overlapping promoters for Klebsiella pneumoniae rpoN.

Activation of gene expression relies on direct molecular interactions between the RNA polymerase and transcription factors. Eubacterial enhancer-binding proteins (EBPs) activate transcription by binding to distant sites and, simultaneously, contacting the sigma(54)-holoenzyme form of the RNA polymerase (Esigma(54)). The interaction between the EBP and Esigma(54) is transient, such that it has been difficult to be studied biochemically. Therefore, the details of this molecular recognition event are not known. Genetic and physical evidences suggest that the highly conserved C3 region in the activation domain of the EBP has major determinants for positive control and for the interaction with Esigma(54). To further investigate the target of this region we searched for extragenic suppressors of some C3 region mutant derivatives of NifA. As a first step we mutagenized Klebsiella pneumoniae rpoN, the gene that codes for sigma(54). A mutant allele, rpoN1320, that suppressed two different NifA derivatives was obtained. Immunodetection of sigma(54) and transcriptional initiation studies demonstrated that the cause of the suppression was an enhanced expression of rpoN. A single point mutation was responsible for the phenotype. It mapped at the -10 region of an unidentified promoter, here denominated rpoNp1, and increased its similarity to the consensus. A second upstream promoter, denominated rpoNp2, was also identified. Its -10 region partially overlaps with the -35 region of rpoNp1. Interestingly, the promoter-up -10 mutation in rpoNp1 caused a reduction in the expression from rpoNp2, likely reflecting a stronger occupancy of the former promoter by the RNA polymerase at the expense of the latter. The presence of two overlapping promoters competing for the RNA polymerase implies a complex regulatory pattern that needs elucidation. The fact that increasing the concentration of sigma(54) in the cell can suppress positive control mutants of NifA adds further evidence for their direct interaction in the activation process. Copyright 1999 Academic Press.