Functionality of purified sigma(N) (sigma(54)) and a NifA-like protein from the hyperthermophile Aquifex aeolicus

The genome sequence of the extremely thermophilic bacterium Aquifex aeolicu s encodes alternative sigma factor sigma(N) (sigma(54), RpoN) and five pote ntial sigma(N)-dependent transcriptional activators. Although A. aeolicus p ossesses no recognizable nitrogenase genes, two of the activators have a hi gh degree of sequence similarity to NifA proteins from nitrogen-fixing prot eobacteria. We identified five putative sigma(N)-dependent promoters upstre am of operons implicated in functions including sulfur respiration, nitroge n assimilation, nitrate reductase, and nitrite reductase activity. We clone d, overexpressed (in Escherichia coli), and purified A. aeolicus sigma(N) a nd the NifA homologue, AQ_218, Purified A. aeolicus sigma(N) bound to E. co li core RNA polymerase and bound specifically to a DNA fragment containing E. coli promoter glnHp2 and to several A. aeolicus DNA fragments containing putative sigma(N)-dependent promoters. When combined with E. coli core RNA polymerase, A. aeolicus sigma(N) supported A. aeolicus NifA-dependent tran scription from the glnHp2 promoter, The E. coli activator PspF Delta HTH di d not stimulate transcription. The NifA homologue, AQ_218, bound specifical ly to a DNA sequence centered about 100 bp upstream of the A. aeolicus glnB A operon and so is likely to be involved in the regulation of nitrogen assi milation in this organism. These results argue that the sigma(N) enhancer-d ependent transcription system operates in at least one extreme environment, and that the activator and sigma(N) have coevolved.