The rpoN gene (encoding the sigma factor sigma 54) of Escherichia coli was cloned and its nucleotide sequence determined. Promoter probe analysis confirmed the presence of a promoter in a 350 bp fragment covering the start of rpoN. The likely promoter was identified. The nucleotide sequence of the region extending 2.1 kb downstream of rpoN was also determined. This region contained four open reading frames encoding potential polypeptides of 10750, 17959, 32492 and 9810 Da; maxicell and T7 promoter studies showed that four polypeptides of similar molecular masses were expressed from this region. The amino acid sequence of the 17959 Da polypeptide showed homology to the enzyme IIA domains of several proteins of the bacterial sugar phosphotransferase system (PTS), and the 9810 Da polypeptide showed homology to the HPr proteins of the bacterial PTS. The proteins encoded downstream of rpoN are known to negatively regulate sigma 54 activity. The homologies therefore suggest that this effect on sigma 54 may be mediated by sequential protein phosphorylation and suggest that there is a link between signal transduction and transcription of sigma 54-dependent genes.