Abstract
Previously isolated promoter mutations that allow expression of the Klebsiella pneumoniae nifHDKY operon in the absence of nifA (R. Bitoun, J. Berman, A. Zilberstein, D. Holland, J.B. Cohen, D. Givol, and A. Zamir, Proc. Natl. Acad. Sci. USA, 80:5812-5816, 1983) were further characterized. pRB1 and pRB5, containing, respectively, point and duplication mutations in the nifHDKY regulatory region, were transformed into Escherichia coli and K. pneumoniae hosts with different nifA and ntrA backgrounds. nif transcription start sites were determined by nuclease S1 mapping. The results indicated that nifA-independent expression from both mutants did not require ntrA. Transcription from pBR5 started 3 base pairs (bp) upstream of the start site of nif-regulated transcription and could stem from a canonical promoter sequence generated at the junction between the two copies of the duplicated sequence. In the presence of nifA-ntrA, transcription from pRB5 started predominantly at the site characteristic of the nif-regulated promoter. The site of constitutive transcription initiation in pRB1 was located 33 bp upstream of the point mutation and 40 bp upstream of the start of nifA-ntrA-activated transcription. Low-level transcription from the upstream site was also evident, in the absence of nifA or nifA or both, with the plasmid containing the wild-type nifHDKY regulatory region. However, when nifA and ntrA were present to activate transcription from the major nif promoter, no activity was evident from the upstream site in either pRB1 or the parental plasmid. Thus, the mutation enhanced the activity of a pre-existing constitutive promoter, the activity of which was repressed on nifA-ntrA activation of the major nif promoter.