Deletion analysis of the Klebsiella pneumoniae nitrogenase promoter: importance of spacing between conserved sequences around positions -12 and -24 for activation by the nifA and ntrC (glnG) products

Abstract

The nitrogen fixation promoters of Klebsiella pneumoniae are atypical procaryotic promoters lacking the usual -10 and -35 elements, requiring instead conserved sequences around -12 and -24 for transcriptional activation. By constructing a set of five deletions between the -12 and -24 elements in the nifH promoter, the spacing between the conserved GC and GG motifs at -12 and -24, respectively, has been reduced from the wild-type 10 bases to 9, 8, 6, 5, and 4 bases. The deletion of a single nonconserved nucleotide was sufficient to eliminate transcriptional activation by either nifA or ntrC (glnG). All deletions relieved the multicopy inhibition of chromosomal nif expression normally shown by the nifH promoter. These results demonstrate a stringent requirement for the 10-base spacing found in ntr-activated promoters. In addition, specific sequences around the invariant GG at -24 were shown to be necessary for activation by either nifA or ntrC, with a minimal requirement for nucleotides through to position -27 for this activation.