Identification of cis- and trans-acting requirements for developmental regulation of thefliF operon of Caulobacter crescentus
Description
The fliF operon of Caulobacter crescentus is near the top of the flagellar gene regulatory hierarchy and it is one of the earliest transcription units to be expressed in the cell cycle. We have identified two cis-acting sequences that are required for cell cycle regulation of fliF transcription. The first sequence was defined by the effect of three two base pair deletions and 21 point mutations which lie between $-48$ and $-18$ from the start site of transcription. We propose that this 18 bp sequence contains all or part of the fliF promoter. We have also identified a second sequence of 17 bp centered at $-8$ which we have designated as ftr4. Most of the point mutations examined in ftr4 resulted in a large increase in fliF operon transcript levels, suggesting a role for ftr4 in negative regulation. A two bp deletion at $-12, -13$ in frt4 altered the cell cycle pattern of fliF operon transcription; the transcript was still expressed periodically, but the period of its synthesis was extended significantly. We suggest that ftr4 may form part of a developmental switch which is required to turn off fliF operon transcription at the correct time in the cell cycle flbD, the last gene of the fliF operon, is required for transcription of flbG and flaN and acts as a repressor of fliF. We cloned and sequenced flbD and found a 455 codon open reading frame whose deduced amino acid sequence is strongly similar over approximately 240 residues to the conserved central domain of the NifA and NtrC proteins. The FlbD amino acid sequence also includes a potential nucleotide binding domain, and a sequence about 20 amino acids from the C-terminus that is similar to the helix-turn-helix DNA binding domains in NifA, NtrC, and other DNA-binding regulatory proteins. Consistent with its predicted size of 48.8-kDa, the cloned flbD gene directed synthesis of a 53-kDa protein in Escherichia coli minicells. The major 3$\prime$ ends of the fliF operon transcript were located by nuclease S1 analysis, and these mapped approximately 149, 164, and 174 nucleotides downstream from the flbD 5$\prime$-UAA termination codon placing them all 5$\prime$ from the flhA operon transcription initiation point