Identification of the promoter and a negative regulatory element, ftr4, that is needed for cell cycle timing of fliF operon expression in Caulobacter crescentus

Abstract

The fliF operon of Caulobacter crescentus, which was previously designated the flaO locus, 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. In this report, we have identified two cis-acting sequences that are required for cell cycle regulation of fliF transcription. The first sequence was defined by the effects of three 2-bp deletions and five point mutations, each of which greatly reduced the level of fliF operon transcript in vivo. These eight mutations lie between -37 and -22 within an 18-bp sequence that matches, at 11 nucleotides, sequences in the 5' regions of the flaQR (flaS locus) and fliLM operons, which are also expressed early and occupy a high level in the regulatory hierarchy (A. Dingwall, A. Zhuang, K. Quon, and L. Shapiro, J. Bacteriol. 174:1760-1768, 1992). We propose that this 18-bp sequence contains all or part of the fliF promoter. We have also identified a second sequence, 17 bp long and centered at -8, which we have provisionally designated ftr4 because of its similarity to the enhancer-like ftr sequences required for regulation of sigma 54 promoters flaN and flbG (D. A. Mullin and A. Newton, J. Bacteriol. 171:3218-3227, 1989). Six of the seven mutations in ftr4 examined resulted in a large increase in fliF operon transcript levels, suggesting a role for ftr4 in negative regulation. A 2-bp deletion at -12 and -13 in ftr4 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 the ftr4 sequence may form part of a developmental switch which is required to turn off fliF operon transcription at the correct time in the cell cycle.