Characterization of the Caulobacter crescentusflbF gene and promoter region and investigation of the membrane topology of FlbF, a member of a new class of proteins that also includes the Escherichia coli FlhA protein
Description
We have investigated the organization and expression of the Caulobacter crescentus flbF gene and have studied the topology of the FlbF protein because the flbF gene occupies a high level in the flagellar gene regulatory hierarchy. The nucleotide sequence comprising the flbF promoter and structural gene and the promoter and structural gene for the gene following flbF (gff) was determined, and the organization of transcription units within this sequence was investigated. The 5$\sp\prime$ end of the flbF transcript was precisely mapped by primer extension analysis and the 5$\sp\prime$ end of gff was located by nuclease S1 protection assay. The nucleotide sequence upstream from the 5$\sp\prime$ end of the flbF transcript contains $-$10 and $-$35 elements similar to those found in promoters transcribed by sigma 28 RNA polymerase in other organisms. Mutations that changed the nucleotides in the $-$10 or $-$35 elements or in the region between them strongly suggest that both the $-$10 and $-$35 elements are essential for flbF transcription. However, nucleotides between the two elements were also found to be important to flbF transcription. We identified a 700-codon open reading frame downstream from the flbF promoter region that was predicted to be the flbF structural gene. The FlbF amino acid sequence is very similar to those of a new class of proteins including the FlhA protein, which is essential for flagella formation in Escherichia coli. The amino-terminal half of the inferred FlbF amino acid sequence contains eight hydrophobic regions that we predicted to be membrane spanning segments, suggesting that the FlbF protein may be an integral membrane protein. A model of the membrane topology of FlbF was constructed and tested by using flbF-phoA gene fusions and measuring the alkaline phosphatase activity levels in cells expressing the chimeric proteins. Nuclease S1 protection assays suggest that gff lies in a transcription unit located downstream from flbF. A 481 codon open reading frame downstream from the gff 5$\sp\prime$ end was predicted to be the gff structural gene. Gff has no amino acid sequence homology to the protein sequences contained in the NCBI databases (July, 1993)