A divergently transcribed open reading frame is located upstream of the Pseudomonas aeruginosa vfr gene, a homolog of Escherichia coli crp.

The Pseudomonas aeruginosa homolog of the Escherichia coli global transcriptional regulator CRP (or CAP) was recently identified and designated Vfr (S. E. H. West, A. K. Sample, and L. J. Runyen-Janecky, J. Bacteriol. 176:7532-7542, 1994). Nucleotide sequence analysis of the region 5' to vfr identified a 423-bp open reading frame (ORF), which was designated orfX. The deduced amino acid sequence of ORFX was 53% identical and 87% similar to a divergent ORF of unknown function located 5' to the E. coli crp gene. When orfX was expressed from a phage T7 promoter in E. coli, a protein with an apparent molecular mass of approximately 18 kDa was produced. We constructed a chromosomal deletion of the region containing the 5' end of orfX (orfX'), vfr, and the 3' end of trpC (trpC') in P. aeruginosa strains PAO1 and PA103. The cloned vfr gene restored Vfr-dependent production of exotoxin A and protease in the PA103 orfX'-vfr-trpC' deletion mutant, suggesting that ORFX is not required for Vfr production or activity. To determine whether transcription of orfX and vfr are controlled by the same mechanisms that control transcription of the region of the divergent ORF (dorf) and of crp, we compared the vfr-orfX and crp-dorf intergenic regions. Using S1 nuclease analysis, we determined that the distance between the orfX and vfr transcriptional start sites was 105 bp. Thus, the P. aeruginosa orfX and vfr promoters are arranged in a back-to-back orientation rather than the face-to-face orientation of the dorf and crp promoters. A CRP recognition site is associated with each promoter in the crp-dorf intergenic region; binding of the CRP-cyclic AMP complex to the stronger dorf CRP recognition site activates transcription from the dorf promoter and represses transcription from the crp promoter. The vfr-orfX intergenic region does not contain an obvious CRP recognition site. In addition, vfr was not required for transcription of orfX. Unlike the dorf and crp mRNAs, the 5' ends of the orfX and vfr mRNAs were not complementary. Thus, the orfX mRNA cannot hybridize to the 5' end of the vfr mRNA to inhibit vfr transcription, a mechanism that has been postulated to control crp transcription in E. coli.