A DIVERGENTLY TRANSCRIBED OPEN READING FRAME IS LOCATED UPSTREAM OF THE PSEUDOMONAS-AERUGINOSA VFR GENE, A HOMOLOG OF ESCHERICHIA-COLI CRP

Citation
Lj. Runyenjanecky et al., A DIVERGENTLY TRANSCRIBED OPEN READING FRAME IS LOCATED UPSTREAM OF THE PSEUDOMONAS-AERUGINOSA VFR GENE, A HOMOLOG OF ESCHERICHIA-COLI CRP, Journal of bacteriology, 179(9), 1997, pp. 2802-2809
Citations number
45
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Microbiology
Journal title
ISSN journal
0021-9193
Volume
179
Issue
9
Year of publication
1997
Pages
2802 - 2809
Database
ISI
SICI code
0021-9193(1997)179:9<2802:ADTORF>2.0.ZU;2-8
Abstract
The Pseudomonas aeruginosa homolog of the Escherichia coli global tran scriptional regulator CRF (or CAP) was recently identified and designa ted VFr (S, E, H, West, A, K. Sample, and L, J, Runyen-Janecky, J, Bac teriol. 176:7532-7542, 1994), Nucleotide sequence analysis of tile reg ion 5' to vfr identified a 423-bp open reading frame (ORF), which was designated at orfX, The deduced amino acid sequence of ORFX was 53% id entical 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 p romoter in E. coli, a protein dth am appal ent molecular mass of appro ximately 18 kDa was produced, We constructed a chromosomal deletion of the region containing tile 5' end of orfX (orfX'), vfr and the 3' end of trpC (trpC') in P, aeruginosa strains PAO1 and PA103, The cloned v fr, 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 tran scription of orfX and vfr are controlled by the same mechanisms that c ontrol transcription of the region of the detergent ORF (dorf) and of crp, we compared the vfr-orfX and crp-dorf intergenic regions. Using S 1 nuclease analysis, we determined that the distance between the orfX and vfr transcriptional start sites was 105 bp, Thus, the P, aeruginos a orfX and vfr, promoters are arranged in a back-to-back orientation r ather than the face-to-face orientation of the dorf and clp promoters, A CRP recognition site is associated with each promoter in the clp-do d intergenic region; binding of the CRP-cyclic AMP complex to the stro nger dorf CRP recognition site activates transcription from the dorf p romoter and represses transcription from the crp promoter, The vfr-orf X intergenic region does not contain an obvious CRP recognition site, In addition, vfr was not required for transcription of orfX. Unlike th e dorf and crp mRNAs, the 5' ends of the orfX and vfr mRNAs were not c omplementary. Thus, the orfX mRNA cannot hybridize to tile 5' end of t he vfr mRNA to inhibit vfr transcription, a mechanism that has been po stulated to control np transcription in E. coli.