TRANSCRIPTIONAL ORGANIZATION OF THE TRANSREGULATORY LOCUS WHICH CONTROLS EXOENZYME-S SYNTHESIS IN PSEUDOMONAS-AERUGINOSA

The transcriptional organization of the exoenzyme S trans-regulatory l ocus was studied by using promoter fusion and transcriptional start si te mapping analyses. The 5' regions flanking open reading frames encod ing ExsC, ExsB, ExsA, and ExsD were cloned in both orientations into t he promoter vector pQF26, which contains the chloramphenicol acetyltra nsferase reporter gene (cat). CAT activity from each promoter fusion t ransformed into Pseudomonas aeruginosa and Escherichia coli was measur ed. The trans-regulatory locus promoters demonstrated low to undetecta ble CAT activity in E. coli regardless of the orientation of the DNA f ragment relative to the reporter gene. In P. aeruginosa two of the pro moter clones containing DNA located 5' of exsC (PC) and exsD (pD) demo nstrated significant CAT activity. Transcriptional initiation from pC and pD was dependent on the orientation of the DNA fragment, the inclu sion of a chelator in the growth medium, and the presence of a functio nal exsA gene. Transcriptional start sites were mapped for the pC and pD promoter regions by using total RNA isolated from P. aeruginosa str ains grown in medium including a chelator. Our data are consistent wit h an operon model for the transcriptional organization of the exoenzym e S trails-regulatory locus. In addition, ExsA appears to be involved in controlling transcriptional initiation from both the trans-regulato ry locus and a region located immediately downstream of the exsA gene.