Localization oftheControl Region forExpression ofExotoxin A inPseudomonas aeruginosa

The2,760-base-pair (bp)PstI-EcoRI segmentofthechromosome ofPseudomonas aeruginosa PA103which carries theexotoxin A structural gene was expressed froman internal promoter whencloned ina pUC9 derivative andtransformed into anontoxigenic mutantofP.aeruginosa PAO1.Theunique terninal EcoRIsite was deleted, anda new EcoRIsite wassubstituted for aPvuIsite located 107bp5'tothetranscription initiation site. Following EcoRIcleavage, Bal31deletions weregenerated fromthis site, andanEcoRIlinker sequence, GGAATTCC,wasinserted inplace ofthedeleted DNA.Mutants withdeletions located 73bpormore upstream ofthetranscription initiation site retained normal expression, whereas inmutants withdeletions extending into theregion 69bporless upstream ofthis site, exotoxin synthesis was greatly reduced. FromaKpnIsite located 473bp3'tothetranscription initiation site, asimilar series ofBal31 deletion mutants weregenerated inwhich theinserted EcoRIlinker sequencewas located within thesame 72-bp region. Pairs ofmutants fromthetwo deletion series wereidentified inwhichtheEcoRIlinker was located atthesame sequence,andthese mutant pairs were ligated toderive a series ofconstructs inwhichtheEcoRIlinker sequenceGGAATTCC was substituted foran 8-bpsequencewithin the72-bpregion. Someofthese linker-substituted mutantsshowed greatly reduced exotoxin A synthesis. Theresults areconsistent with a binding site fora positive activator contiguous withthebinding site for an RNA polymerase.