Transcriptional Analysis ofthePromoter Regionofthe Pseudomonas putida Branched-Chain KetoAcid

Branched-chain ketoacid dehydrogenase isamultienzyme complex produced byPseudomonas putida when itisgrowninaminimal mediumcontaining branched-chain aminoacids. A 1.87-kilobase (kb) DNAfragment wascloned andsequenced whichcontained 0.24kboftheElastructural geneand1.6kbofupstream DNA. There were854basepairs (bp) ofnoncoding DNAupstream ofbkd41, thefirst geneofthebkdoperon, and592 bpbetween thetranscriptional andtranslational starts. TheG+C content ofthenoncoding region was56.7% compared with65.2%forallthestructural genes oftheoperon. A partial openreading framewasfound on thestrand opposite that ofthebkdoperon beginning atbase774.Whenthebkdpromoter wascloned into the promoter probe vector pKT240, streptomycin resistance wasobtained inP.putida butnotEscherichia coli with thepromoter inbothorientations, whichindicates either that thebkdpromoter isbidirectional orthatthere aretwopromoters inthis region. A series ofordered deletions onbothsides oftheproposed site ofthestart oftranscription revealed that almost 700bpupstream ofthestart oftranslation wererequired forexpression. Streptomycin resistance wasalso obtained inanrpoNmutant ofP.putida KT2440containing constructs with theintact bkdpromoter, indicating that thebkdoperon doesnotrequire therpoNsigma factor forexpression. Another construct containing thebkdpromoter, bkdAl, andbkd42inpKT240wasusedtotransform P.putida JS113, amutant whichwasunable toproduce theElsubunits ofthebranched-chain ketoacid dehydrogenase. Inthis case, veryhighinducible expression ofthebkdoperon wasobtained.