Analysis of the promoter and regulatory sequences of an oxygen-regulated bch operon in Rhodobacter capsulatus by site-directed mutagenesis.

inthephotosynthetic bacterium Rhodobacter capsulatus isregulated bytheoxygenconcentration intheenvironment. However, themechanism ofthis regulation hasremained obscure. Inthis study, transcriptional fusions ofthebchCXYZpromoterregion tolacZwere usedtoidentify thepromoterandregulatory sequencesgoverning transcription ofthese bacteriochlorophyll biosynthesis genes.Thepromoterregion was identified invivobymakingdeletions and site-directed mutations. The50bpupstreamofthepromoterregion was showntoberequired forthe oxygen-dependent transcriptional regulation ofbchCXYZ. A previously described palindrome sequence isalso likely involved intheregulation. A gelmobility shift assay further defined theinteraction oftranscription regulators withthese DNA sequenceelements invitro anddemonstrated that a DNA-protein complex isformed atthis promoterregion. Since thesuggested promotersequence andthepalindrome sequence are found upstream ofseveral other bchandcrtoperons,these sequencesmay beresponsible forregulating oxygendependent pigment biosynthesis atthelevel oftranscription inR.capsulatus. Inaddition, these cis-acting DNA elements arenotfoundupstream ofpuh andpufoperons,whichencode thestructural polypeptides ofthe reaction center andlight-harvesting Icomplexes. Thisobservation supports themodelofdifferent regulatory mechanisms forthepigment biosynthesis enzymes andstructural polypeptides required fortheproduction of thephotosynthetic apparatus. Rhodobacter capsulatus isapurple nonsulfur bacterium whichcangrowbymultiple metabolic modes(12, 19). When theconcentration ofdissolved oxygenintheenvironment is low,thisbacterium formsanintracytoplasmic photosynthetic membrane that cantransduce light energy into chemical energy. Thephotosynthetic apparatus consists ofthree major pigment-protein complexes, including areaction centerandtwotypesoflight-harvesting complexes, IandII. Eachofthese complexes iscomposed ofseveral structural polypeptides thatbindbacteriochlorophyll andcarotenoid pigments. Thestructural polypeptides areencoded bythe puf,puh,andpucoperons, andthepigment biosynthesis enzymesareencoded bymultiple bchandcrtoperons. Oxygentension isamajor factor controlling thecoordinateexpression ofthese genes. Atleast partoftheoxygendependent regulation ofpigment biosynthesis occursatthe transcriptional level (6, 20,27). Although thegenesencoding thestructural polypeptides andpigment biosynthesis enzymesarebothtriggered byreduced oxygentension, two lines ofevidence suggest that theregulatory mechanisms for these twoclasses ofoperons maybedifferent. First, Armstrong etal.(1)havenotedthatseveral DNA sequence motifs, a&70-like sequence andapalindrome sequence, are conserved intheregions upstream frommanypigment biosynthesis genesbutnotupstream fromthepufandpuh operons. Second, mutations inanewlydiscovered transregulatory factor, regA, reduce anaerobic induction ofstructural polypeptide biosynthesis significantly butdonothave anysignificant deleterious effect onpigment geneexpression atthelevel oftranscription (20). Thisisthemostdirect