Induction ofAnaerobic GeneExpression inRhodobacter capsulatus IsNotAccompanied bya LocalChangeinChromosomal Supercoiling asMeasured bya NovelAssay (1989)

Inthephotosynthetic bacterium Rhodobacter capsulatus, theenzyme DNA gyrasehasbeenimplicated inthe expression ofgenesforanaerobic metabolic processessuchasnitrogen fixation andphotosynthesis. Toassess theinvolvement ofsupercoiling inanaerobic geneexpression, we havedeveloped an assaytodetect invivo changes insuperhelicity ofsmall regions ofthebacterial chromosome. Ourmethod isbased on thepreferential intercalaction ofpsoralen into supercoiled versusrelaxed DNA,andwe havedemonstrated thesensitivity ofthe assayinvivo onchromosomal regions from2to10kiobases insize. Inexperiments withinhibitors ofgyrase, thereactivity ofindividual chromosomal fragments topsoralen decreases bya factor of1.8compared with DNAfromcontrol cultures. Weused our assaytodetermine whether there isachange insuperhelicity near the genescoding foressential proteins forphotosynthesis upon ashift fromrespiratory toanaerobic photosynthetic growth. Forcomparison, we also examined arestriction fragment containing the Jbcoperon,whichcodesfor thesubunits ofcytochrome bcl, a membrane-bound electron transport complex utilized during bothaerobic andanaerobic photosynthetic growth. During this shift ingrowth conditions, thepufandpuhmRNAs,coding forstructural polypeptides ofthephotosynthetic apparatus, underwent asix- toeightfold induction, while the amountofmRNA fromthejbclocus remained constant. However, we detected nochange inthesuperhelicity ofeither thegenesforphotosynthesis orthose forthebc, complex during this metabolic transition. Ourdata thusdonotsupport a modelinwhichstable changes inchromosomal superhelicity regulate anaerobic gene expression. We suggest instead thattherequirement forDNA gyraseinthetranscription ofphotosynthesis genesresults fromtherequirement fora swivel nearheavily transcribed regions ofthechromosome. A recentmodelforthegeneral control ofgeneexpression foraerobic andanaerobic metabolism haspostulated a role forDNA topology inregulating thetransition between metabolic modes(40). ThemodelproposesthatDNA gyrase,whichintroduces negative supercoils into thechromosome, isessential foranaerobic gene expression, while topoisomerase I,whichrelaxes thechromosome, isrequired forexpression ofgenesforaerobic metabolism. Thishypothesisisbased on theisolation ofobligate aerobic strains of Salmonella typhimurium whichwere showntohavemutations inoneofthegenesforDNA gyrase(gyrA orgyrB) and ofobligate anaerobes withmutations mapping tothegenefor topoisomerase I(topA) (40). Thismodelofagyrase-induced switch inmetabolic modeshasbeenextended toother facultative anaerobes aswellthrough thestudy oftheeffects ofgyraseinhibitors on expression ofessential genesfor anaerobic metabolism. InKlebsiella pneumoniae, expressionofthenifHDKgenes,whichcodefornitrogenase and nitrogenase reductase, isblocked bydrugs whichinhibit gyrase(17). InBradyrhizobium japonicum, expression of enzymesforhydrogen metabolism, an anaerobic process,is alsorepressed bydrugs whichtarget gyrase(22). Forthepurple photosynthetic bacterium Rhodobacter capsulatus, recent papershavedocumented theinhibition of anaerobic geneexpression bydrugs whichinhibit gyrase. Underanaerobic conditions inthelight, R. capsulatus develops an extensive photosynthetic membrane systemin whichpigment-protein complexes carry outlight-driven electron transport togenerate metabolic energy.R.capsu