Induction of anaerobic gene expression in Rhodobacter capsulatus is not accompanied by a local change in chromosomal supercoiling as measured by a novel assay.
In the photosynthetic bacterium Rhodobacter capsulatus, the enzyme DNA gyrase has been implicated in the expression of genes for anaerobic metabolic processes such as nitrogen fixation and photosynthesis. To assess the involvement of supercoiling in anaerobic gene expression, we have developed an assay to detect in vivo changes in superhelicity of small regions of the bacterial chromosome. Our method is based on the preferential intercalaction of psoralen into supercoiled versus relaxed DNA, and we have demonstrated the sensitivity of the assay in vivo on chromosomal regions from 2 to 10 kilobases in size. In experiments with inhibitors of gyrase, the reactivity of individual chromosomal fragments to psoralen decreases by a factor of 1.8 compared with DNA from control cultures. We used our assay to determine whether there is a change in superhelicity near the genes coding for essential proteins for photosynthesis upon a shift from respiratory to anaerobic photosynthetic growth. For comparison, we also examined a restriction fragment containing the fbc operon, which codes for the subunits of cytochrome bc1, a membrane-bound electron transport complex utilized during both aerobic and anaerobic photosynthetic growth. During this shift in growth conditions, the puf and puh mRNAs, coding for structural polypeptides of the photosynthetic apparatus, underwent a six- to eightfold induction, while the amount of mRNA from the fbc locus remained constant. However, we detected no change in the superhelicity of either the genes for photosynthesis or those for the bc1 complex during this metabolic transition. Our data thus do not support a model in which stable changes in chromosomal superhelicity regulate anaerobic gene expression. We suggest instead that the requirement for DNA gyrase in the transcription of photosynthesis genes results from the requirement for a swivel near heavily transcribed regions of the chromosome.
Department of Chemistry, University of California, Berkeley 94720.
D N
DN
Cook
G A
GA
Armstrong
J E
JE
Hearst
D000332:Aerobiosis; D000693:Anaerobiosis; D015139:Blotting, Southern; D002876:Chromosomes, Bacterial / Q000648:ultrastructure; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification / Q000528:radiation effects; D004278:DNA, Superhelical / Q000235:genetics; D005798:Genes, Bacterial; D007700:Kinetics; D005810:Multigene Family; D010788:Photosynthesis; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification; D012241:Rhodopseudomonas / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D014158:Transcription, Genetic
D004269:DNA, Bacterial; D004278:DNA, Superhelical; D012333:RNA, Messenger