Discussion

R.capsulatus carries genes for aerobic and anaerobic metabolic modes，whose expression and regulation are determined by environmental oxygen tension.One of the features of this regulation is the coordinated expression of many groups of genes essential for LH and photochemical reactions［8-12，14，15］upon removal of oxygen.It is reasonable to suggest that a common mechanism for oxygen-specific regulation exists that is superimposed on the individual regulation of some of these genes.Although several hypotheses，such as redox potential control［22］or aporepressor-corepressor interactions［23］，have been proposed as mediators of the environmental signal，the exact mechanism of oxygen regulation has not been clarified.Recent results in Gramnegative bacteria，however，have shed some light on this question［21，24］.Yamamoto and Droffner［24］have hypothesized that the expression of genes required for anaerobic growth in Salmonella typhimurium depends on the supercoiling of chromosomal DNA，whereas the expression of genes required for aerobic growth depends on the relaxation of DNA.Kranz and Haselkorn［21］have shown by lac fusion that the anaerobic expression of the nif operon in R.capsulatus is controlled by the supercoiling of DNA.They also noticed a decrease in cellular pigmentation after gyrase inhibitor treatment.The effect of DNA topology on the transcription of rbcL and atpB in maize［25］and pea［26］and on hydrogenase synthesis in Bradyrhizobium japonica［27］has been recently reported using gyrase inhibitors.The results presented in this report，using the gyrase inhibitors novobiocin，coumermycin，nalidixic acid，and oxolinic acid in vivo，indicate that the regulation by oxygen of the mRNA levels of genes for the LH peptides，RC proteins，and RuP2Case in R.capsulatus is mediated by the supercoiling of DNA.

Superhelical tension may be involved in the regulation of expression of numerous prokaryotic and eukaryotic genes［24-28］.The topology of DNA is mainly determined by two enzymes，topoisomerase I and gyrase，in bacteria.DNA gyrase catalyzes the conversion of relaxed DNA to a superhelical form at the expense of ATP；topoisomerase I catalyzes the reverse reaction，converting supercoiled DNA to a more relaxed form［18，28，29］.There has been speculation that topoisomerase and gyrase might act at specific sites on the chromosome to effect the supercoiling of some genes more than for others［30］.DNA gyrase has been extensively studied in bacteria.It is made up of two subunits，A and B.Novobiocin and coumermycin are specific inhibitors of the bacterial gyrase B subunit，whereas nalidixic acid and oxolinic acid are inhibitors for the A subunit［18，28，29］.We have shown that the ATPdependent gyrase activity in a crude cell extract from R.capsulatus is strongly inhibited by these drugs.The measurement of a decrease in psoralen photoreactivity with the DNA in Bam HI-C，F and K fragments upon treatment of cells with novobiocin has provided direct evidence that novobiocin is effecting the superhelicity of genomic DNA in vivo（D.Cook，G.Armstrong，and J.E.H.，to be published elsewhere）.It is therefore our position that novobiocin and other gyrase inhibitors can be used as useful tools in the study of the superhelicity of the chromosome.

In this study we have shown that novobiocin and coumermycin have strong inhibitory effects on the levels of mRNA for Bchl biosynthesis，LH，and RC complexes and on their respective mRNAs.The levels of mRNA for LH and RC，however，were not reduced in two novobiocin-resistant mutants，RC232 and RC233，following novobiocin treatment（data not shown）.We have also demonstrated a significant inhibition of mRNA from rbcL and the ORFs Q and R.This inhibition occurs on a time scale of minutes and is observed only for oxygen-sensitive genes.The concentrations of the inhibitors used（50 μg/ml）in this study do not irreversibly damage R.capsulatus cells since cell growth is recovered after washing away the novobiocin.The reduction of mRNA for LH-Ⅰ and RC（L，M）has been observed after treatment with novobiocin even at concentrations as low as 5 or 10 μg/ml when the cell growth undergoes only a slight inhibition.We，therefore，believe that the inhibition of transcription upon addition of the antibiotics is a specific consequence of the selective repression of transcription of a large number of photosynthetic genes.

As we reported earlier［9］，the half-lives of mRNAs for LH-Ⅰ，LH-Ⅱ，and RC（L，M，H）in R.capsulatus are 19，24，and 10 min，respectively，when proflavin is used as a specific transcription inhibitor.The half-lives of respective mRNAs in the presence of novobiocin or coumermycin measured in this study agree with these values.The similarity of the kinetics of in vivo mRNA levels of this class of genes in response to the gyrase inhibitors and to proflavin provides strong evidence that the primary effect of the gyrase inhibitors is on the transcription.A second class of genes，whose mRNAs have more or less similar half-lives as the first class does upon addition of proflavin，displays vastly different kinetic response to gyrase inhibitors in comparison to proflavin（see Table 1）.This differential sensitivity of the anaerobic genes tested and constitutive genes to the gyrase inhibitors supports the hypothesis that the gyrase inhibitors selectively alter the transcription of specific genes，presumably by changing the DNA conformation at or near the respective gene.Two classes of genes，therefore，can be distinguished based on their response to gyrase inhibitors：strongly gyrasedependent anaerobic genes，including those for LH and RC complexes，Bchl biosynthetic enzymes，RuP2Case，and the Q and R genes；and genes less dependent on gyrase，such as genes for Crt biosynthesis，cytochromes，and the constitutive genes for LHI and RC complexes that are expressed under aerobic conditions.Thus，we propose that the change in oxygen levels induces changes in DNA topology，perhaps locally，which，in turn，lead to selective changes in transcription of genes.(https://www.daowen.com)

It is interesting to note that the oxygen-regulated and constitutive mRNAs for LH-Ⅰ and RC（L，M）have different sensitivities to novobiocin and coumermycin.The genes for LH-Ⅰ（β and a subunits）and RC（L and M subunits）plus an ORF，C2397，comprise a single operon designated as puf operon［10，11］.Two transcripts（0.5 and 2.6 kb）originate from the puf operon［10，11，13］.The 0.5-kb transcript codes for LH-Ⅰ（β and a）polypeptides，whereas the 2.6-kb transcript codes for LH-Ⅰ（β and α）as well as RC（L，M）and C2397.Two puf promoters，oxygen-regulated and constitu-tive，have been postulated recently in R.caspulatus on the basis of lac fusions.A simple model would be that the different promoters have different sensitivities to DNA topology.The alternative possibility would be a more complex interaction between the promoter，some upstream DNA regulatory sequence［31］，or regulatory protein and gyrase.The different sensitivities of promoters to DNA topology has been demonstrated in other organisms［28，31，32］.The Escherichia coli promoters for DNA gyrase subunits［32］and the Chlamydomonas chloroplast promoter for a dark-induced transcript A［31］are even stimulated by novobiocin.Recently，Menzel and Gellert have found that a DNA sequence 20 bp long is responsible for DNA relaxation-stimulated transcription of the E.coli gyrase gene［33］.The mechanism by which supercoiling of DNA may lead to enhanced transcription during photosynthetic growth in R.capsulatus is unknown.

We thank J.Willison for kindly providing the mutants RC223 and RC233，S.Kaplan for providing his clone pLIl0 as probe for rbcL，and N.Gabellini for providing us with her clone pRSFl for probing fbc.We thank M Gellert，N.Cozzarelli，and J.Wang for their valuable comments and D.Cook，G.Armstrong，M Alberti，F.Leach，and D.Burke-Aguero for their help during writing.Most of this work was supported by the Office of Basic Energy Sciences，Biological Energy Research Division of the Department of Energy under Contract DEACO30-76SF00098.The probes for the Crt genes and BChl genes were obtained from a project supported by National Institutes of Health Grant GM30786.The Biosearch Sam One was leased with Program Development Funds from the Director of Lawrence Berkeley Laboratory.