Differential Inhibition by Novobiocin and Coumermy...
The superhelicity of DNA in bacteria is determined by a dynamic balance between gyrase，which supercoils the DNA，and topoisomerase I，which relaxes the DNA.Thus，inhibition of gyrase leads to a decrease in negative superhelicity［18］.If the expression of the photosynthetic genes in R.capsulatus is dependent on the supercoiling of the DNA，inhibitors of gyrase would repress transcription and reduce the levels of these gene products.We have measured the mRNA levels in vivo of various genes in R.capsulatus cells with or without novobiocin and coumermycin by dot or RNA transfer hybridization using probes for specific genes.To determine whether or not the inhibition is specific to anaerobic genes for photosynthesis，we have also examined the effect of gyrase inhibitors on the levels of mRNA for the cytochrome bc1 complex.The cytochrome bc1 consists of an FeS protein，a cytochrome b，and a cytochrome c1；the genes coding for these three subunits are on the fbc operon［19］.Figure 1（c）shows that the fbc operon is expressed in anaerobic and aerobic conditions.
Dot hybridization（see Figure 1）shows that the levels of the various mRNAs were constant in the absence of the drugs over the course of the 3-h incubation.The levels of various mRNAs，however，changed in different patterns after drug treatment.The response of the various RNA species to gyrase inhibition roughly falls into one of two broad classes：gyrase-dependent，oxygen-regulated RNAs；and gyrase-less dependent，constitutive RNAs.The first group includes the genes for the structural components of the photosynthetic apparatus（LH-Ⅰ，LH-Ⅱ，RC），BChl biosynthesis，and CO2 fixation（RuP2Case）.These mRNAs are immediately and dramatically reduced after the addition of gyrase inhibitors as shown in Figure 1（a）.The patterns of mRNA for LH-Ⅱ，RC-M，and RC-H（data not shown）were similar to LH-Ⅰ and RC-L，respectively.The half-lives of mRNAs for LH proteins following addition of the gyrase inhibitor were≈20 min，and those for RC proteins were about 10 min，a result similar to the half-lives observed following addition of proflavin as an inhibitor of transcription［9］.（See last column，Table 1.）The levels of mRNA from bcJ，E，D in Bam HI-E fragment（see Figure 1（a））and mRNA from rbc L and ORF Q，R（not shown）were also significantly reduced by the inhibitors within 30 min.
Figure 1 Dot blot hybridization of R.capsulatus RNA with gene probes for LH and RC subunits，Bchl and Crt biosynthetic enzymes，RuP2Case，the Q and the R products，and cytochrome bc1complex.（a）Time course of RNA levels in the presence of inhibitors.Novobiocin（50 μg/ml）（marked as C）and coumermycin（50 μg/mg）（marked as B）were added to photosynthetically growing cells.A represents RNA from the photosynthetic cells without the inhibitor treatment.The total RNA was extracted from an equal number of cells at various times（0，30，60，90，120，180 min）as indicated on the left.The RNA was denatured in formaldehyde，blotted onto a GeneScreen membrane，hybridized with32 P-labeled DNA probes，and exposed to X-ray film as described［8］.The probes are marked on the top of each panel.（b）Dot hybridization.The top three rows represent the RNA from photosynthetically growing cells（marked as PS）.The RNA was isolated from the cells 15 min after addition of coumermycin and novobiocin.The bottom three rows represent the RNA isolated from the cells 3 h after a shift from aerobic to anaerobic conditions（marked as O2→PS）.A，control；B，coumermycin（50 μg/ml）；C，novobiocin（50 μg/ml）.Most probes have been described［9，10］.The probe for Q is an M13 clone，ESL1，containing an EcoRI-SalI fragment in the ORF Q from 142 to 342 bp 5'to the pufB gene.The probe for R is an M13 clone，S357，containing an intenal Sau3A insert within the ORF R from 539 to 740 bp 5'to the pufB gene；it may partially overlap with the Q mRNA，based on S1 nuclease analysis.*ESL1 and S357 are within the EcoRI-Q fragment of pRPS404.The probe for the rbcL gene is a plasmid，pLI10，carrying a 0.3-kilobase（kb）heterologous DNA insert coding for form II RuP2 Case of R.sphaeroides［20］.The same size of major transcript of rcbL and its same response to oxygen and light were observed in R.capsulatus（unpublished data）as in R.sphaeroides［8］.
The probe for fbc genes is a plasmid，pRSFl，containing an insert coding for the entire fbc operon of R.capsulatus；only one 2.9-kb transcript from this operon was found［19］.（c）Dot hybridization of mRNAs from fbc and the Q and the R genes.The mRNA was isolated from cells grown under different oxygen and light conditions as described10］.The anaerobic photosynthetic conditions are designated as HL（high light），D（dark），and LL（low light）.The aerobic condition is designated as O2
Table 1 M13 phage，plasmids，or oligonucleotide used as probes
*The sequence addresses of these probes refer to positions on sequences first published by Youvan et al.［34］.
tSpecific for crtA.
The second class of genes that are less sensitive to gyrase inhibitors includes genes for cytochromes and genes for Crt biosynthetic enzymes typified by crtA，I in the BamHI-H fragment［see Figure 1（a）］and CrtE in the BamHI-M fragment（not shown）.The levels of mRNA from these genes and from the fbc genes decreased at a slower rate and to smaller extents，reaching half of their initial values after≈3 h［see Figure 1（a）］.
The reduction of the levels of mRNA from the genes coding for LH-Ⅰ，RC-L，and RC-H and from the genes rbcL，Q，R，and bch（in BamHI-E）under photosynthetic conditions can be detected by dot hybridization in as short a time as 15 min following the inhibitor treatment，as shown in another set of experiments［see top three rows of Figure 1（b），marked as PS］.The sensitivity of different classes of genes to the inhibitors has also been tested in the shift from aerobic to photosyn thetic conditions.The cells were grown aerobically and then shifted to anaerobic photosynthetic conditions in the presence or the absence of novobiocin and coumermycin.RNA was extracted 3 h after the shift.The formation of the photosynthetic membrane is induced in the cells during the transition period characterized by no growth at the early stage.The measurement of mRNA has also revealed the differential sensitivity of different classes of genes to the gyrase inhibitors as shown in the bottom three rows of Figure 1（b），marked as（O2→PS）.The effects of oxygen and light intensity on the mRNA levels from the Q and R genes are shown in Figure 1（c）.
The decrease in levels of mRNA for LH-Ⅰ and RC（L，M）has been observed by using RNA transfer［see Figure 2（a）］and dot［see Figure 2（b）］hybridization even when the novobiocin concentration is reduced to 5 and 10 μg/ml.However，the basal mRNA levels of the same genes from aerobic cells，which is much lower than that in photosynthetic cells，are not affected by novobiocin［see Figure 2（b）］.
Figure 2 RNA transfer（a）and dot（b）hybridization of R.capsulatus RNA with probes for LH-Ⅰ and RC-L.（a）Lanes 1，2，3，and 4，concentrations of novobiocin at 0，5，10，and 50 μg/ml，respectively，under anaerobic photosynthetic conditions.Total RNA was extracted from same number of cells 30 min after drug treatment，fractionated on agarose gel，transferred to GeneScreen，and hybrid-ized with probes for LH-Ⅰ and RC-L as in Figure 1［10］.The 0.5-kb and 2.6-kb transcripts code for LH-Ⅰ（β and α）proteins，whereas the 2.6-kb transcript also codes for RC（L，M）proteins.（b）Dots in rows 1，2，3，and 4，hybridizations to mRNA samples equivalent in source and conditions as lanes 1，2，3，and 4 in（a）.Dots in rows 5 and 6，concentrations of novobiocin at 0 and 10 μg/ml，respectively，under aerobic conditions
