Discussion

Recently a great deal of attention has been paid to the regulation of photosynthetic genes in R.capsulatus and R.sphaeroides［8，9，13，16-19］.In the present study we have used an improved method for RNA isolation［9］and have probed transcripts with DNA fragments specific to particular genes wherever possible.Hybridization of electrophoretically resolved RNAs，in combination with dot blot analysis，was used to measure mRNA quantity and sizes in order to obtain specific information about the regulation of the photosynthetic genes cluster in R.capsutatus in response to light and O2.

Recent studies of physical structure show that the RC subunits L，M，and H comprise a single complex in the intracytoplasmic membrane where the photochemical reaction takes place.Each RC is surrounded by 12—18 LH-Ⅰ（β and α subunits）complexes.The more abundant LH-Ⅱ（β and α subunits）complexes，located peripherally to LH-Ⅰ，harvest light energy and transfer exciton energy to LH-Ⅰ and then to the RC［28］.Bchl and Crt，absorbing light across the visible and near-infrared region，bind to LH-Ⅱ，LH-Ⅰ，and RC［6］.The location of genes coding for these polypeptides parallels the structural and functional association of these polypeptides［7，11］.The structural，functional，and genetic features of these complexes define the patterns of regulation of the genes coding for these polypeptides.Earlier studies on proteins indicated that LH-Ⅱ and LH-I/RC complexes were sequentially accumulated during intracytoplasmic membrane differentiation of R.capsulatus and that they were regulated independently by light and O2［6］.Here we provide evidence for the independent regulation of the genes for LH-Ⅰ and LH-Ⅱ，based on the results from a study of mRNA levels.We found that the level of LH-Ⅱ mRNA is more sensitive to change in O2 concentration，and that it varies more than that of LH-I mRNA.This result agrees with the measurement of absorption spectra of protein-pigment complexes in vivo.Ⅰn addition，we have evidence suggesting that the mRNA specific for LH-Ⅱ is degraded much faster than that of LH-Ⅰ，when the cells are shifted from photosynthetic growth conditions to high O2［21］.Since the gene for the third（14-kDa）subunit of LH-Ⅱ has not yet been isolated，we have no information about the regulation of this gene.

In this study，we found that the ORF C2397 was transcribed，and the level of its mRNA changed in response to light and O2 in a similar way to that of the genes for RC-L and RC-M.Since ORF C2397，236 base pairs long，is located immediately 3'to the gene for RC-M and there is a hairpin structure after the ORF C2397［10］，we suggest that the five structural genes for LH-Ⅰβ and α subunits，RC-L，RC-M，and C2397 comprise a single operon and that the transcription of this operon stops after the gene for C2397.Whether the gene for C2397 codes for a structural protein，or it functions in the assembly of the LH and RC，or it has some other functional role is not known.We assume that it must have some function related to the LH and RC.Although the genes for LH-Ⅰ（β，α），RC（L，M，H），and C2397 generally showed similar regulation by light and O2，some differential regulation was observed.The 2.6-kb mRNA for LH-Ⅰ（β，α），RC（L，M），and C2397 is more responsive to the change in O2 concentration than the 0.5-kb mRNA.Such differential expression of these two transcripts was also observed in R.sphaeroides［9］.

A close correlation between pigment（Bchl，Crt）and LH/RC biosynthesis has been reported in R.capsulatus（3，4，6）.We found a general coordinate regulation between genes for Bchl biosynthesis and the genes for LH and RC in response to light and O2.Some difference was observed，however.The mRNAs of bch H，-K，and-F（in Bam HI-D）and bchJ，-D，and-G（in Bam HI-E）genes increased less than mRNAs for LH and RC when the light intensity decreased.The levels of mRNAs of these bch genes decreased not only when the cells were shifted from photosynthetic growth condition to high O2（data not shown）but also under steady-state low O2 condition（see Figure 2，Table 2），indicating that the genes for Bchl biosynthesis respond more stringently to O2 concentration.The unexpected results were that under steady-state high O2，the level of mRNA homologous to the BamHI-D fragment increased.An O2-shift experiment also showed the induction of an O2-activated gene（data not shown）.We believe that there is an O2-activated gene（s）located in the BamHID fragment.

It is of interest that the genes for Crt biosynthesis are regulated differently from the other photosynthetic genes in response to light intensity and O2.We have found that under steady-state high O2，the level of mRNA from the BamHI-H fragment was relatively high（see Figure 2，Table 2），and it increased when the cells were shifted from anaerobic to aerobic conditions［21］.We have evidence suggesting that crtA in the Bam HI-H fragment is activated by O2.This gene is responsible for the oxidation of spheroidene to spheroidenone.Such regulation in response to O2 may be related to the function of Crt.It has been long known that Crt has two functions：harvesting light and protecting cells including the photosynthetic apparatus from photooxidative damage，which only occurs in the presence of both high light and O2［6］.Activation of crtA and other Crt genes by O2 may be part of the protective mechanism by which Crt scavenges O2 radicals in the cell［21］.Here we have also found another type of protective mechanism in response to high light.That is，in contrast to the decrease in the levels of mRNA for LH，RC，and Bchl biosynthetic enzymes，an increase in light intensity raised the levels of mRNA from a number of crt genes located in the BamHI-M，Bam HI-G，and BamHI-H fragments.Although the increase in transcription of crt genes in response to high light seems to be a plausible protective mechanism，we do not rule out the possibility that regulation may also occur posttranscriptionally，including the activation of Crt biosynthetic enzymes by light and O2.(https://www.daowen.com)

The 0.4-kb transcript from the Bam HI-J fragment also showed a response to high light，in the opposite fashion to the mRNAs for LH and RC［see Figure 4（e）］.It did not hybridize to either regions genetically mapped as crtF or crtE（G.A.Armstrong and J.E.H.，unpublished data）.The response of this transcript to O2 is very similar to that of mRNAs for LH and RC but is very different from that of mRNAs for Crt biosynthesis.On the other hand，transcript level increases in response to high light as do the mRNAs for Crt biosynthesis，except to a greater extent，but it is very different from that of mRNAs for LH and RC in this respect.The nature and function of this small transcript remain to be characterized.

Multiple transcripts for RC-H（1.4 and 1.2 kb）were detected.The 1.2-and 1.4-kb transcripts of the RC-H gene，which is 764 base pairs long［10］，are probably initiated from the middle of ORF F1696.We do not know whether they have different initiation sites or whether the 1.2-kb transcript is the product of processing of the 1.4-kb transcript.The relatively long 5'noncoding region（0.4—0.6 kb）may contain important regulatory sequences.

Assay for transcripts from all of the ORF putative genes near the LH and RC gene clusters in the BamHI-C-EcoRI-B and BamHI-F fragments resulted in the detection of mRNA from only C2397.We suggest that the putative genes in both fragments are either not expressed under the growth conditions used or that their mRNAs，if present，are below the limits of detection by our methods.

We thank D.Cook，F.Leach，G.Armstrong，and N.Stover for helpful discussions；F.Leach for kindly providing plasmids pFL103，-120，-205，and-227；and M.Alberti for technical assistance with phage DNA preparation and DNA sequencing.This work was supported by the Office of Basic Energy Sciences，Biological Energy Research Division of the Department of Energy under Contract DE-ACO30-76SFOOO98，by Department of Agriculture Grant 84CRCR11485，and by NationalInstitutes of Health Grant GM30786.The Biosearch Sam One was leased with Program Development Funds from the Director of Lawrence Berkeley Laboratory.