Discussion

R.capsulatus and other facultative anaerobes such as Salmonella typhimurium and Escherichia coli regulate gene expression for anaerobic and aerobic metabolism depending on environmental conditions，but the events controlling these processes are not well understood.Buck and Ames［6］suggested that tRNA modification，which mediates attenuation of transcription，may play a role in this control in Salmonella species.Yamamoto and Droffner［33］found that activity of topoisomerase I，associated with relaxation of chromosomal DNA in Salmonella species，is necessary for the expression of genes required for aerobic growth，whereas activity of gyrase，associated with supercoiling of chromosomal DNA，is necessary for expression of genes required for anaerobic growth.In this study we have examined the levels and stability of mRNAs from a number of photosynthetic genes and investigated the effects of oxygen on the transcription and the turnover of mRNAs.

Our previous studies on oxygen and light effects［39］and others［3］showed that two transcripts which code for pigment-binding proteins，a short 0.5-kb mRNA from LH-Ⅰ and a longer 2.6-kb mRNA for LH-Ⅰ，RC-L and RC-M polypeptides，have different stabilities.In the present study a thorough investigation of the stabilities of mRNAs for light-harvesting（LH-Ⅰ，LH-Ⅱ）and reaction center（RC-L，RC-M，RC-H）proteins and for some bacteriochlorophyll and carotenoid biosynthetic enzymes has been conducted by inhibiting the initiation of transcription with proflavin and then measuring the decay of the mRNA by means of dot blots and Northern hybridizations.Proflavin is known to inhibit transcription by intercalating with DNA template，a mechanism similar to that of actinomycin D［7，32］.Actinomycin D has been shown to block transcription of the genes coding for photosynthetic membrane proteins in R.capsulatus［13］.The results of half-lives of mRNAs for LH-Ⅰ and RC proteins measured using proflavin as inhibitor of transcription in this study well agree with the previous results of R.capsulatus and R.sphaeroides with rifampin or actinomycin D as an inhibitor of transcription［3，13，40］.The half-lives of mRNAs for LH-Ⅱ and LH-Ⅰ were measured as 24 and 19 min，respectively；the half-lives of mRNAs for RC proteins were about 10 min.Therefore，the LH-Ⅱ mRNA is more stable than the LH-Ⅰ mRNA，and the LH-Ⅰ mRNA（0.5 kb）has greater stability than the RC mRNA under photosynthetic conditions.Comparison of the abundance of the LH-I and LH-Ⅱ mRNAs by using specific oligonucleotide probes of the same length indicates that the LH-H mRNA is more abundant than the LH-Ⅰ mRNA（data not shown），and the ratio of RC mRNA to LH-Ⅰ mRNA is about 1∶10 to 1∶20（［3，39，40］；this study）.The stoichiometry of the RC，LH-Ⅰ，and LH-Ⅱpolypeptides（RC-LH-Ⅰ-LH-Ⅱ，1∶10 to 20∶0 to 80）in the membrane［15］can well be explained by the difference in abundance of the respective mRNAs.The difference in the abundance of mRNAs，however，cannot be accounted for only by the difference in the mRNA decay rates（half-life RC-LH-Ⅰ-LH-Ⅱ，about 1∶2∶2.5）.The data thus suggest that the differential rates of transcription of the genes for these pigment-binding proteins must account for the difference in the abundance of their respective mRNAs and ultimately for the stoichiometry of the assembled protein complexes.

Kinetic studies of mRNA decay demonstrate that the levels of mRNA for LH，RC，and bacteriochlorophyll biosynthesis were immediately reduced after the PS cells were shifted to aerobic conditions.In contrast，the contents of LH，RC complexes，and bacteriochlorophyll and cell growth did not show significant changes during a corresponding short term（1 h）of oxygen shift（data not shown），indicating that the level of mRNAs is more sensitive to oxygen compared with their proteins.It is likely that oxygen inhibits transcription somehow and then，in certain cases，stimulates the degradation of mRNAs.Our data for this argument are twofold.First，for the majority of genes studied，including those for LH-Ⅰ，RC-L，RC-M，and RC-H，the decreases in mRNA levels upon exposure to oxygen are similar to the kinetics observed when proflavin was added to photosynthetic cells.This argues that oxygen probably inhibits the transcription of these genes.Second，in the case of LH-Ⅱ transcripts，the kinetics of the decrease in mRNA was significantly faster than that observed upon addition of proflavin based on both Northern（see Figure 6）and dot hybridization（see Figure 7）.In this case，oxygen probably both inhibits transcription and accelerates the degradation of LH-Ⅱ mRNA，although the mechanism is unknown.There are several possibilities，namely，that either the LH-Ⅱ mRNA becomes more susceptible than the LH-Ⅰ mRNA to RNase attack in the presence of oxygen，or that the LH-Ⅱ mRNA and LH-Ⅰ mRNAs are degraded by two types of RNase whose activities have a different sensitivity to oxygen.Alternatively，it also might be possible that oxygen strongly blocks the translation process for LH-Ⅱ，resulting in release of mRNA from the ribosome where it is attached and increasing its susceptibility to nuclease cleavage.(https://www.daowen.com)

The mRNAs for bacteriochlorophyll and carotenoid biosynthesis show a number of different responses to oxygen.For the Bam HI-E and-J fragments，the response to oxygen was similar to the LH and RC.The Bam HI-J fragment contains a heavily transcribed unidentified gene as well as crtF and crtE，which have previously been mapped［31，34，43］.The size of the unidentified transcript is 0.4 kb（［39］；unpublished data）.The response to oxygen of this fragment is due mainly to this small transcript and is similar to that of the genes for RC and LH polypeptides.The nature and function of this unidentified gene remain to be characterized.For the Bam HI-D probe，the level of mRNA decreased during the first 20 min after the shift and then increased.From this result we assume that there is an oxygen-induced gene（s）located in the BamHI-D fragment which is highly transcribed under steady-state aerobic conditions［39］.When the PS cells were shifted to oxygen，the genes for bacteriochlorophyll biosynthetic enzymes（bch F，bch K，bch H）were repressed，and their mRNAs were degraded at the early stage of aeration，and then unknown oxygenactivated gene（s）in the Bam HI-D fragment was induced.Since the Bam HI D fragment is 7 kb long，it is large enough to accomodate other genes in addition to bchF，bch K，and bch H，which have been only roughly mapped by genetic complementation［31，34］.

For the Bam HI-H fragment，the level of mRNA increased after the O2 shift.The response to oxygen in this fragment，which occurs in an opposite fashion compared with most other photosynthetic genes，is mainly due to the crtA gene.This conclusion is supported by the result that a transposon insertion in crtA abolishes this response.The crtA gene，which probably codes for an oxygenase responsible for the oxidation of spheroidene to spheroidenone［28］，has been mapped to this restriction fragment.Earlier studies［14，29］show that when anaerobic cultures of R.sphaeroides，a closely related species，are aerated，the carotenoid pigment spheroidene is enzymatically converted to spheroidenone.In18O-labeling experiments，the added keto group has been shown to be derived from molecular oxygen［30］.Absorption peaks of spheroidenone are red shifted by about 30 nm compared with spheroidenc［18，22］.Our spectroscopic studies of the pigments（see Figure 2 and Figure 3）reveal that the carotenoid composition rapidly changed when the PS cells were shifted to oxygen，based on absorption shifts to longer wavelengths between 400 and 500 nm.We have confirmed this result by thin-layer chromatography analysis.Under the photosynthetic conditions，the major carotenoid was found to be spheroidene；under aerobic conditions the major carotenoid was found to be spheroidenone.It has long been understood that carotenoids have two functions in photosynthetic bacteria.They serve both to harvest light energy［10］and to protect the photosynthetic apparatus from photooxidative damage which occurs in the presence of light and oxygen［11］.Here we suggest that the increase in expression of the crtA gene in the Bam HI-H fragment as well as other crt genes by oxygen represents one type of protection by carotenoids from the potentially damaging effects of oxygen.The oxidation of spheroidene to spheroidenone，catalyzed by an oxygen-activated oxygenase which is coded by the crtA gene，may scavenge oxygen which can cause damage to the photosynthetic apparatus.