Oxygen

In purple nonsulfur bacteria a decrease in oxygen tension is the signal for a switch of metabolism from aerobic mode to photosynthetic mode.Upon the removal of oxygen，the cells develop an extensive ICM，mainly consisting of LH-Ⅰ，LH-Ⅱ，and RC proteins.This process is accompanied by an immediate biosynthesis of Bchl（Drews and Oelze 1981；Chory et al.1984）.The single chain，however，between variation of oxygen tension in the medium and regulation of gene expression is unknown.

Recent studies（Clark et al.1984）using Southern hybridization have shown that the transcripts from the genes for LH-Ⅰ（β，α），RC-L and RC-M in R.capsulatus are greatly enhanced（fortyfold）in cells grown at low oxygen tension，whereas the transcripts from the genes coding for RC-H and Bchl biosynthetic enzymes respond to a lesser extent，and transcripts encoding several carotenoid biosynthetic enzymes seem unresponsive to changes in oxygen concentration.Lac fusion experiments support the observation that the rate of Bchl synthesis is regulated transcriptionally to a small degree（Biel and Marrs 1983）and that the crt genes are not directly regulated by oxygen tension（Biel and Marrs 1985）.The steady state concentration of mRNA specific for LH-Ⅱ and the level of rRNA also increase after lowering oxygen tension（Klug et al.1984）.Klug et al.（1985）have demonstrated that a maximum in the concentration of mRNA for LH-I and RC proteins occurs 30 minutes after induction under low oxygen concentration，while the maximal expression of mRNA for LH-Ⅱ proteins appears to lag the LH-Ⅰ/RC mRNA maximum by about 25 minutes.This differential expression of the genes for LH-Ⅰ and LH-Ⅱ agrees with the sequential accumulation of LH-Ⅰ and LH-Ⅱ complexes in the membrane of R.capsulalus under decreasing oxygen tension（Schumacher and Drews 1978）.These observations support the transcriptional control of the genes for LH and RC proteins.None of these studies，however，showed discrete mRNA species until Northern hybridization and S-1 nuclease analysis，using highly defined probes，were applied to R.capsulatus（Belasco et al.1985）and R.sphaeroides（Zhu and Kaplan 1985）.

Belasco et al.（1985）have observed two anaerobically induced transcripts of 0.5 kb and 2.7 kb from the puf operon of R.capsulatus.the 2.7 kb transcript coding for LH-Ⅰ（β，a），RC-L，and RC-M，is less stable than the 0.5 kb transcript coding for LH-Ⅰ（3，a）only.Similar observations were made in R.sphaeroides（Zhu and Kaplan 1985）.In addition，we have found that these two mRNAs（0.5 kb and 2.6 kb）respond differently to oxygen concentration and light intensity.The 2.6 kb mRNA is more sensitive to oxygen，whereas the 0.5 kb mRNA is more responsive to light intensity.The response of the 0.5 kb transcript to light intensity can be functionally rationalized，since it acts as a template for LH-Ⅰ antenna proteins.None of these mRNAs are significantly affected by the redox potential of the organic acids used in the medium.In contrast to the membrane proteins，the cellular level of mRNA specific for the large subunit of RuBPCase（form Ⅱ），a cytoplasmic protein and a key enzyme in carbon dioxide fixation，is markedly affected by both oxygen concentration and the redox potential of the substrate.We have found that the expression of the rbcL gene in R.sphaeroides is completely turned off by high oxygen tension under conditions where the mRNAs for LH and RC are still observable.

Figure 4　Northern hybridization of R.capsulatus RNA with cloned probes for LH-Ⅱ，LH-Ⅰ，RC-L，RC-H，and the BamHI-J fragment of pRPS404 containing crtF，crtE，and an unidentified gene.Cells were grown under different conditions.HL，high light（30 W/m2）D，a shift of HL-grown cells to the dark for one hour；LL，low light（5 W/m2）；O2，aerobic conditions；LO2，semiaerobic conditions.Adapted from Y.S.Zhu and J.E.Hearst（1987）.With permission

The regulation by oxygen of the mRNAs for LH-Ⅱ（β，α），LH-Ⅰ（β，α），and RC（-L，-M，-H）and for the Bchl and Crt biosynthetic enzymes in R.capsulatus has recently been investigated in our laboratory using Northern and dot hybridization（Zhu and Hearst 1986 and 1987a；Zhu et al.1986b）（see Figure 4）.We have demonstrated that while the small transcript（0.5 kb）codes only for LH-Ⅰ（β，α），the large transcript（2.6 kb）codes for LH-Ⅰ（β，α），RC-L，and RC-M，as well as an ORF C2397.These five genes thus comprise a single operon.The C2397 gene is contranscribed and coregulated with pufL and pufM in response to oxygen and light.It is 249 bp long and contains the information for a small hydrophobic protein.The function of the C2397 gene product is as yet unknown.However，it may be functionally associated with the LH-Ⅰ and RC genes，based on their tight cotranscription and coregulation as one operon.We have found that the gene for RC-H，puhA，has two transcripts of 1.2 kb and 1.4 kb，that are initiated within the ORF F1696 and whose ratio is light intensity dependent.The puh A gene is coordinately regulated with pufL and pufM in response to oxygen（Zhu and Hearst 1986），an observation different from that of Clark et al.（1984）.It is important to note that the proteins RC-L，RC-M，and RC-H are present in a virtual stoichiometry of 1∶1∶1 in the membrane.Recently，Donohue et al.（1986）made the similar observation in R.sphaeroides that the puhA gene produced 1.13 kb and 1.4 kb transcripts.The mRNA specifying the LH-Ⅱpolypeptides is 0.5 kb long and is more abundant and more sensitive to oxygen change，indicating that the genes for the LH-Ⅱ and LH-Ⅰ/RC are independently regulated（Zhu and Hearst 1986）.Furthermore，we have found that oxygen accelerates the degradation of the mRNA for LH-Ⅱ（Zhu et al.1986a）（see Figure 5）.In R.sphaeroides high oxygen tension（25%）also accelerates the degradation of the mRNAs for LH-Ⅰ，RCL，and RC-M（Zhu et al.1986b）.We hypothesize that oxygen represses the transcription of the photosynthetic genes and stimulates the degradation of their mRNAs.Such a combination of transcriptional and posttranscriptional regulations provides the cells with a mechanism to shift to a new metabolic mode quickly in response to oxygen.Recently，a similar result has been reported for the nif genes of Klebsiella pneumoniae（Collins et al.1986）.

Figure 5　Northen hybridization of R.capsulatus RNA and DNA probes specific for the LH-Ⅰ　polypeptides.The mRNAs are degraded when the photosynthetic cells are exposed to oxygen at various times.

One discrepancy between our observation and that of Clark et al.is that we have found only a threefold to fourfold or a sixfold increase in the mRNA levels for LH-I and RC，depending on light intensity，in contrast to the fortyfold increase they reported when comparing anaerobically and aerobically grown cells.A similar threefold to fourfold increase in mRNA levels has been observed in R.sphaeroides（Zhu and Kaplan 1985）.One argument for such discrepancy is the method utilized to normalize the results to the total RNA.If the rRNA undergoes a dramatic change under different conditions，it would indeed affect the results.Klug et al.（1984）reported that the total rRNA per cell of R.capsulatus increased sevenfold in the transition period of 140 minutes from high to low oxygen tensions.However，it is difficult to understand how they could have gotten sevenfold difference in rRNA amounts when they applied equal amounts of total RNA of which the majority was rRNA for hybridization.As a matter of fact，an early study by Gray（1967）demonstrated that the total RNA，DNA，and protein per cell of R.sphaeroides remain constant during the transtiton from high to low oxygen tesion.Our investigation in R.capsulatus confirms Gray's result.The total RNA amounts for the photosynthetic cells and for the aerobiccells are 2.0×10-2±0.3×10-2 and 2.1×10-2±0.4×10-2pg per cell，respectively，averaged over four experiments.These values are in reasonable agreement with the measurements by Gray（1967）.Since the growth rate is only slightly different under our anaerobic and aerobic conditions.The discrepancy of the rsults among different groups may be due to the experimental differences in methods，including the growth conditions，the accuracy of measurements，and the specificity of the probes used for hybridization.In our experiments we have used specific probes for LH and RC proteins.as well as Northern hybridization to show the discrete mRNA species，which provides direct information about specific mRNA size，quality，etc.Clark et al.used Southern hybridization and a large probe for the entire region，including DNA sequences other than the puf operon.We expect that the difference in methodology has caused many discrepancies between the results.We will not attempt to discuss all of them in this review.

The genes for pigment biosynthesis appear to be regulated by oxygen in somewhat different ways.While the levels of bch mRNA from the bch J，G，D，H，K，and F genes are lower under steady-state aerobic growth conditions than under anaerobic photosynthetic growth conditions，the extent of difference is less than that for the LH and RC mRNAs under those conditions.However，the levels of mRNA from bch genes decrease even more under steady-state low oxygen（semiaerobic）conditions than under aerobic conditions.When the R.capsulatus cells are shifted from anaerobic to high-oxygen conditions，the mRNAs from bch genes decrease rapidly during the first 20 minutes（Zhu et al.1986a）.These results were obtained using several BamHI fragments containing more than one bch gene for studies，as were earlier results（Biel and Marrs 1983；Clark et al.1984）.The same results，however，were observed recently when we used a series of probes specific for bchC and bchA for hybridization（Zhu and Hearst，unpublished observations）.

In contrast to the genes for LH，RC，and Bchl biosynthesis，crt genes respond to oxygen in an opposite fashion.We have found that the amount of mRNA from crt genes，relative to the amounts of mRNA for LH and RC proteins，actually increases during the shift from anaerobic to aerobic conditions（Zhu et al.1986a）（see Table 1）.Although the BamHI-H and the BamHI-J fragments contain several crt genes，the BamHI-M fragment is an internal region specific for crtE gene（Zhu and Hearst 1987）.On the whole the results are consistent.While most photosynthetic genes are turned off by oxygen，the crtA gene responsible for the oxidation of spheroidene to spheroidenone，is activated by oxygen，because a transposon mutation of crtA gene abolished the twofold increase in mRNA level by oxygen.Furthermore，the activation of crtA gene by oxygen and light was also observed when a subfragment of the BamHI-H-fragment specific for crtA gene was used as a hybridization probe（Zhu and Hearst，unpublished observations）.The reaction catalyzed by an oxygenase encoded by crtA may scavenge toxic singlet oxygen，which is formed in the photosynthetic apparatus.These results appear to disagree with Biel and Marrs'argument（1985）that the crt genes are not directly regulated by oxygen.

Table 1　Effects of Light and Oxygen on the mRNA Levels of Genes for LH，RC，Bchl，and Crt Biosynthesis in R.capsulatum

1 Cells were grown under various conditions（HL，D，and LL were anaerobic conditions）and mRNA levels were measured by dot hybridization using various specific probes.The relative amounts of mRNA are expressed as percentages of the mRNA levels for low-light conditions（set as 100%）.HL，high light；D，shift from HL to dark for one hour；LL，low light；O2，aerobic growth.