Proc. Natl. Acad. Sci. USAVol. 88, pp. 1765-1769, March 1991Biochemistry

Endonucleolytic degradation ofpuf mRNA in Rhodobactercapsulatus is influenced by oxygen

(mRNA stability/endonucleolytic cleavage/photosynthetic apparatus/oxygen tension)

GABRIELE KLUGZentrum fuer Molekulare Biologie Heidelberg, Im Neuenheimer Feld 282, D6900 Heidelberg, Federal Republic of Germany

Communicated by Stanley N. Cohen, November 19, 1990 (received for review July 19, 1990)

ABSTRACT The formation of pigment-protein complexesin facultatively photosynthetic bacteria is regulated by theoxygen tension in the culture. It is shown that the degradationof some mRNA species encoding components of the photosyn-thetic apparatus is affected by oxygen. ThepufmRNA segment,encoding the pigment-binding proteins of the reaction center,and the 0.5-kb puc mRNA species, encoding pigment-bindingproteins of the light-harvesting LHII antenna complex ofRhodobacter capsulatus were degraded more rapidly under highoxygen tension than under low oxygen tension. Studies on strainshaving deletions or insertions in the puf operon indicate thatrate-limiting endonucleolytic cleavage in the reaction centercoding region of the polycistronic pufmRNA was influenced bygrowth conditions. However, other mRNA segments, for whichexonucleolytic degradation was postulated to be rate-limiting,decayed with the same rate under either high or low oxygentension. Likewise, the degradation ofthepuhA mRNA, the cycAmRNA, and the cysflcmRNA was found to be independent oftheoxygen tension in the culture. The data strongly suggest thatspecific mRNA sequences or structures are responsible for theobserved oxygen effect on mRNA stability.

The oxygen partial pressure is the major factor regulating theformation of the photosynthetic apparatus in facultativelyphotosynthetic bacteria. The drop of oxygen tension below acertain threshold value results in increased formation ofpigments and pigment-binding proteins and the developmentof intracytoplasmic membrane vesicles, as well as the as-sembly of pigment-protein complexes (reviewed in ref. 1).The increased synthesis of pigment-binding proteins is cor-related with an increase in the cellular concentration ofmRNA encoding these proteins (2-5).

Recently, more intensive studies have been performed onthe oxygen regulation of the puf operon of Rhodobactercapsulatus. The polycistronic puf operon encodes the pig-ment-binding proteins of the light-harvesting (LH) I antennacomplex (genes pufB and pufA) and of the reaction center(RC; genes puJf and puJE) (6). In addition it includes twoopen reading frames pufQ and puJX (refs. 7 and 8; see Fig. 1).It has been shown by fusion of the lacZ gene to the pufpromoter region that the rate of transcription from the pufpromoter increases when the oxygen tension in the culture isreduced (9, 10). The presence of a specific DNA segmentupstream of the puf promoter is necessary for the oxygeneffect on transcription (9, 10).The expression of the pufoperon has also been shown to

be regulated on the posttranscriptional level. The stoichiom-etry ofRC and LHI complexes found in the membrane of R.capsulatus wild-type cells results at least in part from seg-mental differences in decay ofpufmRNA (11). A secondaryhairpin loop structure at the 3' end of the LHI-specific puf

mRNA segment is responsible for its higher stability and itsmolar excess over the less stable RC-specific segment of thepolycistronic pufmRNA (8, 11).

This work shows that posttranscriptional steps in the reg-ulation ofpufgene expression are influenced by oxygen. Datafrom various strains having deletions or insertions in the pufoperon suggest that the rate-limiting endonucleolytic cleavageof the RC-coding mRNA region is affected by oxygen. Deter-mination of the half-lives of different mRNA species encodingcomponents that are involved in photosynthetic energy con-version shows that the decay ofsome mRNAs is influenced bythe oxygen tension, whereas other mRNAs decay with thesame rate under either high or low oxygen.

MATERIALS AND METHODSBacterial Growth. The wild-type strains used in this study

were 37b4 (DSM 938) and B10 (12). The recipient strain forthe plasmids shown in Fig. 1 was ARC6 (14), which has thepuf operon removed from the chromosome. Rhodobacterstrains were grown in a malate-containing minimal medium(15). For growth of ARC6 containing any of the plasmidsshown in Fig. 1, tetracycline was added to the medium at 1.5mg/ml. Growth at high oxygen pressure was performed byincubating 100 ml of culture in 500-ml baffled flasks undervigorous shaking. The optical density of the cultures was0.3-0.45 (1 cm, 660 nm) at the time when rifampicin wasadded and the oxygen partial pressure was determined to be20o by using a Pt/Ag electrode (Bachofer); the doubling timewas 170 ± 5 min. Growth at low oxygen was performed byincubating 40 ml of culture in 50-ml flasks under gentleagitation. The oxygen partial pressure was determined to be1-2%, and the doubling time was 175 ± 6 min.RNA Isolation and Northern Blot Analysis. Total RNA was

isolated from R. capsulatus as described (16) after addition ofrifampicin to a final concentration of 200 ug/ml. Samples(6-10 ug per lane of total RNA) were electrophoresed in 2.2M formaldehyde/1% agarose gels. Transfer to nylon mem-brane (GeneScreen, DuPont) by capillary blot, prehybridiza-tion, and hybridization in 50%o formamide were carried outfollowing the manufacturer's recommendations. DNA frag-ments were labeled with 32P by nick-translation using the kitfrom Boehringer Mannheim. Each filter was hybridized toabout 50 ng of DNA (-4 x 106 cpm).Bands on the autoradiographs were quantified according to

Suissa (17). Where possible, the figures show the linearregression from which the chemical half-life was calculated,using the time point with highest optical density as startingpoint.

RESULTSDecay Rate of the 2.7-kb puJBALMX mRNA but Not of the

0.5-kb puffA mRNA Is Influenced by Oxygen. Cultures ofwild-type strain 37b4 or B10 or of strain ARC6(pTX35), which

Abbreviations: LH, light-harvesting; RC, reaction center.

1765

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Proc. Natl. Acad. Sci. USA 88 (1991)

Q BA L M X

%je .

deletions:BstEII - BstEII

Xv II - KpIinsertions into puf B:185 bpBstEII -Pstl

, - .1;: _,!r' AD .J G. - .l . -. . :- o0 15-I. 7 I._,'I

kb

?5 -

FIG. 1. A partial Xho II fragment from the R. capsulatus chro-mosome containing the complete puf operon was cloned into theBamHI site of a derivative of pTJS133 to generate plasmid pTX35(13). The arrows above the map indicate the 2.7-kilobase (kb) and0.5-kb mRNAs detected on Northern blots by using a 1.7-kb XbaI-Pst I DNA fragment as probe. The dot marks the location of theoxygen-dependent pufpromoter. The pufsequences present in otherplasmids used in this work are shown schematically. Bst, BstEII; K,Kpn I; P, Pst I; X, Xba I; Xho, Xho II; bp, base pairs.

carries the pufoperon on a plasmid, were grown under eitherhigh (20%o) or low (1-2%) oxygen partial pressure. In none ofthe experiments described here did the doubling time of theRhodobacter cells vary by >5% under the different growthconditions. The steady-state level of the 2.7-kb puf mRNAwas about 3.4-fold higher in low-oxygen cells than in high-oxygen cells, whereas the level of the 0.5-kb pufmRNA was2.7-fold higher under low oxygen (Table 1). Different kineticsof decay were observed for the 2.7-kb pufBALMX mRNA incultures grown under different oxygen tension when rifampi-cin was added to the cultures and the decay of puf-specificmRNA was monitored by Northern blot analysis. The aver-age half-life of this mRNA species was about 8 min inlow-oxygen cultures, whereas the half-life of RNA isolatedfrom high-oxygen cultures was only 3-3.5 min (Fig. 2, Table1). The differences in the half-lives for the LHI-specific,0.5-kbpujBA mRNA measured in high-oxygen (average fromseveral experiments, 30.0 min) or low-oxygen (average, 31.5min) cultures were close to the deviation found for severalmeasurements using the identical strain and culture condi-tions (Fig. 2, Table 1). However, the kinetics of decay of the0.5-kb mRNA under high oxygen did not perfectly match alogarithmic curve in all experiments. Especially when RNAwas isolated shortly after the addition of rifampicin (0, 3, 6,and 9 min, data not shown) to high-oxygen cultures, nonlog-arithmic kinetics in the initial phase of decay for the 0.5-kbpufmRNA was observed. The half-life given in Table 1 wascalculated with the assumption of logarithmic decay, and themean from several independent measurements is given.

Half-Life of the 2.7-kbpuJBALMX mRNA Increases After aDrop in Oxygen Tension in the Culture. To study the response

>1(n

-o

C)0

0

0

,01

,001

Aa 2.7 kb

x %

I.-., ., . ., u1 -

0 10 20 30 40 50 60 0 2time, min

20 40 60 80 100

FIG. 2. Ten micrograms of total RNA isolated from high-oxygencultures (doubling time, 169 min) and 6 Ag of RNA isolated fromlow-oxygen cultures (doubling time, 176 min) were run in a formal-dehyde/1% agarose gel, transferred to nylon membrane, and hy-bridized to a 1.7-kb Xba I-Pst I fragment from plasmid pTX35 (Fig.1) that is specific for the pufoperon. The optical density ofthe bandswas plotted against the time of RNA isolation. The half-lives calcu-lated from these blots were 3.5 min for the 2.7-kb mRNA (A) underhigh oxygen ( - -) and 8.0 min under low oxygen (-). For the0.5-kb mRNA (B) the half-life was 27 min in the high-oxygen culture(---) and 31 min in the low-oxygen culture (-).

of the degradation of the 2.7-kb puf mRNA to a suddenchange in growth conditions, aerobic cultures of wild-typestrain 37b4 were shifted to low oxygen tension. Rifampicinwas added to the cultures at various time points after the dropin oxygen tension, and the decay of the puf mRNA wasdetermined by Northern blotting. The half-life of the 2.7-kbpufBALMX mRNA increased significantly after the drop inoxygen tension and reached a maximum (til2 = 10 min) 60 minafter the decrease in oxygen (Fig. 3). When the cultures weregrown at low oxygen for longer times after the drop in oxygentension, the half-life was 8 min, identical to the value mea-sured for cultures constantly grown under low oxygen pres-sure (Table 1). There was no significant change in the half-life

Table 1. Half-lives and steady-state levels of various mRNA species of R. capsulatusmRNA half-life, min Ratio of mRNA

mRNA High Low steady-state levels,Operon Strain(s) size, kb oxygen oxygen low oxygen/high oxygenpuf ARC6(pTX35), 37b4, B10 2.7 3.5 ± 0.6 8.5 ± 0.7 3.42 ± 0.30puf ARC6(pTX35), 37b4, B10 0.5 30.0 ± 3.0 31.5 ± 3.5 2.66 ± 0.25

ARC6(pTX21), ARC6(pARB6)puf ARC6(pARB6) 1.3 18.0 ± 1.8 19.5 ± 1.3 2.10 ± 0.32puf ARC6(pTX21) 1.8 4.0 ± 0.5 4.0 ± 0.4 1.92 ± 0.23puf ARC6(pIB2) 2.9 2.5 ± 0.7 4.0 ± 0.5 2.91 ± 0.26

ARC6(pIB2) 0.7 Nonlogarithmic 11.0 ± 2.0 3.08 ± 0.25puc ARC6(pTX35), 37b4 0.5 18.0 ± 2.0 29.0 ± 1.2 3.30 ± 0.42puh ARC6(pTX35), ARC6(pTX21) 1.4 9.5 ± 0.9 10.5 ± 0.9 2.11 ± 0.18puh ARC6(pTX35), ARC6(pTX21) 1.1 9.0 ± 0.8 10.0 ± 1.0 1.84 ± 0.23Jbc ARC6(pTX35), 37b4 2.9 11.0 ± 1.0 9.5 ± 1.0 0.92 ± 0.09cycA ARC6(pTX35), 37b4 0.68 6.0 ± 0.7 7.0 ± 0.6 0.98 ± 1.10Half-lives and mRNA levels were determined by analysis of at least three different Northern blots. The mean of the

measurements (to the nearest 0.5 min) and the maximal deviation found in a single experiment are listed.

pTX35x

p&RB6

pTX21

s tP Xho Bst K

pIB2

B0.5kb

1 1 \

,1 s

1766 Biochemistry: Klug

0.5VW

Proc. Natl. Acad. Sci. USA 88 (1991) 1767

20 40time, min

FIG. 3. At time zero the oxygen tension was reduced from 20oto 1-2%. The growth rate of the cells remained constant for 90 minfollowing the shift (doubling time, 175 min). At 0, 15, 30, or 60 minafter the drop in oxygen tension, rifampicin was added and thehalf-lives of the 2.7- and 0.5-kb puf mRNAs were determined asdescribed in the legend to Fig. 2.

of the 0.5-kb pufBA mRNA after a shift-down of oxygentension in the culture (Fig. 3).Oxygen Affects the Rate of Endonucdeolytic Cleavage of the

RC-Specific puf mRNA Segment. Previous studies on thedegradation of the pufmRNA led to the hypothesis that thedecay of the 2.7-kbpufmR.NA is initiated by endonucleolyticcleavage within a 1.4-kb segment of the RC coding region(18). To test whether the oxygen-dependent degradation ofthe 2.7-kb pufmRNA was correlated with the presence ofthepostulated sites of initial endonucleolytic cleavage, the ki-netics of mRNA decay in strain ARC6(pARB6) was studied

(Fig. 1). This mutant has the 1.4-kb BstEII fragment removedfrom the RC coding region and shows a 20-min half-life of theremaining RC-specific mRNA as a result of the deletion (18).When RNA was isolated from high- or low-oxygen culturesof ARC6(pARB6), both puf-specific mRNAs detected onNorthern blots showed similar kinetics ofdecay, independentof the growth conditions (Fig. 4, Table 1). The steady-statelevel of the 1.3-kb puf mRNA was 2.1-fold higher in low-oxygen cells than in high-oxygen cells (Table 1).

In strain ARC6(pTX21) (Fig. 1), which has the 3' part of thepuf operon deleted, a decreased half-life (3.5 min) of theRC-specific pufmRNA segment has been described (18). Itwas suggested that the decrease in the half-life of the RC-specific mRNA was due to exonucleolytic degradation of thesegment caused by the removal of the two secondary struc-tures normally localized just downstream of the stop codonof pufX. When the stability of the puf mRNA of strainARC6(pTX21) was studied in cultures grown under eitherhigh or low oxygen, the same kinetics ofdecay were found forboth pufmRNA segments, independent ofgrowth conditions(Table 1).

Strain ARC6(pIB2) (Fig. 1) has a 185-bp BstEII-Pst Ifragment from the RC coding region, suggested to carry sitesfor rate-limiting endonucleolytic cleavage, inserted into thepufB gene, and the degradation of the resulting 0.7-kb and2.9-kb pufmRNAs is faster than the decay of the respective0.5-kb and 2.7-kb wild-type pufmRNA segments (18). Thesteady-state levels ofthe 0.7- and 2.9-kbpufmRNAs were 3.1and 2.9 times higher, respectively, in low-oxygen cells thanin high-oxygen cells (Table 1). The kinetics of decay of the0.7-kb mRNA in strain ARC6(pIB2) was not logarithmic inhigh-oxygen cultures (Fig. 4). It was clearly faster for theinitial 10 min after the addition of rifampicin to aerobiccultures than for the following time, when the 0.7-kb pufmRNA from aerobic and low-oxygen cultures showed thesame rate ofdecay corresponding to a half-life ofabout 13 min(Fig. 4). The 2.9-kb mRNA was degraded more rapidly in

high oxygen I low oxygen0 5 10 20 45 90 0 5 10 20 45 90 mmn

high oxygen U low oxygen0 3 6 9 20 601 0 3 6 9 20 60 min

kb

2 9-

0 -i-:.0.7- * *

*l

I A1.3 kb

u3 .1

f)O

a). I

0

r a. ----ITtUU

0 20 40 60 80 100 0 2time. min

'0 40 60 80 100.001 01

0 1 0 20 30 0 20 40 60 80time, min

FIG. 4. Ten micrograms of total RNA isolated from high-oxygen cultures and 6 pg of total RNA isolated from low-oxygen cultures fromstrains ARC6(pARB6) (I) and ARC6(pIB2) (II) at various time points after the addition ofrifampicin were analyzed on Northern blots as describedfor Fig. 2. The half-lives ofpuf-specific mRNAs were calculated from the decrease ofthe optical density ofthe RNA bands. For the 1.3-kb mRNAof strain ARC6(pARB6) (A) the half-life was 20 min under high oxygen tension and 18.5 min under low oxygen. The half-life for the 0.5-kb pufmRNA of ARC6(pAR"96) (B) was determined to be 31 min in the high-oxygen culture and 32 min in the low-oxygen culture. In ARC6(pIB2) the2.9-kb mRNA (C) decayed with a half-life of 2.5 min under low oxygen tension and 4 min under low oxygen. The 0.7-kb mRNA of ARC6(pIB2)(D) showed nonlogarithmic decay in the high-oxygen culture and a half-life of 13 min in the low-oxygen culture. - - -, RNA from high-oxygencultures; -, RNA from low-oxygen cultures.

kb

1 .3-

0.5 -

B0.5 kb

.11

Biochemistry: Klug

Proc. Natl. Acad. Sci. USA 88 (1991)

high-oxygen cultures (2.5 min) than in low-oxygen cultures(4.0 min) (Fig. 4, Table 1).

Effect of Oxygen on the Decay of Other mRNAs EncodingComponents Involved in Photosynthetic Energy Conversion.As shown above, the kinetics of decay of the RC-specificsegment of the puf mRNA was influenced by oxygen. Theaccelerated turnover ofthepufmRNA at high oxygen tensioncould be due to a general increase in RNase activities underhigh oxygen tension. Alternatively, only certain steps inmRNA degradation or specific endonucleases could be af-fected by oxygen. To decide between these possibilities thedegradation of mRNA encoding other components of thephotosynthetic apparatus was studied. The same Northernblot filters used in the experiments for measuring pufmRNAdecay were also hybridized to DNA fragments derived fromthe other genes.

In the first experiment a 4.0-kb BamHI fragment fromplasmid pRPS404 (19) containing the puhA gene was used asa probe. This DNA fragment encodes the non-pigment-binding protein of the RC (6) that shows only little increaseafter a drop in oxygen tension in the culture compared to LHproteins (4). Two RNAs, 1.1 kb and 1.4 kb in length, weredetected on Northern blots, as described earlier for a puhA-specific probe in Rhodobacter sphaeroides (18), and theirsteady-state level was about 2-fold higher under low oxygen(Table 1). The rates of decay of these mRNAs were verysimilar in cultures grown under high or low oxygen tension.The half-life of the 1.4-kb mRNA was determined to be 9.5 ±0.9 min in high-oxygen cultures and 10.5 ± 0.9 min in cellsgrown under low oxygen tension (Table 1). The half-life ofthe1.1-kb mRNA was 9.0 ± 0.8 min in cells grown under highoxygen pressure and 10.0 ± 1.0 min in low-oxygen cultures(Table 1).A 0.6-kb Apa I fragment derived from the puc operon of

strain 37b4 encodes the pigment-binding proteins of the LHIIcomplex (20). The formation of the LHII complex is stronglyinduced in low-oxygen cultures. When the 0.6-kb Apa Ifragment was hybridized to the RNA isolated from high- orlow-oxygen cultures of strains 37b4 or ARC6(pARB6), asignificant difference in the half-lives of a 0.5-kb mRNAdetected on Northern blots was found (Fig. 5, Table 1). Thehalf-life for this mRNA was 29 ± 1.2 min in low-oxygencultures but only 19.0 ± 2.0 min in high-oxygen cultures.The cytochrome Jbc complex and the cytochrome c2 pro-

tein are involved in photosynthetic and chemotrophic energyconversion. A 2.9-kb mRNA hybridized to a 1.3-kb EcoRIfragment derived from thefbc operon on plasmid p14-3 (21).The levels offbc-specific mRNA were identical under high orlow oxygen tension, and the half-life of this mRNA was 11.0

0

co

a

0

,01 --

0

Is 0A

-kb

1-

ni... . . . . ~~~~~~~~~~~..

-20 40 60 80 100 (time, min

B2.9 kb

10 20 30

FIG. 5. Decay of a 0.5-kb puc-specific mRNA (A) and a 2.9-kbmRNA transcribed from thejbc operon (B) determined from North-ern blot analysis as described for Fig. 1. The half-lives for the 0.5-kbpuc mRNA (A) were 17 min under high oxygen tension and 28 minunder low oxygen. The 2.9-kb Jbc-specific mRNA (B) showed ahalf-life of 10 min under high oxygen and 9.5 min under low oxygen.- - -, RNA from high-oxygen cultures; , RNA from low-oxygencultures.

± 1.0 min under high oxygen tension and 9.5 ± 1.0 min underlow oxygen tension (Table 1).A 1.7-kb Bgl II fragment from plasmid pB/115-5 that

contains 320 bp of the 3' sequence of the cycA gene (22) wasused to detect mRNA transcribed from cycA. A 0.6-kb majorRNA band and two minor bands of 0.52 and 0.42 kb weredetected, which showed the same intensity and turnover incells from high- or low-oxygen cultures (Table 1). The size ofthe mRNAs detected with the cycA-specific probe is not inaccordance with the cycA-specific mRNAs described for R.sphaeroides (23).The Northern blots used to determine the rates of decay of

the various mRNAs were also hybridized to radiolabeledplasmid pRC1 (24). This plasmid contains rRNA genes of R.capsulatus. Because the level of rRNA did not change for atleast 90 min after the addition of rifampicin, we could be surefrom this internal standard that equal amounts of total RNAfrom each time point had been present on the filter. In allRNA preparations from low-oxygen cultures we detected a0.45-kb band hybridizing to pRC1, which was not present inRNA isolated from aerobic cultures (data not shown).

DISCUSSIONThe formation of the photosynthetic apparatus in R. capsu-latus is regulated by the oxygen partial pressure in nonpho-totrophic cultures. A decrease in oxygen tension in culturesof R. capsulatus results in an increase ofmRNA specific forgenes involved in the formation of the photosynthetic appa-ratus (2-4, 25), and at least part ofthe increase inpufmRNAis due to an increased rate of transcription under low oxygentension (9, 10). The results presented here show that theoxygen tension in the culture also affects the kinetics ofdecayof certain mRNA segments encoding components of thephotosynthetic apparatus.The RC-specific segment of the pufmRNA was degraded

about twice as fast under high oxygen as under low oxygenin the culture. The steady-state level of puf mRNA inlow-oxygen cells was only about 3.5 times that in high-oxygen cells. This suggests that RNA degradation contrib-utes considerably to the increased puf mRNA levels inlow-oxygen cells. The effect of oxygen on the degradation ofthe RC-specific mRNA segment was dependent on the pres-ence of a 1.4-kb fragment within the RC coding region,suggested to contain sites at which rate-limiting endonucle-olytic cleavage occurs. Insertion of 185 bp of this fragmentinto the pufB segment resulted in a 0.7-kb mRNA thatdecayed more rapidly than the 0.5-kb mRNA of the wild-typepuf operon. Further, the 0.7-kb mRNA isolated during thefirst 20 min after the block of transcription from high-oxygencultures showed a much faster decay than the 0.7-kb mRNAisolated from low-oxygen cultures. The nonlogarithmic ki-netics of decay may result from the overlapping of thekinetics of multiple mechanisms involved in the degradationof the 0.7-kb mRNA.

In strain ARC6(pTX21) the two secondary structures at the3' end of the puf operon had been removed, resulting inaccelerated degradation ofthe RC-specificpufsegment, mostlikely due to exonucleases (17). In this strain the half-life ofthe RC-specific segment was not dependent on the oxygentension. These data suggest that oxygen does not affect theexonucleolytic degradation of pufmRNA segments.

In none of the strains was the degradation of the 0.5-kbmRNA markedly influenced by the oxygen tension. Thisimplies that the pufBA mRNA segment is degraded mainlyeither by exonucleases or by an endonucleolytic mechanismthat is different from the mechanism responsible for rate-limiting cleavage of the RC-specific pufmRNA segment. Inagreement with this hypothesis, the effect of ribosome cov-

1768 Biochemistry: Klug

Proc. Natl. Acad. Sci. USA 88 (1991) 1769

erage on mRNA degradation was found to be different for thepufBA and the pufLMX segments (30).An effect of cell growth conditions on the decay of puf

mRNA was not observed in earlier experiments (4, 8). In onework (4) the levels ofpufmRNA were quantified by dot blothybridizations that did not discriminate between the 2.7- and0.5-kb puf mRNAs. In other experiments involving thegrowth of cultures under high- or low-oxygen conditions,oxygen tension in the culture was not directly measured (8).

Earlier studies proved that the changes in the relativelevels of the 2.7-kb and the 0.5-kb pufmRNA species in thecells are followed by a change in the stoichiometry of the RCand LHI complexes (11). Therefore, regulation of the levelsof the pufmRNA segments is an important step in the overallregulation of the formation of the photosynthetic apparatus.In the study presented here, however, only chemical half-lives ofmRNA were determined and discussed. The effect ofoxygen on the biological half-lives ofthese mRNAs cannot beinvestigated in vivo by, for example, measuring the amountofmembrane-bound proteins (26), as the effects of oxygen onpufmRNA translation or on Puf protein incorporation into ortransport across the membrane are unknown. To establish anin vitro system to study the effect of oxygen on the biologicalhalf-life ofRhodobacter mRNA species, the characterizationand purification of enzymes involved in mRNA degradationand the identification of the cell components sensing andtriggering the oxygen tension in the culture will be necessary.

Effects ofgrowth conditions on the stability ofmRNA havealso been described for other bacterial systems. The stabilityof the Escherichia coli ompA mRNA was found to depend onthe rate of cell growth (27), and the half-life of the sdh mRNAin Bacillus subtilis varies in different phases of growth (28).In the experiments described here the doubling times of thecells were almost identical under high and low oxygentension. Therefore, the different rates of mRNA decay arenot related to growth rate. An oxygen-dependent mRNAdegradation was previously observed in Klebsiella pneumo-niae, where oxygen causes a faster decay of certain nif-specific mRNAs (29). It is not known for any of the previ-ously studied systems which steps in mRNA degradation areinfluenced by growth conditions, and it was not observedpreviously that the decay of certain segments of a polycis-tronic transcript was affected by growth conditions, whereasthat of other segments was not. The results presented in thiswork suggest that the step in pufmRNA degradation influ-enced by the oxygen tension in the culture is endonucleolyticcleavage within the 1.4-kb BstEII-BstEII segment of the RCcoding region. The rate of exonucleolytic degradation, sug-gested to be rate-limiting for the decay of the 1.8-kb pufmRNA in strain ARC6(pTX21) (18), is not influenced byoxygen. Likewise, oxygen does not affect the rate ofmRNAdegradation in R. capsulatus in general. That only certainmRNA species or segments show faster turnover under highoxygen tension strongly indicates that specific sequences orstructures need to be present in the mRNA molecule in orderto cause this effect. It is not known whether oxygen affectsendonucleolytic cleavage of mRNA in Rhodobacter in gen-eral or whether different endonucleases that respond tooxygen differently are present. Further information on thedecay of other mRNA species of R. capsulatus is necessaryalso to decide whether oxygen affects the concentration oractivity of ribonucleases or whether it changes ribosomeloading or mRNA structure.The accelerated decay under high oxygen that was ob-

served for thepufLMXmRNA segment but not for the pufBA

mRNA segment shows that bacteria can achieve differentialexpression of polycistronic genes in response to an externalfactor by using different mechanisms to degrade segments ofa polycistronic transcript. This step in gene expression maybe of similar importance for the regulation of the geneproducts of a polycistronic transcript as translational controlmechanisms.

I thank F. Daldal for plasmids p14-3 and pB/115-5, H. V. Tichy forplasmid pG3-EP4.5, and R. Liebetanz for the subcloned BamHIfragment from plasmid pRPS404. I gratefully acknowledge the tech-nical assistance of S. Jock and thank R. Dierstein for reading themanuscript. Part of the experiments were carried out in the labora-tory of S. N. Cohen, who supported the work financially and bymany valuable discussions. I also thank H. Schaller for financialsupport of this work.

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Biochemistry: Klug