Vol. 171, No. 5JOURNAL OF BACTERIOLOGY, May 1989, p. 2827-28340021-9193/89/052827-08$02.00/0Copyright © 1989, American Society for Microbiology

Microaerophilic Growth and Induction of the PhotosyntheticReaction Center in Rhodopseudomonas viridis

FRIDL S. LANGt AND DIETER OESTERHELT*

Max-Planck-Institut fur Biochemie, D-8033 Martinsried, Federal Republic of Germany

Received 11 October 1988/Accepted 26 January 1989

Rhodopseudomonas vinidis was grown in liquid culture at 30°C anaerobically in light (generation time, 13 h)and under microaerophilic growth conditions in the dark (generation time, 24 h). The bacterium could becloned at the same temperature anaerobically in light (1 week) and aerobically in the dark (3 to 4 weeks) ifoxygen was limited to 0.1%. Oxygen could not be replaced by dimethyl sulfoxide, potassium nitrate, or sodiumnitrite as a terminal electron acceptor. No growth was observed anaerobically in darkness or in the light whenair was present. A vai-iety of additional carbon sources were used to supplement the standard succinatemedium, but enhanced stationary-phase cell density was observed only with glucose. Conditions for inductionof the photosynthetic reaction center upon the change from microaerophilic to pliototrophic growth conditionswere investigated and optimized for a mutant functionally defective in phototrophic growth. R. viridisconsumed about 20-fold its cell volume of oxygen per hour during respiration. The MICs of ampicillin,kanamycin, streptomycin, tetracycline, l-methyl-3-nitro-1-nitrosoguanidine, and terbutryn were determined.

The purple bacterium Rhodopseudomonas viridis wasfound to contain a new type of bacteriochlorophyll (type b)in 1963 (10) and was characterized in more detail andclassified 3 years later (9). R. viridis is an anaerobic, photo-synthetic bacterium with microaerophilic growth capacity(28), in contrast to the aerobic species Rhodobacter capsu-latus and Rhodobacter sphaeroides, but no detailed investi-gation on the microaerophilic properties of R. viridis hasbeen reported until now.Under anaerobic conditions, R. viridis forms intracyto-

plasmic membranes, called thylakoids, which contain thephotosynthetic reaction center (8). The integral membraneprotein-reaction center complex catalyzing light-inducedelectron transport through the photosynthetic membrane hasbeen isolated and crystallized (18). By X-ray diffraction ofcrystals, an electron density map at a resolution of 3 A (0.3nm) could be calculated (6); this, together with elucidation ofthe primary structure of the reaction center by a combinationof protein and DNA sequencing methods (19, 20, 34),enabled for the first time a complete understanding of thestructure of a membrane protein complex at atomic resolu-tion.To study the structure-function relationship in the photo-

synthetic reaction center of R. viridis, site-specific mutagen-esis, homologous expression, and investigation of the spe-cifically modified gene products are ideal tools. To applythese methods, however, an understanding of photosyn-thetic and microaerophilic growth behavior and of the con-ditions for induction of the photosynthetic process is neces-sary. In addition, an effective gene transfer system and anappropriate reaction center-negative mutant for use as a hostmust be available. Here we report on microaerophilic growthconditions and induction of the reaction center independentof its functional role.

* Corresponding author.t Present address: Boehringer Mannheim GmbH, D-8132 Tutz-

ing, Federal Republic of Germany.

MATERIALS AND METHODSMaterials. R. viridis DSM 133 (ATCC 19567) was obtained

from H. Scheer. The cells were kept on agar plates at 4°C orin suspension with 50% glycerol at -20°C. Yeast extract andBacto-Agar were from Difco Laboratories (Detroit, Mich.);1-methyl-3-nitro-1-nitrosoguanidine (MNNG) was fromServa (Heidelberg, Federal Republic of Germany); ampicil-lin, kanamycin, tetracycline, streptomycin, sodium pyru-vate, maltose, fructose, and fluorescein isothiocyanate-la-beled goat antibodies directed against rabbit immunoglobulinG were from Sigma Chemical Co. (Munich, Federal Repub-lic of Germany); protein A-Sepharose CL-4B was fromPharmacia (Freiburg, Federal Republic of Germany); L-[35S]methionine (1,005 Cilmmol) was from Amersham-Buchler (Braunschweig, Federal Republic of Germany);complete and incomplete Freund adjuvants were from Be-hring (Marburg, Federal Republic ofGermany); and GasPak-BBL Microbiology Systems were from Becton Dickinsonand Co. (Paramus, N.J.). All other chemicals were fromMerck AG (Darmstadt, Federal Republic of Germany), andthe oxygen-nitrogen gas mixtures were obtained from Linde(Munich, Federal Republic of Germany).Growth of R. viridis in liquid culture. R. viridis was grown

in sodium succinate medium 27 (N medium) (4) with aninoculum of 5% of total volume from an early-stationary-phase phototrophic culture at 30°C. Cell densities weremeasured with a photometer (model 800-3; Klett Manufac-turing Inc., New York, N.Y.) equipped with a red filter andexpressed as Klett units. One Klett unit corresponds toabout 105 CFU/ml in a phototrophically grown R. viridis cellculture but to about 106 CFU/ml in a microaerophilicallygrown culture. The difference is due to the absorption ofbacteriochlorophyll in the phototrophic cultures.Anaerobic conditions for phototrophic growth were cre-

ated by bubbling nitrogen through the medium for 3 min afterinoculation. The cultures were stirred in white light at anirradiance of 5 to 10 mW/cm2.For microaerophilic growth, cells were suspended in 50 ml

of medium in 100-ml Erlenmeyer flasks plugged with cottonand grown in the dark on a rotary shaker at 50, 100, or 150rpm. Growth conditions were varied by addition of the

2827

2828 LANG AND OESTERHELT

following supplementary carbon sources: 0.6 or 1% glucose,1% fructose, 1% maltose, 0.5 or 1% sodium pyruvate, and1% sodium malate. Alternative electron acceptors duringanaerobic growth in the dark were tested with parallelcultures with the same compositions under microaerophilicgrowth conditions as a control: dimethyl sulfoxide (50 mM),potassium nitrate (0.01, 0.03, 0.1, 0.3, 1, and 3%), andsodium nitrite (0.01, 0.03, 0.1, 0.3, 1, and 3%). Furtherexperiments on growth behavior under standard microaero-philic conditions were done with media of different compo-

sition, including RAH (7) or RCV (32), Sistrom (26), andLuria-Bertani (LB) (21) media.Colony growth of R. viridis. Portions of 100 ,ul from R.

viridis cultures were plated after appropriate dilution on

N-medium agar plates containing 1.5% Bacto-Agar andincubated at 30°C. For phototrophic growth, the agar plateswere incubated in transparent Plexiglas boxes (22) in whichan anaerobic environment was generated by purging with N2and by use of the GasPak system. The plates were irradiatedthrough the agar during growth under light from 50-W whitelight halogen lamps (Osram) at 15 to 20 mW/cm2. Formicroaerophilic growth conditions, the Plexiglas boxes were

purged in the dark for 5 min every 24 h with mixtures ofoxygen and nitrogen (0.01, 0.05, 0.1, 0.3, 0.8, and 1%oxygen; relative accuracy, ±+-2%).As an alternative to growth on agar plates, cell suspen-

sions (100 ,ul) were mixed with 1% Bacto-Agar in 10 ml of Nmedium at 40°C and poured into sterile glass tubes, whichwere sealed with a cotton plug. The cultures were incubatedat 30°C under illumination with white light from 150-W lamps(Osram) at a distance of 50 cm (15 to 20 mW/cm2) or at 30°Cin darkness.

Induction of the photosynthetic reaction center. R. viridiswas grown microaerophilically in darkness at 30°C in 250 mlof N medium with or without 1% glucose in 500-ml Erlen-meyer flasks on a rotary shaker at 100 rpm. In the latelogarithmic growth phase, oxygen was removed by bubblingnitrogen through the cell suspension. The culture was thenincubated at 30°C with stirring with or without illuminationby white light (5 to 10 mW/cm2). Portions of 10 ml were

sampled for cell density and bacteriochlorophyll b determi-nation.

Bacteriochlorophyll b determination. Cells from the 10-mlsamples were collected by centrifugation, and the cell pelletwas weighed. Bacteriochlorophyll b was extracted with 2.0ml of methanol in the dark (25), and extinction of the clearedmethanol extract was measured at 785 nm. With a molarextinction coefficient of 96,000 liters mol-V cm-' (25), thebacteriochlorophyll b concentration in nanomoles per gramof cells (net weight) was calculated.

In vivo labeling of R. viridis cells with L-[355]methionine.Microaerophilically grown cells were induced by transfer tonitrogen atmosphere, and 20-ml samples taken every 24 h.The cells were pelleted and suspended in 3 ml of N mediumwithout yeast extract. After 2 h of anaerobic incubation at30°C under illumination (5 to 10 mW/cm2), during which timethe cells consumed residual nutrients, the cells were resus-

pended in 1 ml of N medium without yeast extract, and 12[Ci of L-[35SJmethionine (1,005 Ci/mmol) was added. Thesuspension was incubated for an additional 15 min under thesame conditions. The cells were then pelleted and suspendedin 250 pL1 of extraction buffer (0.2 M Tris hydrochloride [pH7.5], 1% sodium dodecyl sulfate [SDS], 40 mM dithiothrei-tol). After 30 min at 100°C, insoluble cell fragments were

separated by centrifugation. Trichloroacetic acid-insolublelabeled material was precipitated from duplicate 2-p.d sam-

ples, and [35S]methionine incorporation was determined (seebelow). From a volume of extract containing 30,000 cpm ofacid-insoluble 35S radioactivity, protein was precipitated byaddition of 7 volumes 25% trichloroacetic acid at 4°C. Thepellet was washed with 5% trichloroacetic acid and ethanol,solubilized in 24 pil of extraction buffer at 60°C, mixed with6 p.1 of sample buffer (0.5 M Tris hydrochloride [pH 7.8], 5%SDS, 50% glycerol, 5% 2-mercaptoethanol) and applied to anSDS-polyacrylamide gel.

Analysis of incorporated radioactivity and of labeled pro-teins. The incorporated 35S radioactivity was determined induplicate 2-,lI samples as described previously (2). Proteinmixtures were separated in SDS-polyacrylamide gels (14)and blotted to nitrocellulose as described previously (3).Fluorography of polyacrylamide gels was performed asdescribed elsewhere (16).

Determination of respiratory activity. Respiration of R.viridis cells from a microaerophilic culture in late logarithmicphase was measured manometrically as 02 consumption in aWarburg apparatus (31) at 30°C in the dark under normalatmosphere.MIC determinations. N medium (10 ml) containing increas-

ing amounts of antibiotics (1, 5, 10, 25, 50, and 100 ,ug/ml)was inoculated with 0.5 ml of a stationary phototrophicculture of R. viridis and incubated under microaerophilicconditions at 30°C in the dark. Cell densities were measuredafter 4 days. The herbicide terbutryn was mixed into agarplates at concentrations of 10-2, 10-3, 10-4, 10-5, and 10-6M, and growth was examined under phototrophic condi-tions.

Survival in the presence of mutagens. A cell suspension (10ml) in the logarithmic phase was irradiated at room temper-ature in an open petri dish by UV light from above (DesagaMinivis, 254 nm, 1 mW) (23). After 0, 5, 10, 20, 30, 45, 60, 90,120, 180, and 300 s of illumination, equal portions werediluted and mixed in sterile glass tubes with N mediumcontaining 1% Bacto-Agar. The tubes were incubated at30°C in the light. The chemical mutagen MNNG was addedto a final concentration of 10 ,ug/ml in 10 ml of the cellsuspension, and the tubes were incubated for 0, 2, 5, 10, 20,30, 45, 60, 90, 120, and 150 min at 30°C in the dark. Thesamples were then treated as described above.For mutagenesis, cells were treated for 35 s with UV light

or for 30 min with MNNG (10 p.g/ml) as described above.Both mutagenized cell suspensions were diluted, plated on Nmedium, and incubated with 0.1% oxygen at 30°C in thedark.

Immunological methods. Adult rabbits were injected withpartially solubilized reaction center crystals (0.5 mg) in 1.2%SDS-50% complete Freund adjuvant and reinjected after 4weeks with the same amount in 50% incomplete Freundadjuvant. After 10 more days, blood samples were taken andthe immunoglobulin G fraction was isolated from the serumby ammonium sulfate precipitation and affinity chromatog-raphy on protein A-Sepharose CL-4B (11). Antibody titerswere determined by dot blot analysis with the help of anitrocellulose-fixed reaction center. The reaction center-antibody complex was visualized with fluorescein isothiocy-anate-labeled goat antibodies against rabbit immunoglobulinG as described previously (27).

RESULTS

Growth of R. viridis in liquid culture. The standard growthmedium (N medium) for R. viridis contains 0.1% sodiumsuccinate and 0.1% yeast extract as carbon sources. R.

J. BACTERIOL.

PHOTOSYNTHETIC REACTION CENTER IN R. VIRIDIS

TABLE 1. Growth of R. viridis in liquid culture under conditionsof various combinations of light and 02 at 30°C

Condition Designation Growth Generation time (h)

+ Light, -02 Photosynthetic Yes 13 (5 to 10 mW/cm2)+ Light, +02 No-Light, -02 Fermentative No-Light, +02 Microaerophilic Yes 39 (50 rpm)

24 (100 rpm)44 (150 rpm)

viridis shows photosynthesis in the light under nitrogen andmicroaerophilic growth in darkness under low oxygen ten-sions. Therefore, the different requirements for growth inlight and darkness in the presence and absence of oxygenwere investigated and optimized (Table 1; Fig. 1). Optimalconditions for growth of R. viridis are a temperature of 25 to30°C and a pH of 6.5 to 7.0 (9). Under photosynthetic growthconditions, the bacteriochlorophyll b concentration, whichreflects the sum of the concentrations of light-harvestingcomplexes and reaction centers, is inversely proportional tolight intensity. Under microaerophilic growth conditions, theconcentration of oxygen in the medium determines thegrowth rate, with high concentrations inhibiting growth andlow concentrations limiting respiration. Under standardgrowth conditions in Erlenmeyer flasks, the oxygen supplycan best be regulated by rotary frequency, and this led tooptimized (100 rpm) growth conditions for R. viridis DSM133. Strain DSM 134, described originally as an oxygen-tolerant strain, showed growth behavior identical to that ofDSM 133 and was not used further. Generation times andcell densities in the stationary phase were reproduced with acoefficient of variation of less than 5%. In any case, only

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cultures with cell concentrations of higher than 106 cells perml after inoculation grew under microaerophilic conditions(Fig. 2a), and this per se prevented cloning of R. viridisunder aerobic conditions. Attempts to stimulate microaero-philic growth of R. viridis in liquid culture by use ofadditional carbon sources or alternative media normallyused for other photosynthetic bacteria failed. Inhibition ofgrowth occurred in N medium with 0.5 or 1% sodiumpyruvate, with 1% malate, and in LB medium. Growth wasreduced in N medium with 1% fructose or 1% maltose. Onlythe addition of 0.6 or 1% glucose to N medium increased thestationary-phase cell density (growth at 100 rpm) from 65 toabout 90 Klett units but did not reduce the generation time.A spontaneous mutation of R. viridis to a phenotype capableof aerobic growth has been reported for another R. viridisstrain (24) but was not observed for R. viridis DSM 133.Furthermore, fermentation of organic substrates does notallow substantial growth ofR. viridis (29), and illumination inthe presence of oxygen inactivates photosynthetic pigmentsby photooxidation and inhibits growth. Oxygen has alsobeen shown to prevent the formation of thylakoid mem-branes in R. viridis (33).

Cloning of R. viridis. Since cloning of R. viridis underphotosynthetic and microaerophilic growth conditions is aprerequisite for gene transfer, mutagenesis, and complemen-tation experiments, single cells were exposed to controlledoxygen conditions by one of two methods. R. viridis cellswere mixed with agar, packed into glass tubes, and thenincubated either in the dark or in light. In the dark, a lightyellow band forms about 5 mm distant from the agar surface,indicating the area of tolerable oxygen concentration. Abovethis area, because of high oxygen concentration, and belowit, because of low oxygen concentration and lack of fermen-

.5 1 1.5 2 2.5 3 3.5 4

Time (days)FIG. 1. Photosynthetic and microaerophilic growth of R. viridis in liquid culture. Symbols: 0, phototrophic growth in stirred cultures at

30°C in the absence of 02, illumination with white light at an incident intensity of 5 to 10 mW/cm2; A, microaerophilic growth in darkness at30°C with 50 ml of medium in 100-ml Erlenmeyer flasks at 100 rpm.

2829VOL. 171, 1989

2830 LANG AND OESTERHELT

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FIG. 2. Dependence of microaerophilic growth of R. viridis on the number of cells used for inoculation and growth of R. viridis in agar.(a) Microaerophilic growth at 30°C as a function of cell concentration; (b) growth as inclusion colonies at 30°C (A, without light after 4 weeks;B, with light [5 to 10 mW/cm2] after 10 days).

tation, no growth occurs. The illuminated tubes showed, bythe green color of the colonies starting about 6 mm below theagar surface, the anaerobic zone where photosyntheticgrowth could occur (Fig. 2b). This growth method was alsoused for viable count assays of R. viridis. For cloning of R.viridis under photosynthetic or microaerophilic conditions,cells were streaked on agar plates and incubated either in anilluminated anaerobic chamber or under microaerophilicgrowth conditions by exposure in the same chamber indarkness to define mixtures of oxygen (0.05 to 0.30%) andnitrogen. Under optimal conditions of microaerophilicgrowth (0.1% oxygen), single cells grew into colonies inabout 4 weeks; under photosynthetic growth conditions,colonies formed in about 1 week.

Alternative electron acceptors. Since microaerophilicgrowth of R. viridis colonies is time consuming and requiresmechanical constructions, alternative electron acceptors tooxygen were investigated. Neither dimethyl sulfoxide (35),potassium nitrate, nor sodium nitrite mediated anaerobicgrowth in the dark. In parallel experiments, the toxicity ofthese compounds was tested under microaerophilic growthconditions; sodium nitrite at any concentration and potas-sium nitrate at concentrations of more than 1% were foundto be toxic.

Induction of the photosynthetic reaction center. For geneticmanipulation of R. viridis and specific mutagenesis of itsreaction center, induction of the photosynthetic mechanismindependent of its functional state is essential but difficult.For the purple bacterium Rhodobacter capsillatlus, the in-duction signal was shown to be the drop in oxygen levelbelow a certain threshold (38). The problem with R. viridis isthat if a functionless reaction center is to be induced by thecell, the requirement to turn off oxygen in the dark leaves the

cell with no metabolic energy, neither from light nor fromoxygen or fermentation; therefore, protein synthesis de-pends on endogenous energy reserves. In a first experiment,a microaerophilically grown wild-type culture from the latelogarithmic phase was illuminated under anaerobic condi-tions. This stopped growth but induced the synthesis ofphotosynthetic proteins, as seen by the fact that the bacte-riochlorophyll concentration in the cells increased until itreached the level in a photosynthetic culture. Subsequently,cell density also increased to the value of such a culture (Fig.3). The same experiment carried out in darkness did notreveal a change in bacteriochlorophyll b concentration orcell density, as expected on the basis of the lack of an energysource (Fig. 3).As mentioned above, 1% glucose added to N medium had

some stimulating effect, and this proved to be the solution tothe problem. Fermentative metabolism in R. viridis does notallow growth but produces energy at a low level (29, 30).Addition of glucose stimulated the production of energyenough to drive protein synthesis, seen as the induction ofthe photosynthetic process upon addition of 1% glucose tothe medium (Fig. 3). Production of metabolic energy, how-ever, was not high enough to allow further growth afterinduction of the photosynthetic process, as in the experi-ment carried out under illumination. To correlate the mea-surable increase of bacteriochlorophyll concentration andsynthesis of photosynthetic reaction center subunits, in vivolabeling experiments with R. viridis cells upon inductionunder anaerobic illumination were carried out with L-[35S]methionine. Starting at about 24 h after removal ofoxygen from the cell suspension, a band correspondingspecifically to the H subunit became labeled with [35S]methionine, and maximum incorporation occurred 3 to 4

J. BACTERIOL.

PHOTOSYNTHETIC REACTION CENTER IN R. VIRIDIS

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Time (days)FIG. 3. Induction of the photosynthetic process in R. viridis. Microaerophilic cultures were grown at 30°C with shaking at 100 rpm for 4

days. The reaction centers were induced by replacement of oxygen with nitrogen. Bacteriochlorophyll b concentration (filled symbols) andturbidity (open symbols) were measured. Symbols: +, phototrophic growth (see Fig. 1); 0, *, induction with white light (5 to 10 mW/cm2)in N medium; A, A, induction in the dark in N medium; 0, 0, induction in the dark in N medium-1% glucose.

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FIG. 4. In vivo labeling of R. viridis cells with L-['5S]methionine during induction of the photosynthetic reaction center, with onset ofillumination on the day 4 of growth. (a) Growth, total "5S incorporation, and bacteriochlorophyll b concentration in cells; (b) autoradiographyof SDS-polyacrylamide gel electrophoresis of samples from cells grown for 10 days and induced on day 4 (N2, light). 35S labeling of sampleswas for 15 min. H, Position of the reaction center subunit H (lane 4, microaerophilic control; 30,000 cpm of 35S per lane).

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VOL. 171, 1989 2831

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2832 LANG AND OESTERHELT

TABLE 2. Cell poison resistances of R. viridis

Cell poison MIC Concn'

Ampicillin 25 ,ug/ml 100 ,ug/mlChloramphenicol 50 ,ug/mlKanamycin 5 ,ug/ml 20 ,ug/mlStreptomycin 4 ,ug/mI 50 jig/mlTetracycline 4 ,ug/ml 20 ,ug/mlMNNG 20 ,ug/mlTerbutryn 1o-4 M lo-4 M

a Concentration used in selection experiments (15).

days after induction began (Fig. 4b). Protein synthesis activ-ity started to decrease at day 4, apparently because station-ary levels of reaction centers and accessory pigments nec-essary for effective photosynthesis were reached.These results were confirmed by measuring the total

incorporation of [35S]methionine per gram of cells afterinduction of the reaction center (Fig. 4a). Again, at day 4after induction, maximum radioactive labeling occurred be-fore the biosynthetic activity decreased to the level of astandard microaerophilic culture. The increase in methio-nine incorporation resulted only from synthesis of proteinsof the photosynthetic unit.The labeled band on the SDS-gel (Fig. 4b) was identified as

the H subunit by Western blotting (immunoblotting) (notshown). For this procedure, electrophoretically separatedcell proteins were transferred to nitrocellulose and incubatedwith rabbit antibodies directed against reaction centers. Therabbit antibodies were then visualized by using fluoresceinisothiocyanate-labeled goat antibodies against rabbit immu-noglobulin G. The finding that the rabbit antibodies recog-nized mainly the H subunit is explained by the fact that withcrystalline reaction centers as antigens, most of the antibod-ies produced are against the hydrophilic H subunit andcytochrome c. In addition, the L and M subunits of thereaction center together with cytochrome c usually forminsoluble aggregates when heated and are lost during samplepreparation.

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TABLE 3. Statistical mutagenesis of R. siridis withUV light and MNNG

Resultant % Mutagenesis Phenotype'mutant type UV MNNG Ind Phot

1 35 50 - -2 60 40 + +3 5 10 + -

Ind, Induction of photosynthetic reaction center without 02 in darkness;Phot, photosynthetic growth.

Determination of respiratory activity. Since respirationcould be involved in the phenomenon of microaerophilicity,the respiratory activity of R. viridis was measured in aWarburg apparatus. Microaerophilically cultivated R. viridiscells showed normal respiratory activity, consuming about20-fold their cell volume per hour.

Sensitivities to antibiotics and mutagens. Several antibioticswere tested for inhibition of R. viridis growth (Table 2). Theeffective antibiotics were those that could be inactivated byproducts of resistance genes that are frequently used invector constructions. The cells were also sensitive to themutagen MNNG and the herbicide terbutryn (Table 2).For isolation of R. viridis mutants unable to express the

photosynthetic reaction center, statistical mutagenesis withUV light (254 nm) and MNNG at a concentration of 10 p.g/mlwas used. Figure 5 shows the survival curves of R. viridiscells upon treatment with these mutagens.

Statistical mutagenesis. Mutagenesis with UV light andMNNG produced three types of phenotypically distinctmutants (Table 3). Type 1 were almost colorless coloniesthat grew in N medium-1% glucose and remained colorlessupon removal of oxygen in the dark, i.e., could not inducebactefiochlorophyll b synthesis and therefore functionalreaction centers. Although these mutants were phototrophicgrowth negative (Phot-), they were useless for experimentswith mutagenized reaction center genes. These experimentsrequired an intact biosynthetic pathway of bacteriochloro-phyll and proper incorporation into the reaction center

10

TimeFIG. 5. Survival curves of R. viridis after UV light and MNNG treatment. Proportion of surviving cells (as percentage of untreated control

cells) after exposure to UV light and MNNG is plotted versus incubation time. Symbols: 0, illumination with UV light (254 nm, 1 mW/cm2)at room temperature (time in seconds); K, incubation with MNNG (10 jig/ml) at 30°C in the dark (time in minutes).

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PHOTOSYNTHETIC REACTION CENTER IN R. VIRIDIS

complex. Type 2 colonies also were nearly colorless whengrown on N medium-1% glucose, but they displayed induc-ible bacteriochlorophyll b biosynthesis and were Phot+.These were either wild-type cells or mutants with a defectnot in the photosynthetic system. Type 3 colonies were alsoalmost colorless when grown on N medium-1% glucose anddisplayed inducible bacteriochlorophyll b synthesis but werenot able to grow phototrophically and thus were Phot-mutants. This group included reaction center-negative mu-tants and light-harvesting complex-defective mutants thathad a dramatic increase in generation time and thereforecould be considered Phot-. Assembly mutants of the reac-tion center would also be expected to fall into this category.Therefore, screening of this group for the appropriate reac-tion center-negative cells is necessary.

DISCUSSION

R. viridis can be cloned in the dark only with oxygenconcentrations of 0.05 to 0.3%. The biochemical reason forthis microaerophilic property remains unknown. Limitationon the respiratory rate could be excluded because therespiratory rate was found to be comparable to that ofaerobic bacteria. Another explanation not investigated herecould involve enhanced sensitivity to toxic forms of oxygen,such as superoxide, hydrogen peroxide, or hydroxyl radi-cals, caused by either production or slowed enzymaticdegradation of these species. Other possibilities are theoccurrence of oxygen-sensitive proteins, such as cy-tochromes, flavoproteins, hydrogenases, or nitrogenases(13). A most unlikely possibility is the dependence of growthon oxygen-sensitive substrates.

Microaerophilic growth suppresses in R. viridis, as inRhodobacter capsulatus (37), the synthesis of bacteriochlo-rophyll b, the reaction center, soluble cytochrome c2 and thebe complex (12). A soluble cytochrome of the b type,however, which does not occur in phototrophically growncells could be identified (12). As mentioned above, lack ofthe bec complex does not prevent effective respiration;therefore, one must assume that in R. viridis as in Rhodobac-ter capsulatus, a branched pathway makes respiration inde-pendent of the active bec complex and also of cytochromec2. Such a branched pathway, which uses terminal oxidasesthat transfer electrons directly from ubiquinone to oxygen,was first discussed 1973 (17) and summarized 1988 (5) forRhodobacter capsulatus. In conclusion, the be1 complexseems to be obligatory for photosynthetic growth but ap-pears not to be essential for respiration in R. viridis.

All possible aerobic and anaerobic combinations of lightand dark growth conditions for R. viridis were investigatedduring this work. No significant fermentative growth withoutlight and oxygen was observed (29). R. viridis, in contrast toRhodobacter capsulatus, was not able to use dimethylsulfoxide (35), potassium nitrate, or sodium nitrite as analternative terminal electron acceptor to oxygen. None ofthe usual carbon sources could be used for fermentativegrowth, and no other media improved microaerophilic oreven phototrophic growth. Only the addition of glucose to Nmedium resulted in slightly enhanced cell densities at thestationary phase of a microaerophilic culture.Use of glucose in N medium was essential to find induc-

tion conditions for functionally defective reaction centers.Although the mechanism by which glucose increases thecellular energy level is unknown, the fermentative produc-tion of energy at low levels (30) is a good candidate. Thisexperimental condition proved to be essential for all further

investigations on the reaction center in R. viridis becausereaction centers are induced only by a drop in oxygentension in the medium. Since microaerophilic growth occursat 0.05 to 0.3% oxygen, the signal apparently is between0.05% and zero oxygen; thus, the total arrest of respiratoryactivity is a necessary prerequisite and consequence. Sinceno fermentation process was found to energize the cellsenough for growth, the addition of glucose is so far the onlyway to induce reaction centers.For expression of reaction center genes by complementa-

tion, an appropriate reaction center-negative mutant must beproduced. This could be done in alternative ways. Here wedescribe standard methods of chemical or UV mutagenesisto isolate Phot- mutants unable to grow phototrophically.Phot- mutants could derive their negative properties from avariety of gene defects, including defects of genes encodingenzymes of ubiquinone, menaquinone, carotenoid, heme,and bacteriochlorophyll b biosynthesis or defects of genes ofthe be1 complex. The probability that an organism with aPhot- phenotype has a puf mutation can be estimated as10-2. Unfortunately, for technical reasons the method ofenhanced fluorescence in mutant colonies with inactivereaction centers, as has been used for Rhodobacter capsu-latus (36), could not be applied to R. viridis cells showingfluorescence at 1,063 nm (1). Therefore Phot- mutants willhave to be screened for expression of reaction center genesby immune hybridization, and negative colonies will need tobe checked further for mutations in the structural genes toexclude regulatory mutants. This procedure might in thelong run be more time consuming than construction specif-ically of mutants of the reaction center genes with help of thegene transfer system to be described.

ACKNOWLEDGMENTS

We thank G. Krippahl for expertise in carrying out the Warburgexperiments and H. Michel for the reaction center crystals. We alsothank J. W. Farchaus for reading the manuscript and improving theEnglish.

LITERATURE CITED1. Angerhofer, A., J. U. von Schutz, and H. C. Wolf. 1984. Fluo-

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