Vol. 154, No. 2JOURNAL OF BACTERIOLOGY, May 1983, p. 748-7550021-9193/83/050748-08$02.00/0Copyright © 1983, American Society for Microbiology

Isolation and Characterization of Enhanced FluorescenceMutants of Rhodopseudomonas capsulata

DOUGLAS C. YOUVAN,l* JOHN E. HEARST,1'2 AND BARRY L. MARRS3Melvin Calvin Laboratory, Division of Chemical Biodynamics, Lawrence Berkeley Laboratory,' and

Department of Chemistry,2 University of California, Berkeley, California 94720; and E. A. Doisy DepartmentofBiochemistry, St. Louis University School of Medicine, St. Louis, Missouri 631043

Received 21 June 1982/Accepted 4 February 1983

After enrichment by a tetracycline suicide under conditions nonpermissive forthe growth of mutants defective in photosynthesis, colonies were screened forenhanced fluorescence in near-infrared light by using high-speed infrared photog-raphy. Twenty mutants were isolated, and the chromatophore membranes wereanalyzed by a new, rapid microprocedure that revealed many different pheno-types among the mutants. The enhanced fluorescence mutants typically possesseda functional light-harvesting II antenna, but showed reduced or absent light-harvesting I. Twelve isolates were also defective in reaction center polypeptides.An R-prime plasmid that bears 50 kilobases of Rhodopseudomonas capsulataDNA coding for components of the photosynthetic apparatus (B. L. Marrs, J.Bacteriol. 146:1003-1012, 1981), pRPS404, complemented all 20 enhanced fluo-rescence mutants as demonstrated by the quenching of fluorescence in mutantsthat had received the R-prime plasmid by conjugation. Fluorescence was regainedupon loss of the 50-kilobase insert. Complementation of the fluorescent lesionsimplies that most or all of the genes necessary for the expression of the reactioncenter and the light-harvesting antennae are carried by the R-prime plasmid andthat these genes are actively transcribed in the homologous organism. All 20mutants are complemented by one of two pBR322 subclones of the R-primeplasmid, pRPSEB2 or pRPSE2. pRPSEB2 bears a 4.5-kilobase fragment of R.capsulata DNA including the rxcA locus, and pRPSE2 is a pBR322 derivativebearing a 7.5-kiobase R. capsulata DNA fragment bearing the rxcB locus. Thesefragments therefore carry sequences necessary for the normal synthesis of thelight-harvesting and reaction center polypeptide complexes.

The bacterial photosynthetic apparatus veryefficiently transduces the energy of absorbedvisible and near-infrared (IR) photons into thehigh-energy bonds of ATP. Carotenoids andbacteriochlorophyll are the chromophores of thelight-harvesting (LH) antennae. Energy effi-ciently migrates from the antennae to the reac-tion centers (RC), where a special pair of bacte-riochlorophyll molecules is photooxidized. Theenergetic photoelectron drives a cyclic series ofredox couples consisting of quinones and cyto-chromes. Protons are components of the vec-torial redox reactions that are pumped acrossthe membrane to generate an electric field. Cou-pling factor ATPase driven by the proton gradi-ent regenerates ATP. The bacterial photosyn-thetic apparatus has been reviewed by Drews(2).At least eight polypeptides are involved in the

light-harvesting, charge separation, and photo-

electron transport functions within the photo-synthetic apparatus. These include three reac-tion center polypeptides (21, 24, and 28kilodaltons [kd]), three LH II polypeptides (8,10, and 14 kd), and two LH I polypeptides (8 and12 kd) (2). In Rhodopseudomonas capsulatavisible light is absorbed by the carotenoid pig-ments, and near-IR light is absorbed by thebacteriochlorophyll components of the twolight-harvesting antennae. LH I has maximalabsorption at 860 nm, and LH II absorbs at 800and 850 nm. When a photosynthetic bacteriumabsorbs light, but cannot utilize the energythrough photochemical paths, fluorescence mayresult (4). Such a situation arises when there is afunctional light-harvesting antenna and a defec-tive reaction center. Fluorescence emissionfrom these antennae is red shifted by 10 to 20nm. Hence, observing enhanced light-harvestingfluorescence is a potential method for screening

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FLUORESCENCE MUTANTS OF R. CAPSULATA 749

TABLE 1. Bacterial strains and plasmidsa

Strains Relevant properties Source orreference

E. coliHB101 recA pro leu thr lacY Strr Res-(K) Mod-(K) S. CohenBEC404 HB101(pRPS404) Taylor et al.TEC5121 HB101(pDPT51/pRPSE2) Taylor et al.TEB2 HB101(pDPT51/pRPSEB2) Taylor et al.

R. capsulataSB1003 Rife (7)Y142 RC- LH I- Strr (7)

PlasmidspDPT51 R751 derivative with pBR322-mobilizing activity Taylor et al.pRPS404 RP1 derivative, Kmr, Tcar, Apam, bears PSA genes, crtD223 (7)pRPSE2 EcoRI-F fragment of pRPS4W4 cloned in pDPT44, Kmr Taylor et al.pRPSEB2 BamHI-C-EcoRI-B cloned pBR322 derivative, Kmr Taylor et al.a Abbreviations: Km, kanamycin; Tc, tetracycline; Ap, ampicillin; Rif, rifampicin;

amber; PSA, photosynthetic apparatus.

photosynthetic bacteria for defective or missingpolypeptides involved in energy transfer andphotochemistry.

Most, if not all, of the genes coding for thedifferentiation of the anoxygenic photosyntheticapparatus are clustered on the R. capsulatachromosome and have been isolated on the R-prime plasmid, pRPS404 (7; W. G. Clark, J. M.Shivily, D. P. Taylor, S. N. Cohen, and B. L.Marrs, submitted for publication). Thirteengenes involved in carotenoid and bacteriochlo-rophyll biosynthesis and two genes affecting theexpression of the reaction center and LH Ipolypeptides have been mapped to the section ofR. capsulata DNA carried by pRPS404. In thisstudy mutants with hitherto unknown pheno-types are described, and the mutations causingthese phenotypes are mapped to the same sec-tion of R. capsulata DNA. These observationsstrengthen the suggestion that most, if not all, ofthe genes coding for the photosynthetic appara-tus are found in one large cluster in the genomeof this organism. The enhanced fluorescencemutants should provide valuable experimentalmaterial for future studies on the mechanism ofenergy transfer among components of the photo-synthetic apparatus.

MATERIALS AND METHODSBacterial cultivation and conjugations. Cultivation

and mating ofR. capsulata strains and the Escherichiacoli donor (BEC404) carrying the R-prime plasmidpRPS404 were performed as previously described (11).Briefly, R. capsulata organisms were grown and spotmated on PYE medium (11) at 32°C and transconju-gants were selected and repurified on RCV medium(10) containing 30 ,ug of kanamycin per ml; the donorwas counterselected by auxotrophy. Tetracycline sui-cides were performed as previously described (8).Photosynthetic growth assays were conducted as pre-viously described (11) by spotting mutants on PYE

Str, streptomycin; am,

plates and testing for growth within an anaerobic jar inthe light. Table 1 lists the relevant properties of thebacterial strains and the plasmids used in this study.Marker rescue crosses were performed as previous-

ly described (9a). Recombinant plasmids pRPSEB2and pRPSE2, which bear the rxcA and rxcB genes,respectively, were each introduced separately intoeach UFT strain of R. capsulata by conjugal mobiliza-tion from the appropriate E. coli strains. Recombinantplasmid-bearing exconjugants were identified by selec-tion for kanamycin-resistant R. capsulata colonies.Those colonies were picked and respread to test forcomplementation. UFT strains that were incapable ofphotosynthetic growth (Psg-) before introduction ofthe recombinant plasmids were respread under photo-synthetic growth conditions. For Psg+ strains, colo-nies were respread, grown aerobically, and then exam-ined for fluorescence levels as described below. Ineach case the presence of one of the recombinantplasmids quenched the fluorescence of the UFT mu-tants, whereas the other plasmid had no effect onfluorescence.Chromatophore preparation. R. capsulata cultures

were grown semiaerobically in supplemented RCVmedium (10). After 3 to 4 days of incubation, 3.0 ml ofbacterial culture was harvested with a Beckmannmicrofuge by two consecutive 3-min spins in 1.5-micentrifuge tubes. The bacterial pellet was washed bysuspension and centrifugation in B buffer (50 mMglucose, 10 mM EDTA, 25 mM Tris-hydrochloride,pH 8.0). The bacteria were suspended in B buffercontaining 10 ,ug of lysozyme per ml and incubated at37°C for 30 min. Each sample was sonicated with amicrotip for 10 s or until the solution cleared. Thislysate was centrifuged for 10 min in the microfuge, thecrude chromatophores from the supernatant weretransferred to microcentrifuge tubes (Beckmann; cel-lulose propionate no. 341288), and 400 ,ul was centri-fuged in two consecutive 7-min spins in an Airfuge A-100/30 rotor at 90,000 rpm. The crude chromatophorepellet was suspended in W buffer (10 mM potassiumphosphate [pH 7.35], 1 mM EDTA). This suspensionwas again centrifuged in the Airfuge, and the finalchromatophore pellet was redissolved in 15 ,ul of water

VOL. 154, 1983

750 YOUVAN, HEARST, AND MARRS

Film 7250- 900nm

> 780 nm z j

870 nm

Petri Dish

400-560 nm

A- ____

Plexiglas

1 cm 1 M CuSO4Glass Tray

FluorescentLight Box

J. BACTERIOL.

suitable for photography of bacterial colonies on fromone to six 100-mm petri dishes or for photography ofcultures in microtiter trays. An 80-W fluorescent lightbox with an 18- by 24-in. (ca. 45- by 60-cm) surfacewas masked with black tape so as to illuminate thebottom surface of the cupric sulfate filter. This filterconsisted of 1.5 liters of 1.0 M cupric sulfate in a 13.5-by 8.75- by 1.75-in. (ca. 33.75- by 21.88- by 4.38-cm)Pyrex baking tray which was covered with a sheet ofPlexiglas. Samples were photographed with 35 mmKodak HIE135 film through a 50-mm Macro lens atf3.5 with a Kodak Wratten 87C IR gelatin filter.Typical exposures for a field filled by one 100-mmpetri dish were 1.0 min for IR fluorescence with the87C filter and 1/60 s for visible photography withoutthe 87C filter. Two photographs were taken for eachpetri dish: one with the 87C filter (fluorescence mode)and one without the filter (absorption mode). Theenlargement factors were held constant such that thetwo prints are superimposible, facilitating the localiza-tion of fluorescent colonies. HIE135 high-speed IRfilm requires no special processing and can be devel-oped in the laboratory with Kodak D-76 developer asdescribed by the company.

RESULTSXBL 823-8827 Detection of IR fluorescence from single colo-

FIG. 1. Photographic apparatus used to record nies on petri dishes. A photographic comparisonnear-IR fluorescence from bacterial colonies. Petri of the fluorescence emission from two strains ofdishes are irradiated with blue light, and photons R. capsulata is shown in Fig. 1. SB1003 is theemitted due to fluorescence with a wavelength be- wild-type strain; Y142 lacks all RC and LH Itween 780 and 900 nm from bacterial colonies are polypeptides, but has functional LH II (4).imaged by the camera and recorded by high-speed IR These strains streaked PYE plate and

film. Components of the apparatus have been separat- grown aerobically in the dark. The plates were

ed for display; in use the petri dish, Plexiglas sheet grown o n the dark.aTheaplatus wereand cupric sulfate filter are stacked. As drawn, the placed on the photographic apparatus shown in

apparatus is in the fluorescence mode, and without the Fig. 1 as described above. This device illumi-

Wratten 87C filter the bacterial colonies are photo- nates the colonies with light emitted from fluo-graphed in an absorption mode. rescent tubes and filtered through cupric sulfate.

Photons in the range of 330 to 560 nm are

transmitted through the filter. The spectral char-and stored at -20°C until solubilization for sodium acteristics of the fluorescent tube and a Plexiglasdodecyl sulfate-polyacrylamide gel electrophoresis support above the cupric sulfate filter result in(SDS-PAGE). exciting light in the range of 400 to 560 nm. TheSDS-PAGE of chromatophore polypeptides. For frequency of this blue light matches the carot-

SDS-PAGE, we used a minislab gel electrophoresis enoid absorption spectra of the R. capsulataapparatus available from Idea Scientific Co., Corval- bacteria. With Kodak HIE135 high-speed IRlis, Oreg., with a gel mold that is 10 by 17 by 0.08 cm. film (sensitive from 250 to 900 nm), the petri dish

The gel composition was essentially that of Laemnmli was photographed through a Macro lens. This is(6), formed from 9 and 18% acrylamide solutions in a referred as the absorption mode, and a photo-linear gradient with a 6% stacking gel. The running raphic t is she in moden a Kodakbuffer was 0.05 M Tris-0.38 M glycine-0.1% SDS. The graphic print is shown in Fig. 2B. When a Kodak

2x sample buffer was 0.125 M Tris-hydrochloride (pH Wratten 87C gelatin filter is placed on the Macro6.8)-4% SDS-20% glycerol-0.002% bromophenol lens, only photons with a wavelength longerblue-100 mM dithiothreitol. Equal volumes of the than 780 nm are transmitted (5), resulting in a

aqueous chromatophore suspension and the 2x sam- photographic sensitivity in the range of 780 to

ple buffer were combined and heated at 65°C for 1 min; 900 This includes the wavelengths charac-

10 Rg of protein was loaded (usually about 6 RI of the teristic of LH II fluorescence from B850 at 864

loading mix). The gel was run at a constant power of 5 nm and the LH I B875 fluorescence at 891 nm

W until the buffer front reached the bottom of the gel. (4). A photographic print in theThe gel was stained and destained as previously de-

mode (Wratten 87C filter) of SB1003 and Y142 isscribed (1).

Fluorescence IR photography. The photographic ap- shown in Fig. 2A. Although both strains areparatus used to screen for enhanced IR fluorescence approximately equal in absorption, Y142 ismutants is diagrammed in Fig. 1. This device is much more highly fluorescent. By comparing a

High Speed Infrared FilmHIE 135

50 mm Macro Lena

Wratten 87C IR Filter

FLUORESCENCE MUTANTS OF R. CAPSULATA 751

B *~~~~~~~~~~~~~~~~~~~~~~. ..,..................

* 4

.......

FIG. 2. IR fluorescence from wild-type and an en-

hanced fluorescence mutant. (B) Wild-type SB1003and Y142 (RC- LH I-) streaked on an RCV plate andphotographed in the absorption mode. (A) Same platephotographed through the Wratten 87C filter in thefluorescence mode.

time course of exposures (data not shown), we

have estimated that Y142 is approximately 50times more fluorescent than the wild type. Theseobservations are consistent with the enhancedlevels of LH II fluorescence previously mea-

sured from LH I-negative, reaction center-nega-tive (LH 1- RC-) mutants as determined byspectrofluorometry of isolated chromatophores

(4). The enhanced fluorescence of Y142 relativeto SB1003 can also be observed using the samefilters and an IR viewing scope.A notable feature of the low-level fluores-

cence from the wild-type colonies is that thefluorescence is emitted principally from the pe-rimeter of the colony. Since the perimeter con-tains the younger, less induced colonies, thissuggests that there is a stage in the differentia-tion of the photosynthetic apparatus when pho-tons are harvested, but the energy is not asefficiently transferred to reaction centers as inthe fully induced state. An alternative explana-tion is that the fluorescence photons are reab-sorbed in the central area of the colony, and thatthe fluorescence photons emitted near the edgeof the colony (which is less pigmented) escapereabsorption.

Isolation of enhanced fluorescence mutants.Mutants that are enhanced in near-IR fluores-cence have been isolated by using a tetracyclinesuicide enrichment of photosynthetically defec-tive spontaneous mutants from a wild-type cul-ture (8) and by screening the surviving coloniesfor fluorescence by using IR photography. Atetracycline suicide was performed on SB1003under conditions (anaerobic, light) nonpermis-sive for the growth of mutants defective inphotosynthetic growth, and the survivors wereplated on RCV medium and photographed after5 days of growth under permissive conditions(aerobic, dark). Colonies with enhanced fluores-cence were observed with a frequency of ap-proximately 1 fluorescent mutant in 30 mutantssurviving the tetracycline suicide. This fractioncan be substantially smaller if the suicide is notcomplete. Figure 3 shows several enhanced fluo-rescence mutants on an RCV plate on which 100,ul of the suicide culture was spread. From platessimilar to this one, 20 fluorescent colonies werepicked and purified. The purifications weremonitored by IR photography, and the enhancedfluorescence phenotype was found to be stable.These strains are designated UFTI throughUFT20.

Characterization of enhanced fluorescence mu-tants. One indication of the diversity of thelesions generating the enhanced fluorescence isthe variously altered abilities of the mutants togrow photosynthetically. Strains UFT1 throughUFT20 were assayed for photosynthetic growthby comparing growth on dark, aerobic plateswith growth on plates grown photosynthetically(anaerobic, light). All 20 strains grew equallywell under the permissive conditions (aerobic,dark); however, after 6 days of photosyntheticgrowth five categories of photosynthetic growthwere observed as judged qualitatively from spot-ted inocula. Out of the 20 strains, 13 strainsshowed little or no growth (Psg-), and the

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752 YOUVAN, HEARST, AND MARRS

FIG. 3. IR fluorescence photography of a bacterialpopulation enriched for photosynthetically defectivemutants. Wild-type bacteria were subjected to a tetra-cycline suicide under conditions nonpermissive for thegrowth of photosynthetically defective spontaneousmutants. The suicide culture was plate on RCV andphotographed in the absorption mode (B) and thefluorescence mode (A). Several enhanced fluores-cence mutants are visible.

remaining strains grew at various rates rangingfrom slow to wild-type rates (Psg+). These dataare summarized in Table 2.Chromatophores were prepared from all 20

UFT strains plus SB1003 and Y142. SDS-PAGEof the chromatophore polypeptides on a 9 to

18% acrylamide gradient gel is shown in Fig. 4.There are several general features in the poly-peptide composition of the UFT strains: the 8-kdand 10kd polypeptides are present in all 20strains, and the 12-kd polypeptide is significant-ly reduced or absent in 19 of 20 strains. All threereaction center polypeptides are expressed atne stoichiometric ratios to each other: in sevenstrains they are missing or are undetectable, infour strains they are significantly reduced fromwild-type levels, and in nine strains the threepolypeptides are present at near wild-type lev-els. The LH II polypeptides are present in 17 of20 straing and reduced in the other 3. The sevenstrains that lack reaction centers are Psg- asexpectqd, but four strains with wild-type levelsand two with reduced levels of reaction centerpelypeptides are also Psg-. Five of the latter sixstrains lack LH I.By photographing cultures grown under iden-

tical conditions in microtiter tray wells contain-ing RCV plus medium, we have observed thatthe UFT strains fluoresce at various intensities(data not shown). The most fluorescent strainscorrelate well with those lacking all of the reac-tion center polypeptides as indicated by SDS-PAGE. Strains with reduced reaction centerpolypeptides fluoresce less intensely than thoselacking reaction centers, and strains with wild-type levels of reaction center polypeptides showthe lowest levels of fluorescence in the UFTstrains.Compiementation of enhanced fluorescence

mutants by R-prime pRPS404. The R-prime plas-mid pRPS404 complements lesions which gener-ate the enhanced fluorescence in all 20 of the

TABLE 2. UFT strains

UFT Phenotype' Genotypebstrain no. Psg RC LH I LH II PRPSEB2 pRPSE2

13 + + + + - +1,12,17 + + ± + - +

6 + + ± + + _2,16 + + - + - +15 - + + + - +18 - + - + - +

3,5,20 - + - + + -11 - + - + + -

4,8,19 - - + + + -7 - - + + - +

9,10,14 - - - + + -a Determined from photosynthetic growth assays

and polypeptide analysis by SDS-PAGE. Abbrevia-tions: Psg, photosynthetic growth ability; RC, LH I,and LH II, relative amount of the reaction centers andlight-harvesting antennae I and II, respectively, inCoomassic blue-stained polyacrylamide gels.

b Determined from marker rescue assays withpRPSEB2 and pRPSE2 (see text).

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FLUORESCENCE MUTANTS OF R. CAPSULATA 753

Y 1 2 3 4 5 6 7 8 9 10 S Y 11 12 13 14 15 16 17 18 19 20 S

28 _..24

21

-14 .`i-t2-10- j

I-8-I

FIG. 4. SDS-PAGE of chromatophore membranes from 20 enhanced fluorescence mutants. Strains UFTIthrough 20 (lanes 1 through 20, respectively) are compared with wild-type chromatophores from SB1003 (lane S)and a well-characterized mutant (Y142) lacking reaction center and LH I polypeptides (lane Y). Common to theenhanced fluorescence mutants are the 8-kd and 10-kd LH II polypeptides and the reduction or loss of the 12-kdLH I polypeptide. The three reaction center polypeptides (21, 24, and 28 kd) are reduced or missing in some ofthe mutants.

UFT strains. pRPS404 was conjugated into theUFT strains from HB101 and merozygotes wereselected on RCV plates containing 30 ,ug ofkanamycin per ml. The E. coli donor was coun-terselected by auxotrophy. The transfer efficien-cy is approximately 10-2 per recipient. Inoculafrom each spot mating on PYE plates werestreaked on the selective medium and incubatedaerobically in the dark for 5 days and comparedwith UFT strains streaked on RCV plates byinfrared photography. Absorption and fluores-cence photographs were compared for the UFTstrains with and without the R-prime plasmid.The results for UFT14 (LH I RC-) are shownin Fig. 5. In all 20 strains the R-prime plasmidquenched the fluorescence of the isolated mero-zygotic colonies, whereas there was no detect-able reversion of the fluorescence phenotype inthe control streak. Psg- UFT strains bearingpRPS404 were restreaked on PYE plates andfound to be Psg+.Complementation rather than recombination

is implied for the mechanism of the fluorescencequenching in the UFT merozygotes because of

the isotropic nonfluorescent morphology of thecolonies. Previously studied rates of recombina-tion between homologous chromosomal and R-prime alleles (11) suggest that only infrequentand narrow nonfluorescent sectors would bepresent in the merozygotic colonies if the mech-anism involved recombination, as the case is forthe crtD mutation. Additional evidence in favorof complementation is that the merozygotic col-onies regain their fluorescence upon restreaking.Fluorescence reappears in greater than 95% ofthe colonies during the first repurification of theUFT strains on RCV plates containing 30 jxg ofkanamycin per ml. This rate is consistent withthe previously observed (11) flanking IS21-medi-ated intramolecular recombination and deletionof the prime DNA from pRPS404 in wild-type R.capsulata and in RecA+ E. coli. This stronglysuggests that the transient nonfluorescent stateinvolves complementation of the fluorescencemutations in trans by actively transcribed R-prime plasmid genes.Marker rescue crosses. Plasmids pRPSEB2

and pRPSE2 carry fragments of the photosyn-

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754 YOUVAN, HEARST, AND MARRS

D

FIG. 5. Complementation of an enhanced fluorescence mutant with the R-prime plasmid pRPS404. Plasmid

pRPS4O4 was conjugated into UFT14 (RC- LH I-), and merozygotes were streaked on a selective plate. UFT14

without the R-prime plasmid (A and C) is compared with UFT714 with the R-prime plasmid (B and D) in both the

absorption mode (C and D) and the fluorescence mode (A and B). UFT`14 without the R-prime plasmid is highlyfluorescent, and this fluorescence is quenched by the R-prime plasmid. The fluorescence quenching of each UFT7strain by the conjugal introduction of the R-prime plasmid was verified in this manner.

thetic region that are known to carry the rxcAand rxcB genes, respectively. Mutations in theseloci result in high fluorescence phenotypes, andmost affected mutants previously describedwere incapable of photosynthetic growth byvirtue of the absence of reaction centers (9a).These plasmids were tested for their ability tocomplement the mutations carried in each of theUFT strains (Table 2). Of the seven UFT strainsthat are Psg+, six were conplemented by the R.capsulata DNA carried on pRPSE2, and theother was complemented by pRPSEB2,pRPSEB2 complemented 10 of the 13 Psg-strains, and pRPSE2 complemented the other 3.

DISCUSSION

A common feature of R. capsulata mutantswith enhanced fluorescence is that they have afunctional LH II antenna and are lacking or have

reduced LH I and reaction center polypeptides.Mutations affecting the transfer and photochem-ical dissipation of light energy gathered by LH IIresult in fluorescence. Light energy gathered bythe 8-kd and 10-kd polypeptides and associatedchromophores is emitted as fluorescent light inthe near-IR at wavelengths that we can detect byphotography with appropriate filters and high-speed IR film. This interpretation is in agree-ment with what has been previously observedwith isolated chromatophores and spectrofluo-rometry (4): LH I- RC- mutants are morefluorescent than the wild type which is in turnmore fluorescent than LH II- crt- mutants.

Mutations affecting energy transfer and photo-chemical utilization of absorbed light energymay include regulatory or polar mutations thatresult in the loss of expression of polypeptides,point mutations that result in nonsense muta-tions terminating polypeptides, and point muta-

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FLUORESCENCE MUTANTS OF R. CAPSULATA

tions that result in missense mutations and poly-peptide analogs that may be either functional ornonfunctional. Assembly complicates this analy-sis, since some polypeptide analogs with singleamino acid substitutions may not be incorporat-ed into the membrane, or they may be incorpo-rated at reduced levels. Some of the mutantsthat we have characterized as having reducedlevels of particular polypeptides may be in thislatter category. Assembly groups inferred fromstoichiometric ratios of their component poly-peptides at various levels of expression in the 20enhanced fluorescence mutants that we haveanalyzed include the reaction center protein(21-, 24-, and 28-kd polypeptides) and two of thethree LH II polypeptides (8 and 10 kd). We havepreviously observed LH II mutations whereonly the 14-kd polypeptide is missing (11). Muta-tion of a polypeptide in each of these twoassembly groups may reduce the amount ofincorporation of the other polypeptides withinthat group into the photosynthetic membrane.Testing this hypothesis will require further ex-periments.

Since the R-prime pRPS4O4 plasmid comple-ments and transiently quenches all 20 enhancedfluorescence mutants, we deduce that most orall of the genes necessary for the differentiationand expression of the reaction center and LH Ipolypeptides are carried on the R-prime plasmidand that these genes can be expressed in trans inthe homologous organism. This assumes that thespontaneous mutations which are enriched by atetracycline suicide and screened by enhancednear-IR fluorescence are randomly distributedthrough the regulatory and structural genes forthe photosynthetic apparatus. The diversity ofmutant phenotypes obtained by the enrichmentand selection procedure supports this assump-tion. Site-directed transposon mutagenesis withTn7-mutagenized R-prime plasmids indicatesthat some LH II genes are also carried bypRPS404 (11).The marker rescue results demonstrate that

the genes affecting the synthesis and assembly ofeach pigment-protein complex are located in oneof the two subregions of the cluster of genescoding for the photosynthetic apparatus. Thesesubregions are separated by genes concernedwith pigment biosynthesis. In several cases thesame phenotype can be generated by a mutationin either of the two subregions. For example, themutation in UFT6 maps near rxcA, but it isindistinguishable from UFIl, -12, or -17, whichhave mutations mapping near rxcB. Similarly,UFT7 has the same phenotype as UFT4, -8, and-19, but maps near rxcB, in contrast to the otherthree.

The fact that the genes carried by pRPSEB2and pRPSE2 were expressed in trans, and there-fore were capable of complementing chromo-somal lesions, might be due to promoters on thevectors or on the inserts. The R. capsulatainsert in pRPSE2 is downstream from the Plpromoter of pBR322 (9), so further work will benecessary to distinguish the alternatives. The Plpromoter is deleted from pRPSEB2 (9a), so itseems likely that an indigenous promoter hasbeen cloned in that case. The study of thepromoter that drives transcription of the pig-ment-binding protein should reveal much aboutthe regulation of synthesis of the photosyntheticapparatus.

ACKNOWLEDGMENTS

This work was supported by the Director of Energy Re-search, Biomedical and Environmental Research Division ofthe U.S. Department of Energy under contract number W-7405-ENG48, by Public Health Service grant GM-20173 fromthe National Institutes of Health, and by grant PCM 78-27938from the National Science Foundation.We thank Ken Sauer for helpful discussions.

LITERATURE CITED

1. Chua, N.-H. 1980. Electrophoretic analysis of chloroplastproteins. Methods Enzymol. 69:434-446.

2. Drews, G. 1978. Structure and development of the mem-brane system of photosynthetic bacteria. Curr. Top.Bioenerget. 8:161-207.

3. Drews, G., R. Dierstein, and A. Schumacher. 1976. Genet-ic transfer of the capacity to form bacteriochlorophyll-protein complexes in Rhodopseudomonas capsulata.FEBS Lett. 68:132-136.

4. Feick, R., R. Van Grondelle, C. P. Rigersberg, and G.Drews. 1980. Fluorescence emission by wild-type andmutant strains of Rhodopseudomonas capsulata. Bio-chim. Biophys. Acta 593:241-253.

5. Kodak filters for scientific and technical users. KodakTechnical Publication no. B-3H. Eastman Kodak Co.,Rochester, N.Y.

6. Laenmili, U. K. 1970. Cleavage of structural proteinsduring the assembly of the head of bacteriophage T4.Nature (London) 227:680-685.

7. Marrs, B. L. 1981. Mobilization of the genes for photosyn-thesis from Rhodopseudomonas capsulata by a promiscu-ous plasmid. J. Bacteriol. 146:1003-1012.

8. Marrs, B. L., S. Kaplan, and W. Shepard. 1980. Isolationof mutants of photosynthetic bacteria. Methods Enzymol.69:29-37.

9. Stuber, D., and H. Bujard. 1981. Organization of tran-scriptional signals in plasmids pBR322 and pACYC184.Proc. Natl. Acad. Sci. U.S.A. 78:167-171.

9a.Taylor, D. P., S. N. Cohen, W. Gregg Clark, and Barry L.Marrs. 1983. Alignment of genetic and restriction maps ofthe photosynthesis region of the Rhodopseudomonas cap-sulata chromosome by a conjugation-mediated markerrescue technique. J. Bacteriol. 154:580-590.

10. Yen, H.-C., and B. L. Mam. 1977. Growth ofRhodopseu-domonas capsulata under anaerobic dark conditions withdimethyl sulfoxide. Arch. Biochem. Biophys. 181:411-418.

11. Youvan, D. C., J. T. Elder, D. E. Sandlin, K. Zsebo, D. P.Alder, N. J. Panopoulos, B. L. Marrs, and J. E. Hearst.1982. R-prime site-directed transposon Tn7 mutagenesisof the photosynthetic apparatus in Rhodopseudomonascapsulata. J. Mol. Biol. 162:19-41.

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