JOURNAL OF BACTERIOLOGY, Dec. 1971, p. 1348-1356Copyright O 1971 American Society for Microbiology

Vol. 108, No. 3Printed in U.S.A.

Mutants of Rhodospirillum rubrum ObtainedAfter Long-Term Anaerobic, Dark Growth

R. L. UFFENI, C. SYBESMA, AND R. S. WOLFEDepartment of Physiology and Biophysics and Department of Microbiology, University of Illinois, Urbana,

Illinois 61801

Received for publication 6 July 1971

Rhodospirillum rubrum S, cells were grown for more than 100 generationsunder strict anaerobic, dark conditions in liquid medium with sodium pyruvate.During this time, growth became nonpigmented. When cells were streaked onto thesurface of solid growth medium in anaerobic bottles and placed in the dark, a fewlight-red colonies developed, but the majority was nonpigmented. Mutants wereobtained from colonies selected on the basis of pigmentation and bacteriochlor-ophyll a content. The growth, ultrastructure, and light reactivity of two mutantswere examined. Mutant C synthesized bacteriochlorophyll a (7.2 itmoles per mg ofprotein), altered membrane structures, and chromatophores during dark growth.Examination of light-induced changes of the absorption spectrum of this mutantsuggested that only an electron transport pathway, which included the low poten-tial cytochrome-like pigment C428, could be detected. Mutant C grew anaerobi-cally in the light, whereas mutant GI was light sensitive and produced only traceamounts of bacteriochlorophyll a (0.6 gmole per ml of protein). Poorly pigmentedG1 cells contained unusual membrane structures. When dark-grown G1 colonieswere placed in the light, deep-red colored papillae developed in the nonpigmentedcolonies. During anaerobic, dark growth with sodium pyruvate, both C and G1synthesized poly-,3-hydroxybutyrate and produced acetate, carbon dioxide, andhydrogen gas.

We reported recently that light was not re-quired for the growth of purple nonsulfur bac-teria under strict anaerobic conditions (32);when anaerobic, light-grown Rhodospirillumrubrum S1 cells were placed in a dark environ-ment, growth proceeded with no significant lagin protein synthesis. During 10 to 15 generationsin the dark, pigments and associated membranestructures were synthesized which appeared to befunctionally and structurally similar to thoserequired for photosynthetic metabolism. Basedupon these observations, it was suggested that innature the retention of a preformed photosyn-thetic apparatus would be an advantage for thecell in a transitory anaerobic, dark environment.The present study is the first investigation of

structural and functional changes in photosyn-thetic purple nonsulfur bacteria after long-termanaerobic, dark growth.

MATERIALS AND METHODSOrganism, medium, and growth conditions. R.

rubrum S1 was kindly supplied by H. Gest. Cells were

' Present address: Department of Microbiology and PublicHealth, Michigan State University, East Lansing, Mich. 48823.

grown at 37 C in a complex medium which containedsodium pyruvate as previously described (32). Sodiumphosphate buffer (pH 7.0) was sterilized and addedseparately to liquid medium to produce a final concen-tration of 0.1 M. For anaerobic growth, medium wasprepared under an argon atmosphere by a modificationof the Hungate technique (3, 16). Light or dark growthconditions were achieved as previously described (32).

Selection of mutant strains. Cells grown for 24 hr indark conditions were spread over the surface of solidgrowth medium in anaerobic bottles (32); certain bot-tles were placed in light and others in dark conditions.Cells were obtained from isolated colonies selected onthe basis of pigmentation and were subcultured twiceunder the same growth conditions on solid mediumbefore preparation of stock cultures. Mutant cells re-mained viable after 1 year when placed in glycerol so-lution (I ml per 100 ml of bacterial culture) and main-tained at -70 C.

Determination of cell numbers. An anaerobic bottlewas inoculated by spreading a 0.1-ml sample from aserial dilution of a culture onto the surface of solidgrowth medium; viable cell numbers were determinedby colony count. Cells were counted directly with aPetroff-Hauser bacterial cell counting chamber.

Bacteriochlorophyll. Cells grown in liquid mediumwere collected by centrifugation and washed in distilledwater. Bacteriochlorophyll a was extracted as pre-

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MUTANTS OF R. RUBRUM

viously described (32), and the concentration was de-termined spectrophotometrically by use of the molarextinction coefficient (E = 75 mM-' cm -') at 770nm by the method of Clayton (5).

Protein. Samples were digested with I M NaOH atroom temperature for 24 hr. Protein content was esti-mated by the method of Lowry et al. (20). Bovineserum albumin was used as a standard.

Gas chromatography. Hydrogen and carbon dioxidewere detected with a Packard gas chromatographequipped with a silica gel column connected to anargon ionization detector (32). Volatile fatty acidswere determined with a gas chromatograph by themethod of Erwin, Marco, and Emery (9).

Poly-#-hydroxybutyric acid. Cells were hydrolyzedwith 5.25% sodium hypochlorite or Clorox, and poly-13-hydroxybutyric acid (PHB) was extracted from the res-idue with hot chloroform by the method of Law andSlepecky (19). The extracted PHB was hydrolyzedwith concentrated sulfuric acid, and the concentrationwas calculated from the molar extinction coefficient at235 nm.

Spectroscopy. The characterization of bacteriochloro-phyll a and carotenoids was determined with aCary 14 recording spectrophotometer with whole cellsuspensions in 60% sucrose (24).

Light-induced changes in absorbance were measuredin a split-beam absorption difference spectrophotometerdescribed earlier (28). For these measurements, wholecells were collected by low-speed centrifugation andsuspended in a salts solution (pH 6.8) which containedno organic substrates (28).

Photomicroscopy. Photographs of colonies weretaken with a Zeiss photomicroscope equipped with aI x objective lens.

Electron microscopy. Cells grown for 24 to 36 hr un-der anaerobic, dark conditions were fixed by a modifica-tion of the method of Kellenberger, Ryter, and Sechaud(17). A 1% (w/v) solution of osmium tetroxide at pH7.0 was used. Agar blocks were dehydrated, and thinsections were prepared and stained as previously de-scribed (32). Sections were examined with a SiemensElmiskop I electron microscope operating at 60 kv,equipped with a 50-am objective aperture unless other-wise indicated.

RESULTSR. rubrum cells, maintained for more than 100

generations under anaerobic, dark growth condi-tions, remained scotophobic and the bacterio-chlorophyll a concentration slowly decreased tolevels of 0.3 to 0.5 ,umole per mg of protein. [In-terruption of the light for approximately 1 sec ina light microscope causes a phototrophic bac-terial cell to reverse its directional movement.Contrary to the phototactic response in greenplants, the bacterial response is oriented nottoward a light source but rather away from dark-ness (8, 21, 23). For this reason we propose theterm scotophobic response (Gr. skotos darkness+ phobos fear). We suggest that scotophobic isan adjective which describes the specific response

or "Schreckbewegung" (8) in the photosyntheticbacteria.] If cells were streaked onto the surfaceof solid growth medium in an anaerobic bottleand placed in the dark, colonies 2 to 3 mm indiameter formed that differed in pigmentation.Most of the colonies were white or faint pink;however, a few developed light-red pigmentation.Mutant strains C and GI were obtained fromcolonies selected on the basis of pigmentation.

Colony pigmentation. Dark-grown mutant Cdeveloped light-red colonies. Cells were scoto-phobic and synthesized approximately 7.2Amoles of bacteriochlorophyll a per mg of pro-tein (Table 1). Similar values were obtained pre-viously for cells grown anaerobically in the darkfor 10 to 15 generations after transfer from lightconditions (32). GI cells formed white or slightlypink-colored colonies in the dark and synthesizedtrace amounts of bacteriochlorophyll a (Table 1).Poorly pigmented G I cells were scotophobicduring early logarithmic growth. No photosyn-thetic pigments were synthesized during aerobic,dark growth of C or GI (Table 1), and a scoto-phobic response could not be demonstrated.

Response to light. Growth of mutants C and G 1in light conditions was determined by measuringincrease in protein (Fig. 1). During the first 12hr after transfer into light, the protein concentra-tion decreased, perhaps a result of cell lysis. Cellprotein in C, however, increased fivefold duringthe following 36 hr, and the ratio of bacterio-chlorophyll a to protein increased to 16.2 (Table1). Cell protein in GI doubled and after 48 hr de-creased as the cells lysed. No growth of G1 wasdetected during 4 additional days in the light.After 36 hr growth in the dark, GI reached adensity of 5 x 108 cells per ml.A suspension of dark-grown mutant C was se-

rially diluted under anaerobic conditions, and asample of each dilution was spread onto the sur-face of solid growth medium in a separate anaer-obic bottle. One set of anaerobic bottles, inocu-lated with each dilution, was placed in the light;a second set was placed in the dark. During 7days in dark conditions, colonies 2 to 3 mm indiameter formed. Anaerobic bottles placed in thelight were examined for colony formation, andan equal number of C colonies developed in lightor dark conditions (Table 2, experiments 1 and2). Under dark conditions C colonies were lightred, and those formed in the light were dark red.When dark-grown C colonies were transferredinto the light, pigmentation increased; if light-grown colonies were placed in the dark, no lossin pigmentation was observed. The number ofcolonies did not increase when anaerobic bottleswere switched from dark to light conditions orvice versa.

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1350 UFFEN, SYBESMA, AND WOLFE J. BACTERIOL.

TABLE 1. Bacteriochlorophyll a produced by mutants of Rhodospirillum rubrum S, under different growthconditions in liquid medium

Period of Bacteriochlorophyll Protein BacteriochlorophyllMutant strain Growth condition incubation (zmoles) (mg) per mg of protein

(hr) (Mmls)(g)(moles)

33 20.80 2.44 8.1C Dark, anaerobic 36 25.20 3.42 7.4

38 14.02 2.26 6.253 13.44 0.83 16.2

C Light,a anaerobic 53 14.84 0.98 15.153 16.00 0.89 18.026 0.00 1.14 0.0

C Dark, aerobic 36 0.00 1.28 0.048 0.00 1.05 0.036 1.79 3.02 0.6

G I Dark, anaerobic 38 1.00 1.52 0.738 0.48 1.42 0.338 20.48 1.25 16.4

G2b Light, anaerobic 46 20.43 1.19 17.2_72 20.70 1.00 20.7

24 0.00 2.14 0.0G I Dark, aerobic 25 0.00 1.12 0.0

27 0.00 2.05 0.0

a Approximately 60 foot candles.I Photosynthetic revertant obtained from light-sensitive G I.

7

6

5

4

3

2,

12 24 36TIME (hours)

48

FIG. 1. Increase of protein in Rhodospirillumrubrum S, mutant C (0) and GI (-) transferredfromanaerobic, dark into anaerobic, light (55 to 60 footcandles) conditions.

When anaerobic bottles were inoculated (asdescribed above) with dark-grown GI and were

placed in the light, only 2 to 3 out of 101 cellsgrew and formed deep-red pigmented colonies(Table 2, experiments 3 and 4). When these bot-

TABLE 2. Growth of mutants of Rhodospirillumrubrum S1 under anaerobic, dark or light

conditions on solid medium

Colonies countedbMutant GrowthExpt strain conditiona Dilution Pig- Nonpig-

mented mented

I C Dark 105 0 230Light" 105 280 0

2 C Dark 106 0 100Light 106 70 0

3 GI Dark 105 0 250Light 10' 48 0

4 GI Dark 106 0 59Light Undiluted 156 0

a Colonies were allowed to develop for 9 days at 37C under the respective growth condition.

'Colonies were assayed visually. Nonpigmented col-onies were faint red (strain C) or faint pink-white(strain GI). Pigmented colonies were dark red.

c Approximately 60 foot candles.

tles were placed in the dark for 14 days, no addi-tional colonies developed. Accordingly, whenbottles containing nonpigmented, G I dark-formed colonies were placed in the light, no in-crease in isolated colonies occurred. Under theseconditions, however, deep-red pigmented papillaeformed within or on the periphery of 80 to 90%of the colonies (Fig. 2). An average of one totwo papillae developed per colony. In one case,however, this number reached as high as 11.

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MUTANTS OF R. RUBRUM

Mutant G2 was obtained from a papilla whichdeveloped under these conditions. Photosyn-thesizing G2 cells were scotophobic and pro-duced bacteriochlorophyll a at concentrationssimilar to those observed for light-grown C cells(Table I ).

Internal membrane structure. Mutant C pro-duced a variety of internal structures during darkgrowth. Chromatophores were formed whichappeared similar to those in light-grown cells.Figure 3 is an electron micrograph of a thin lon-gitudinal section of a dark-grown cell in whichchromatophores were aligned along the cellmembrane. In a few sections they extendedthroughout the cytoplasm; in others, they wereabsent. Thin sections of light-grown C were filledwith chromatophores and appeared similar to G2(see Fig. 9) and to normal R. rubrum S (32).

Figure 4 is a thin transverse section through acomplex membrane structure; these structureswere observed frequently in dark-grown C. Thisstructure was composed of a series of concentricmembrane vesicles. Each vesicle enclosed anelectron-translucent area with a density similarto that inside chromatophores (see Fig. 9). Cyto-plasmic material appeared to be deposited in thecenter of the structure and in the spaces betweenthe encircling vesicles. Similar membrane config-urations have been observed in R. rubrum (26)and Rhodopseudomonas palustris (30, 31) placedunder different conditions. In R. palustris theyappeared between the cytoplasmic membraneand the cell wall and were suggested to representflagellar basal bodies (30, 31). This, however,does not seem likely in C (6). Concentric mem-brane structures have not been observed in G1.

Internal vesicles composed of a unit membranethat contained an electron-dense material wereobserved in dark-grown C (Fig. 5) and G1. Thevesicles remained attached to internal membranematerial in lysed cell preparations (Fig. 6 and 7),together with chromatophores, and probablydeveloped from membrane invaginations.Schmidt and Kamen (25) noted granules withsimilar staining properties in Chromatium strainD cells. These granules, however, were not sur-rounded by a unit membrane and probably rep-resented glycogen deposits. The function andchemical composition of the electron-dense vesi-cles in R. rubrum is not known.The internal structure of G I was different

from C; GI synthesized only trace amounts ofphotosynthetic membrane, and a typical chro-matophore was rarely observed in thin sections(Fig. 8). On the other hand, mutant G2, a photo-synthetic revertant, was filled with chromato-phores (Fig. 9) and appeared similar to wild-typecells (32). G I cells contained electron-dense

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Os4FIG. 2. Rhodospirillum rubrum S1 GI nonpigmented

colony formed during anaerobic, dark growth contain-ing a pigmented papilla (P) which developed after trans-fer into light. x 10.

areas of myelin-like material, or membrane ag-gregates, which extended through the cytoplasmin an unordered manner. The structures appearedto be formed from unit membranes (Fig. 10),approximately 9 nm across, that were continuouswith the cytoplasmic membrane (Fig. 11). Sim-ilar structures have been observed in dark-grownC. Myelin-like deposits have been reported in R.rubrum maintained under different conditions(15, 26). Schoen and Ladwig (26) suggested thatthese deposits resulted from the deterioration ofchromatophores. Membrane aggregates in GIcells are probably formed by invaginations of thecytoplasmic membrane, independent from chro-matophore formation. The function of thesestructures and their relationship to photosyn-thetic membranes is not known. Similar mem-branous configurations have been reported innonphotosynthesizing cells such as Vibrio mar-inus (10) and Escherichia coli (33).

Light absorption. Dark-grown mutant C dem-onstrated an absorption spectrum similar tolight-grown R. rubrum (32). To observe thespectrophotometric absorption properties ofpigments in mutant G 1, it was necessary to use asensitive instrumental range of 0.0 to 0.2 opticaldensity units. Cells grown under aerobic, darkconditions, where no photosynthetic pigmentswere synthesized (Table 1), were suspended insucrose and placed in the reference beam. Anaer-obic-grown G 1 cells, observed under these condi-tions, demonstrated absorption maxima for bac-teriochlorophyll a at 890 and 806 nm with a light

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FIG. 3. Electron micrograph of longitudinal thin section of anaerobic, dark-grown mutant C that containedchromatophores and a poly-$l-hydroxybutyrate (PHB) storage granule. x88,000.

FIG. 4. Electron micrograph of thin section through anaerobic, dark-grown mutant C showing a concentricallyaligned membrane structure (MS) and chromatophores (Chr). x80,000.

FIG. 5. Electron micrograph of thin section of anaerobic, dark-grown mutant C containing membrane-bound,electron-dense vesicles (ED). x 144,000.

FIG. 6. Electron micrograph of thin section through vesicles (ED) attached to membrane material inside mu-

tant C lysed by osmotic shock treatment (22). Cells were fixed with glutaraldehyde (3% w/v) followed by osmiumtetroxide. (Phillips 300 EM electron microscope operating at 80 kv, 30-a,m objective aperture.) x82,000.

FIG. 7. Enlargement of the vesicle in the lower right portion of Fig. 6. x164,000.1352

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MUTANTS OF R. RUBRUM

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FIG. 8. Electron micrograph of thin longitudinal section of anaerobic, dark-grown mutant GI. x60,000.FIG. 9. Electron micrograph of thin longitudinal section ofanerobic, light-grown revertant G2. x64,000.FIG. 10. Electron micrograph of thin section through a membrane aggregate observed in anaerobic, dark-

grown mutant GI. Loops of membrane (arrow) extend into the surrounding cytoplasm. x 144,000.FIG. 11. Electron micrograph of thin section of anaerobic, dark-grown mutant GI showing a membrane aggre-

gate continuous with (arrow) the cell membrane (operating at 80 kv, 35-,um objective aperature). x440,000.

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UFFEN, SYBESMA, AND WOLFE

absorption ratio of approximately 2: 1, respec-

tively. Only trace amounts of carotenoids weredetectable.

Light-induced reactions. Photosynthetic ac-

tivity of both Gl and C was examined by meas-

uring light-induced changes in the absorptionspectrum. In GI cells, no light-induced reactionof cytochromes could be detected. Small light-induced changes in the near-infrared spectral re-gion, however, indicated some photo-oxidation ofreaction center bacteriochlorophyll a. The light-minus-dark difference spectrum (Fig. 12) showeda bleaching centered at about 870 nm and a blueshift of an absorption band at about 800 nm.These spectral features are characteristic for thephoto-oxidation of the high-potential (+440 mv)reaction center P870 (4). The extent of the ab-sorption change was small, and the reaction was

sluggish.In contrast, C cells showed marked and much

more efficient photosynthetic activity. The photo-oxidation of only one cytochrome, even at ac-tinic intensities as high as 7 nano-Einstein percm2 per sec, could be detected. Figure 13 showsthat this cytochrome is the low-potential, cyto-chrome-like pigment C428, which was previouslyfound to be photo-oxidized at low actinic lightintensities (28). Mutant C did not show absorb-ance changes characteristic for the oxidation ofP870 photoreaction center. The light-minus-darkspectrum (Fig. 14) in the near-infrared spectralregion showed features which could be inter-preted as a red shift of an absorption band atabout 865 nm (11, 14). The light-induced absorb-ance changes in the near-infrared spectral regionwere rapid and monophasic and were the onlyones measured in dark-grown C in the near-in-frared spectral region.

FIG. 12. Light-minus-dark difference spectrum of

absorbance changes in mutant GI. Intensity of actiniclight at 586 nm was 3.5 nano-Einstein per cm2 per sec.Whole cell suspension; approximately 0.6 nmole ofbacteriochlorophyll a per mg ofprotein.

360 380 400 420 440 500 520 540 560 580

FIG. 13. Light-minus-dark difference spectra ofabsorbance changes in the visible spectral region ofmutant C. Actinic light at 874 nm (I874) was at intensi-ties of 0.4 (O ) and 7 (0) nano-Einstein per cm2 per

sec. Whole cell suspension; approximately 8.0 ,umolesofbacteriochlorophyll a per mg ofprotein.

FIG. 14. Light-minus-dark difference spectrum ofabsorbance changes in the near-infrared spectral regionin mutant C. Intensity of actinic light at 586 nm was

2.4 nano-Einstein per cm2 per sec. Whole cell suspen-sion; approximately 8.0 umoles of bacteriochlorophylla per mg ofprotein.

Metabolism of pyruvate. R. rubrum grew rap-idly in the dark under an argon atmosphere inthe presence of sodium pyruvate and producedacetate, carbon dioxide, and hydrogen gas. Inaddition, cells synthesized poly-,B-hydroxybutyricacid storage granules (Fig. 3). The synthesis ofthis product is presented as a function of cellprotein in Table 3. After 65 hr of dark growth, Ccells contained approximately twice as muchpoly-,B-hydroxybutyric acid as cells grown in thelight for 72 hr. Light-sensitive G1 accumulatedalmost four times as much poly-f-hydroxybu-tyric acid after 24 hr of growth than did photo-synthesizing G2 cells after 72 hr. The metabo-lism of sodium pyruvate and synthesis of poly-,B-hydroxybutyrate during dark growth is beinginvestigated.

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TABLE 3. Poly-f3-hydroxybutyric acid (PHB) production by mutants of Rhodospirillum rubrum S, underanaerobic, dark or light growth conditions in liquid medium

Mutant Growth Growth period PHB Protein PHB per mg ofstrain conditiona (hr) (pmoles) (mg) protein (gmole)

C dark 24 0.53 2.44 0.2248 0.74 1.80 0.4165 0.84 1.42 0.58

C lightb 48 0.25 1.32 0.19______________ 72 0.82 2.88 0.28

GI dark 24 2.13 2.60 0.8232 1.84 2.00 0.92

G2 light 48 0.27 3.60 0.0872 0.80 3.87 0.21

a Cells grown under strict anaerobic conditions.Approximately 60 foot candles.

It was noted that cells produced hydrogen sul-fide during dark growth. Cystein, supplied as areducing agent in the crude growth medium (3,32), may be metabolized to pyruvate with therelease of H2S.

DISCUSSIONIn these initial studies on cells which have been

transferred and grown for long periods of timeanaerobically in the dark, the amount of bacte-riochlorophyll a synthesized during growth grad-ually decreased, and after more than 100 genera-tions only trace amounts were detected. Observa-tions of thin sections of these cells demonstratedtwo general types of internal structure: strain C,which formed chromatophores, and strain GI, inwhich chromatophores were difficult to find inthin sections.The function of a photosynthetic apparatus

during light growth has been extensively studied;it is now possible to study its activity during an-aerobic, dark growth. The photosynthetic appa-ratus present in C appears different from normalcells. Characteristics of cyclic light-induced elec-tron transport (the photo-oxidation of the reac-tion center bacteriochlorophyll P870 and cyto-chrome c2) are missing (cf. reference 29). Onlythe low-potential pigment C428, which has beenproposed to function during noncyclic electrontransport (14, 28), is oxidized by light (Fig. 13).No indication of light-induced oxidation of reac-tion center bacteriochlorophyll P870 is seen. In-stead, light-induced absorbance changes in thenear-infrared spectral region showed a red shiftof an absorption band at about 865 nm (Fig. 14).Similar reactions are present in normal photo-synthesizing cells in a late stage of development(1 1, 14). The light-induced, near-infrared absorb-ance changes may reflect the photo-oxidation ofa low-potential reaction center component, dif-ferent from P870, which is re-reduced by the

subsequent oxidation of pigment C428. The pres-ence of only pigment C428 and a reaction centercomponent different from P870 may mean thatmutant C synthesizes a low-potential electrontransport system which can function during darkmetabolism. Pigment-producing cells that de-velop during long-term dark growth in low redoxconditions may be selected for their ability torepress synthesis of the high-potential cyclic elec-tron transport pathway required in photophos-phorylation (4, 29). In the light, P870 becomesfunctional during photosynthetic growth. Theabsence of P870 reaction center in these dark-grown cells cannot be stated with certainty, how-ever. It is possible that the reaction betweenC428 and high-potential P870 is so fast that itcannot be observed in experiments with contin-uous actinic light even at high intensities;flashing light should give results which are con-clusive in this respect.

In poorly pigmented GI, no light-induced cy-tochrome reactions could be detected. The light-induced absorbance changes measured in thenear-infrared spectral region (Fig. 12) show areversible photo-oxidation of high-potential P870reaction center component. The absorbancechanges, however, were small and sluggish. Be-cause of this, the lack of cytochrome activity,and the discovery that G1 is light-sensitive, therudimentary remains of a P870 photoreactivecenter may not be physiologically functional. Thetransport of electrons in these nonpigmentedcells is probably different from the noncyclicphotosynthetic mechanism suggested for C.The production of poly-,B-hydroxybutyric acid

and hydrogen during dark fermentation of pyru-vate is difficult to reconcile with published data(1, 2, 12, 13, 18, 27; K. T. Shanmugam and D. I.Arnon, Fed. Proc., p. 1136, 1971). To ourknowledge, this is the first report of poly-,B-hy-droxybutyric acid synthesis in fermentative me-

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UFFEN. SYBESMA, AND WOLFE

tabolism. The data suggest that an

pyruvate clastic reaction may function

dark growth, electrons being transferred

drogenase as well as to the polymer-forming

system (2, 7, 27).

Continued study of purple nonsulfur

during anaerobic, dark growth may provide

tions to a few of the unresolved questions

garding the metabolic activities of this

group of microorganisms. Hopefully,

strict anaerobic, dark growth techniques

additional opportunities to obtain mutant

which will contribute to our understanding

control and function of the photosynthetic

ratus in light-grown cells. The observation

R. rubrum becomes diverse under

anaerobic, dark growth (in the absence

selective pressure of radiant energy)

that a similar process could occur as

natural aquatic environment.

ACKNOWLEDGMENTS

We thank H. Gest, E. E. Ishiguro, and

helpful discussions. The electron micrograph

6 and 7 was kindly provided by S. Pankratz.

This investigation was supported by

fellowshipAl-33148 from the National Institute

Infectious Diseases to R.L.U.

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