Vol. 56, No. 11

Bacteriochlorophyll and Photosynthetic Reaction Centers inRhizobium Strain BTAi 1

WILLIAM R. EVANS,lt* DARRELL E. FLEISCHMAN,2 HARRY E. CALVERT,lt PADMA V. PYATI,1§GERALD M. ALTER,2 AND N. S. SUBBA RAO3II

Battelle-C. F. Kettering Research Laboratory, Yellow Springs, Ohio 453871; Department of Biochemistry,Wright State University, Dayton, Ohio 454352; and Division of Microbiology,

Indian Agricultural Research Institute, New Delhi-i 10 012, India3

Received 9 May 1990/Accepted 10 September 1990

Rhizobium strain BTAi 1, which nodulates both stems and roots of Aeschynomene indica L., formedbacteriochlorophyll and photosynthetic reaction centers resembling those of purple photosynthetic bacteriawhen grown aerobicaHly ex planta under a light-dark cycle. Bacteriochlorophyll formation was not observedunder continuous dark or light growth conditions. The amount of pigment formed was similar to thatpreviously found in aerobic photosynthetic bacteria. Stem nodules appear to fix nitrogen photosynthetically, as

illumination of A. indica stem nodules with near-infrared light resulted in an enhanced rate of acetylenereduction. Near-infrared light did not enhance acetylene reduction when either A. indica or soybean rootnodules were illuminated. The BTAi 1 isolate can be differentiated from members of the family Rhodospiril-laceae by several criteria.

Nitrogen-fixing symbioses between legumes and soil bac-teria occur almost exclusively in nodular tissue on plantroots. Notable exceptions have been reported in Neptuniaoleracea (25), Sesbania rostrata (8), and Aeschynomeneindica (4, 35), in which rhizobium-induced nodules alsodevelop on stems. A cytological examination of a collectionof stem and root nodules ofA. indica obtained near Madurai,India, revealed the presence of an unusual coccoid endo-phyte with an elaborate internal membrane system resem-bling that of purple photosynthetic bacteria. Although thesenodules had been fixed in the field at the time of collection,endophytes from fresh A. indica nodules had previouslybeen identified as a Rhizobium sp. (35). Similar observationshave been made by Vaughn and Elmore with stem nodules ofA. indica collected in Mississippi (33).

Endophytes subsequently isolated from a few unfixed A.indica stem nodules collected near Chidambaram, India,displayed fluorescence excitation and emission spectra re-

sembling those of purple photosynthetic bacteria (D. Fleisch-man, W. R. Evans, A. R. J. Eaglesham, H. E. Calvert, E.Dolan, Jr., N. S. Subba Rao, and S. Shanmugasundaram, inS. K. Dutta and C. Sloger, ed., Proceedings of the Interna-tional Symposium and Workshop on Biological NitrogenFixation Associated with Rice Production, in press). Theseobservations seemed to suggest that a coccoid endophytehaving photosynthetic properties may occupy stem noduleson A. indica plants growing throughout India and perhaps inthe United States as well. However, efforts to culture thenodule endophytes anaerobically in the light were unsuc-

cessful.

* Corresponding author.t Present address: Department of Biological Sciences, Wright

State University, Dayton, OH 45435.t Deceased.§ Present address: Hipple Cancer Research Center, Dayton, OH

45439.|| Present address: Department of Agricultural Microbiology, Uni-

versity of Agricultural Sciences, Indian National Academy of Sci-ences, Bangalore-560 065, India.

Concurrent with our cytological and spectrophotometricstudies of the endophytes collected in India, experimentswere also being carried out with Rhizobium strain BTAi 1,previously isolated at the Boyce Thompson Institute byEaglesham and Szalay (10).The purpose of this study was to determine whether the

BTAi 1 isolate can form the photosynthetic system ex plantaand whether illumination of A. indica BTAi 1 stem noduleswith light which can drive photosynthetic electron transportin the endophytes but not in the host chloroplasts has anyeffect on the rate of acetylene reduction.

MATERIALS AND METHODSElectron microscopy. A. indica nodules were collected

from plants growing near Madurai, India, and immediatelyfixed in 3% glutaraldehyde in 0.5 M phosphate buffer, pH6.9. The nodules were brought to the United States, wherethey were cut into smaller pieces (approximately 2 mm3),rinsed briefly in buffer, and fixed in 2% osmium tetroxide in0.05 M sodium-potassium phosphate buffer (pH 6.9) for 4 hat room temperature. After several short rinses in buffer, thetissue was dehydrated in a graded water-ethanol series andembedded in Spurr's epoxy resin. Thin sections were cutfrom the polymerized tissue blocks with a Sorvall MT2ultramicrotome, stained with 2.5% aqueous uranyl acetate,counterstained in alkaline lead citrate and examined with a

Philips 200 transmission electron microscope.Bacterial strain. Rhizobium strain BTAi 1 isolated from A.

indica was obtained from A. R. J. Eaglesham of the BoyceThompson Institute.Media and culture conditions. Growth experiments were

conducted in a gyratory shaker (150 rpm) at 26 to 28°C undera bank of incandescent lights (6.2 W/m2) on a 16-h/8-hlight-dark cycle. A mineral salts medium (12) supplementedwith various carbon and nitrogen sources and 40 mM MOPS(morpholinepropanesulfonic acid) or MOPSO (3-[N-morpho-lino]-2-hydroxypropanesulfonic acid) buffer at pH 6.8 was

employed. Malate (0.15%) was routinely used as the carbonsource, and yeast extract (0.05%) was routinely used as thenitrogen source. Inocula, usually 1%, were obtained from

3445

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1990, p. 3445-34490099-2240/90/113445-05$02.00/0Copyright © 1990, American Society for Microbiology

on August 5, 2020 by guest

http://aem.asm

.org/D

ownloaded from

APPL. ENVIRON. MICROBIOL.

cultures grown in the medium of Stowers and Eaglesham(29). Routinely, 250-ml Erlenmeyer flasks containing 100 mlof medium were used. In most of the experiments, thebacteria were allowed to grow for 5 to 7 days before pigmentanalysis.Pigment content of cells. To determine the pigment com-

position, the cells were centrifuged and washed twice with0.05 M Tris-0.005 M EDTA (pH 8) buffer. The pellet wasextracted with acetone-methanol (7:2, vol/vol) for 30 min inthe dark. The supernatant was analyzed spectrophotometri-cally with a Varian 2200 spectrophotometer. The dry weightof the extracted pellet was determined, and the pigmentcontent was calculated on a dry weight basis by using 76mM-1 cm-1 as the extinction coefficient of bacteriochloro-phyll a at the 767-nm absorption maximum (6).

Spectrophotometric analyses. The absorption spectrum ofintact cells was recorded with a Cary 14 spectrophotometerequipped with a scatter attachment which allowed the cu-vette to be placed adjacent to the photomultiplier in order tominimize loss of scattered light.The membrane fraction was prepared by suspending cells

in 0.05 M Tris hydrochloride (pH 7.5), breaking them with aFrench pressure cell, centrifuging the resultant extract at15,000 x g for 15 min to remove whole cells and debris, andfinally centrifuging the supernatant for 1 h at 100,000 x g.The pellet was resuspended in the Tris hydrochloride buffer.

Light-induced absorbance changes were measured with anAminco-Chance dual beam spectrophotometer as describedin reference 14. For the kinetic trace (see Fig. 4), thespectrometer was operated in a split beam mode; excitinglight was passed through a Corning 4-96 glass filter, and thephotomultiplier was protected by a 780-nm interference filterin order to minimize detection of fluorescence.

Acetylene reduction. A section of A. indica stem (2.5 cmlong and 6 mm in diameter), previously inoculated withstrain BTAi 1 and containing 25 nodules, was bisectedlongitudinally. The halves were placed in a transparentplastic flask containing 10% acetylene in air in an orientationsuch that all of the nodules faced a light source (a 1,000-Wtungsten halogen lamp). The flask was wrapped in aluminumfoil and incubated in the dark. At the time indicated by thearrow in Fig. 6, the foil was removed and the nodules wereilluminated with an intensity of 800 WIm2; the light wasfiltered through 3 cm of water and a heat-rejecting filter witha cutoff at about 950 nm and through a RG-780 glass filter(transmittance of <1% at wavelengths below 730 nm) toremove light which could drive electron transport or oxygenevolution in the cortical chloroplasts. For comparison, fivesoybean nodules (each about 1.5 mm in diameter) wereexcised from a root and similarly incubated and illuminated.Gas samples were periodically removed, and ethylene accu-mulation was measured by gas chromatography as describedin reference 13.

RESULTS AND DISCUSSION

Ultrastructure of nodule cells and coccoid endophytes fromA. indica plants collected near Madurai, India. The cytoplasmof nodule cells containing the coccoid endophyte appearsnormal, suggesting that the unusual endophyte has elicitedno pathogenic response in the host (Fig. 1A). Indeed,ultrastructural examination shows that the coccoid bacte-rium occupies the same topological space in the host cell asthe Rhizobium sp. Each bacterial cell is surrounded by aperiplasmic space which contains fine fibrillar material (Fig.1B). The periplasmic space is limited by a periplasmic

FIG. 1. Structure of nodules and endophytes from A. indicaplants growing near Madurai, India. (A) Transmission electronmicrograph of an A. indica nodule cell infected with the sphericalendophyte. This host cell shows an infection density typical for thisorganism. Samples were fixed in 3% glutaraldehyde at the time ofcollection and returned to our laboratory, where conventionalprocessing was completed. Bar = 10 ,um. (B) A transmissionelectron microscope close-up of a single endophyte cell showing itsconspicuous central nucleoplasm (N), numerous membrane-boundvesicles in the peripheral cytoplasm (V), and the gram-negative cellwall. Each endophyte cell is enclosed by a peribacteroid membrane(arrowheads). Fibrillar material can be seen in the peribacteroidspace (double arrowheads). Bar = 1 ,um.

membrane which is continuous with the plasmalemma of thehost cell.The coccoid endophyte is a spherical procaryote 2.5 to 3.5

,um in diameter. It has a trilayered cell wall typical ofgram-negative bacteria and a conspicuous central nucleo-plasm containing darkly-stained chromatin bodies. The re-mainder of the cytoplasm is filled with membranous inclu-sions, generally in the form of small spherical vesicles.Ultrastructurally, this organism bears a striking resemblanceto the purple photosynthetic bacteria of the genusRhodobacter.Growth and pigment formation in BTAi 1 cultures. Colo-

nies of BTAi 1 grown on agar in a mineral medium (12)containing 0.15% malate and 0.05% yeast extract werepinkish red in color, while colonies grown either in themedium employed by Stowers and Eaglesham (29) or in ayeast extract-mannitol medium (5) were creamy white incolor. These agar plates had been subjected to the normallight-dark cycle of the laboratory. Spectrophotometric ex-

3446 EVANS ET AL.

on August 5, 2020 by guest

http://aem.asm

.org/D

ownloaded from

PHOTOSYNTHETIC RHIZOBIUM STRAIN 3447

0.8 higher, whereas members of the Rhodospirillaceae grow

0.7 -\ under those conditions. BTAi 1 does not grow on plates ofsoybean tryptone, while Rhodospirillum rubrum does grow

0.6 - on this medium. Repeated attempts were made to growBTAi 1 anaerobically in the light or dark, in medium utilized

0.5 to grow members of the family Rhodospirillaceae (28), or incQ0.4 \ the medium used to grow the bacteria aerobically. However,

co \ Agrowth was never obtained either in liquid culture in com-O 0.3 - pletely filled glass-stoppered bottles or on plates in anaerobiccn< 0.2 jars.

Until Sato (24) and Harashima et al. (18) reported theo, presence of bacteriochlorophyll in obligately aerobic facul-oo0 . . . . ffitative methylotrophs and aerobic marine bacteria, this pig-

400 500 600 700 800 900 1000 ment had been found only in photosynthetic bacteria capableWavelength, nm of growing anaerobically in the light. The "aerobic photo-

synthetic bacteria" described by Sato and Harashima et al.,FIG. 2. Absorption spectrum of BTAi 1 cells. Cells were grown lik BTA 1, ar unbet rwaarbcly vnihin malate-containing medium under a 16-h/8-h light-dark cycle. The like BTAi 1, are unable to grow anaerobically, even in thecells were collected by centrifugation and resuspended in 0.05 M light, in the absence of auxiliary electron acceptors such asTris hydrochloride buffer, and their absorption spectrum was re- trimethylamine-N-oxide (3, 27). Photosynthetic ATP forma-corded with a Cary 14 spectrophotometer equipped with a scatter tion and the photochemical oxidation of reaction centerattachment. bacteriochlorophyll and cytochrome have been demon-

strated in membrane preparations of Protaminobacter ruberNR-1 (30) and Erythrobacter sp. strain OCh 114 (16, 21, 22),

amination of the pigmented colonies revealed the presence but only under aerobic conditions. The properties of theof carotenoidlike absorption bands in the visible region as aerobic photosynthetic bacteria have been reviewed in awell as an A8870 characteristic of members of the family recent monograph (17).Rhodospirillaceae. Both the pigmented and white colonies Pigment formation in liquid cultures of BTAi 1 was ob-were capable of nodulating seedlings of A. indica grown in served only with intermittent lighting; under continuousplastic pouches. illumination or continuous darkness no pigmentation was

Further spectrophotometric analyses of BTAi 1 were found. With intermittent periods of light and darkness, BTAicarried out with bacteria grown in liquid culture. Figure 2 is 1 produced 0.40 nmol of bacteriochlorophyll per mg (dryan absorption spectrum of whole cells of BTAi 1 showing an weight) of cells (average of three experiments). This prop-A870 band similar to that of the long-wavelength antenna erty is similar to that observed by Sato (24) for Pseudomonasbacteriochlorophyll protein (LHC I) of bacteriochlorophyll spp., in which either continuous illumination or continuousa-containing purple photosynthetic bacteria (15, 31). Endo- darkness inhibited pigment formation. However, this is notphytes isolated from stem nodules of A. indica inoculated the case with marine bacteria, which synthesize bacterio-with BTAi 1 also exhibited an absorption spectrum similar to chlorophyll in the dark (18). Preliminary results also indicatethat of bacteria grown ex planta, but with a smaller A800 band that pigment formation may be controlled by the carbon(Fleischman et al., in press). source (data not shown). The amount of bacteriochlorophyll

Figure 3 is a typical spectrum of an acetone-methanol (7:2, synthesized aerobically by BTAi 1 with malate as the carbonvol/vol) extract of BTAi 1 cells. Acidification of the extract source was similar to those previously observed in aerobicresulted in the characteristic replacement of the A770 band of photosynthetic bacteria, 0.045 to 0.16 nmol per mg (drybacteriochlorophyll a by the A750 band of bacteriopheophy- weight) of cells for methylotrophs (24) and 0.9 to 5.5 nmoltin a. per mg (dry weight) for marine bacteria (27). However, theThe BTAi 1 isolate can be distinguished from the members amount of bacteriochlorophyll formed by BTAi 1 was much

of the Rhodospirillaceae by several criteria. Growth of BTAi less than that normally observed in anaerobically grown1 was inhibited by malate at concentrations of 0.5% or members of the Rhodospirillaceae (11 to 13 nmol per mg [dry

weight] [20]). Members of the Rhodospirillaceae grownaerobically in the dark contain 1 to 5% of the amount found

0.4. a in anaerobically grown cells (19).Photochemical activity. Membranes isolated from BTAi 1

0.3 - displayed reversible light-induced absorbance changes sim-ilar in kinetics (Fig. 4) and spectrum (Fig. 5) to those due to

/l.{ l electron transfer within the photosynthetic reaction centersDo 0.2 of members of the Rhodospirillaceae (7) or aerobic photo-

synthetic bacteria (16). If it is assumed that the peak extinc-tion coefficients of the longest-wavelength absorption bands

0.1 l of the reaction centers and the light-harvesting chlorophyllprotein are similar to those in R. rubrum (143 mM' cm

0.0 §S*4 ' rns hs ecto ete ieis r hwnii.[32]and 153 mM-1 cm-1 [6], respectively), then the mem-50 450 550 e50 5 branes whose reaction center kinetics are shown in Fig. 4Wavelength, nm contain about 80 mol of light-harvesting bacteriochlorophyll

FIG. 3. Absorption spectrum of an acetone-methanol (7:2, vol/ per mol of reaction centers. This ratio is typical of purplevol) extract of BTAi 1 cells. Symbols: , before addition of HCI photosynthetic bacteria (7).to convert bacteriochlorophyll to bacteriopheophytin; ---, after Enhancement of acetylene reduction by light. Most photo-addition of HCl. synthetic bacteria fix nitrogen photosynthetically (36). To

VOL. 56, 1990

on August 5, 2020 by guest

http://aem.asm

.org/D

ownloaded from

APPL. ENVIRON. MICROBIOL.

CO 0.004.0D0.002t r0A

* o 5 10 15Time, seconds

FIG. 4. Kinetics of light-induced A780 change in BTAi 1 mem-branes. Exciting light was turned on and off at the times marked byupward- and downward-pointing arrows, respectively. The mem-branes were suspended in 0.05 M Tris hydrochloride buffer to givean optical A873 of 0.490.

determine whether the Aeschynomene endophytes can fixnitrogen photosynthetically under the microaerobic condi-tions present in the nodule, the effect of light on acetylenereduction was examined in nodulated stem sections. Eardlyand Eaglesham had previously shown that white light stim-ulates acetylene reduction by BTAi 1-containing Aeschy-nomene stem nodules and speculated that the effect might bedue to oxygen evolved by the illuminated chloroplasts in thenodule cortex (11). This speculation was supported by theirobservation that increased oxygen levels indeed accelerateacetylene reduction in the dark. In order to assure that lightwould be absorbed only by the endophytes and not bychloroplasts in the outer nodule cortex (to preclude artifactsresulting from oxygen evolution or other light-dependentchloroplast functions), filters which transmitted light only inthe spectral region between 730 and 940 nm were employed.In each of about a dozen experiments, illumination resultedin at least a doubling of the rate of acetylene reduction (Fig.6). Because light only beyond 730 nm was absorbed by thenodule sections, the increase in the rate of acetylene reduc-tion could not be due to enhanced oxygen diffusion resultingfrom photosynthesis by chloroplasts in the nodule cortex.A telethermometer probe attached to an unilluminated

side of the incubation flask indicated a temperature increaseof about 3°C during the first few minutes of illumination; thetemperature increase within the illuminated nodules wasundoubtedly greater. In order to obtain some informationabout whether such heating might influence acetylene reduc-tion rates, similar experiments were performed with soybean(Fig. 6), alfalfa, and Aeschynomene root nodules (data not

0.002

0.001C

co 0.000D0

D -0.001

< -0.002

-0.003

400 500 600 700 800 900

Wavelength, nm

FIG. 5. Light-minus-dark difference spectrum of light-inducedabsorbance changes in BTAi 1 membranes. The spectrum is a plot ofthe wavelength dependence of the amplitude of kinetic traces suchas that in Fig. 4. The membranes were from the same preparation as

those of the experiment whose results are shown in Fig. 4, but at a

2.4-fold higher concentration.

co00E

0ILE00

.-C

'54 E

3 @cE0

2uLa)

1 O-w

Time, HoursFIG. 6. The effect of near-infrared illumination on acetylene

reduction by A. indica stem nodules (a; left ordinate scale) and bysoybean root nodules (0; right ordinate scale). Arrows indicatebeginning of illumination. An A. indica stem section, previouslyinoculated with BTAi 1 and containing 25 nodules, was bisectedlongitudinally and placed in a flask containing 10% acetylene in air.At the time marked by the arrow, the sides containing the noduleswere illuminated through filters transmitting light of wavelength>730 nm. Five soybean nodules were incubated and illuminated ina similar manner.

shown). No light stimulation of acetylene reduction occurredin these experiments.Although these results are preliminary, they suggest that

the endophytes are photochemically active in situ. Theability of the endophytes to use light in a wavelength regionwhich cannot be used by chloroplasts could be of value tothe plant, since it would diminish competition betweencarbon and nitrogen fixation for energy. In addition, nitrogenfixation using ATP generated within the endophytes byphotophosphorylation would decrease the demand for avail-able chloroplast-produced photosynthate, which is thoughtto limit symbiotic nitrogen fixation (23). The capability ofAeschynomene stem nodules to reduce acetylene persists fora number of days, even after the plant has been defoliated(11).The photosynthetic system may also help the rhizobium

survive in the environment outside the plant. Adebayo et al.(1) have recently reported that rhizobia which form stemnodules on A. indica are found on the leaves of their hostsand other plants in much higher concentrations than arerhizobia which are capable only of root nodulation. Illumi-nation of BTAi 1 cultures substantially prolongs their viabil-ity (A. R. J. Eaglesham, personal communication), as doesillumination of cultures of the aerobic photosynthetic bacte-rium Erythrobacter sp. strain OCh 114 (26).

Possible phylogenetic implications. On the basis of thesimilarity of their 16S rRNA sequences, Woese (34) hasproposed that purple photosynthetic bacteria are closelyrelated to Rhizobium spp. and, in fact, may have been theirevolutionary precursors. The data obtained in this studysupport that proposal, as the Rhizobium strain used hasattributes of both the purple bacteria and Rhizobium spp.These include the capacity to synthesize bacteriochlorophylland photosynthetic reaction centers along with the ability toinfect and produce nitrogen-fixing nodules on a legume. It isof interest that the rhizobium which forms stem nodules onS. rostrata has been reported to share more phenotypiccharacteristics with photosynthetic bacteria than with otherrhizobia and has been assigned to a separate genus,Azorhizobium (9). Unlike the rhizobia which form only rootnodules on Aeschynomene and Sesbania spp., the rhizobiawhich form stem nodules on these plants are able to fixnitrogen in pure culture (2, 9; M. Hungria, J. M. Ellis, A. R.J. Eaglesham, and R. Hardy, unpublished data).

.. -.1

j 'IV

I

3448 EVANS ET AL.

on August 5, 2020 by guest

http://aem.asm

.org/D

ownloaded from

PHOTOSYNTHETIC RHIZOBIUM STRAIN 3449

We have attempted to demonstrate the presence of bacte-riochlorophyll in Azorhizobium caulinodans ORS 571, inAeschynomene americana Rhizobium strain USDA 3412,and in two carotenoid-containing Rhizobium strains, USDA3412 and USDA 3413, which nodulate Lotonis bainseiiBaker but have been unsuccessful. Nevertheless, it seemsworthwhile to examine other stem- and leaf-nodulating bac-teria for the potential to synthesize the photosyntheticapparatus.

ACKNOWLEDGMENTS

This study was supported in part by the Indo-U.S. Science andTechnology Initiative and the Program for Science and TechnologyCooperation of the United States Agency for International Devel-opment.We thank A. R. J. Eaglesham for cultures of Rhizobium sp. strain

BTAi 1, A. indica seeds, and valuable discussions.

LITERATURE CITED1. Adebayo, A., I. Watanabe, and J. K. Ladha. 1989. Epiphytic

occurrence of Azorhizobium caulinodans and other rhizobia onhost and nonhost legumes. Appl. Environ. Microbiol. 55:2407-2409.

2. Alazard, D. 1990. Nitrogen fixation in pure culture by rhizobiaisolated from stem nodules of tropical Aeschynomene species.FEMS Microbiol. Lett. 68:177-182.

3. Arata, H., Y. Serikawa, and K. Takamiya. 1988. Trimethyl-amine N-oxide respiration by aerobic photosynthetic bacterium,Erythrobacter sp. OCh 114. J. Biochem. 103:1011-1015.

4. Arora, N. 1954. Morphological development of the root andstem nodules of Aeschynomene indica L. Phytomorphology4:265-272.

5. Bhuvaneswari, T. V., K. K. Mills, D. K. Crist, W. R. Evans, andW. D. Bauer. 1983. Effects of culture age on symbiotic infectiv-ity of Rhizobium japonicum. J. Bacteriol. 153:443-451.

6. Clayton, R. K. 1966. Spectroscopic analysis of bacteriochloro-phylls in vitro and in vivo. Photochem. Photobiol. 5:669-677.

7. Clayton, R. K. 1980. Photosynthesis: physical mechanisms andchemical principles. Cambridge University Press, Cambridge.

8. Dreyfus, B., and Y. R. Dommergues. 1981. Nitrogen-fixingnodules induced by Rhizobium on the stem of the tropicallegume Sesbanis rostrata. FEMS Microbiol. Lett. 10:313-317.

9. Dreyfus, B., J. L. Garcia, and M. Gillis. 1988. Characterizationof Azorhizobium caulinodans gen. nov., sp. nov., a stem-nodulating nitrogen-fixing bacterium isolated from Sesbaniarostrata. Int. J. Syst. Bacteriol. 38:89-98.

10. Eaglesham, A. R. J., and A. A. Szalay. 1983. Aerial stem noduleson Aeschynomene spp. Plant Sci. Lett. 29:265-272.

11. Eardly, B. D., and A. R. J. Eaglesham. 1985. Fixation ofnitrogen and carbon by legume stem nodules, p. 324. In H. J.Evans, P. J. Bottomley, and W. E. Newton (ed.), Nitrogenfixation research progress. Martinus Nijhoff, The Hague.

12. Evans, W. R., and D. K. Crist. 1984. The relationship betweenthe state of adenylation of GS1 and the export of NH4 infree-living, N2-fixing Rhizobium. Arch. Microbiol. 138:26-30.

13. Evans, W. R., and D. L. Keister. 1976. Reduction of acetyleneby stationary cultures of free-living Rhizobium sp. under atmo-spheric oxygen levels. Can. J. Microbiol. 22:949-952.

14. Fleischman, D. E., and J. A. Cooke. 1971. Electron transport inRhodopseudomonas viridis at low temperatures. Photochem.Photobiol. 14:71-83.

15. Glaser, A. N. 1983. Comparative biochemistry of photosyntheticlight-harvesting systems. Annu. Rev. Biochem. 52:125-157.

16. Harashima, K., M. Nakagawa, and N. Murata. 1982. Photo-chemical activities of bacteriochlorophyll in aerobically growncells of aerobic heterotrophs, Erythrobacter sp. (OCh 114) and

Erythrobacter longus (OCh 101). Plant Cell Physiol. 23:185-193.17. Harashima, K., T. Shiba, and N. Murata. 1989. Aerobic photo-

synthetic bacteria. Japan Scientific Societies Press, Tokyo.18. Harashima, K., T. Shiba, T. Tatsuka, U. Simidu, and N. Taga.

1978. Occurrence of bacteriochlorophyll a in a strain of anaerobic heterotrophic bacterium. Agric. Biol. Chem. 42:1627-1628.

19. Lascelles, J. 1959. Adaptation to form bacteriochlorophyll inRhodopseudomonas spheroides: changes in activity of enzymesconcerned in pyrrole synthesis. Biochem. J. 72:508-518.

20. Lascelles, J. 1963. Tetrapyrroles in photosynthetic bacteria, p.35-52. In H. Gest, A. San Pietro, and L. P. Vernon (ed.),Bacterial photosynthesis. The Antioch Press, Yellow Springs,Ohio.

21. Okamura, K., F. Mitsumori, 0. Ito, K.-I. Takamiya, and M.Nishimura. 1986. Photophosphorylation and oxidative phos-phorylation in intact cells and chromatophores of an aerobicphotosynthetic bacterium, Erythrobacter sp. strain OChll4. J.Bacteriol. 168:1142-1146.

22. Okamura, K., K. Takamiya, and M. Nishimura. 1984. Photo-synthetic and respiratory electron transfer systems in an aerobicphotosynthetic bacterium, Erythrobacter sp. strain OCh 114, p.641 644. In C. Sybesma (ed.), Advances in photosynthesisresearch. Martinus Nijhoff, The Hague, The Netherlands.

23. Rawsthorne, S., F. R. Minchin, R. J. Summerfield, C. Cookson,and J. Coombs. 1980. Carbon and nitrogen metabolism inlegume root nodules. Phytochemistry 19:341-355.

24. Sato, K. 1978. Bacteriochlorophyll formation by facultativemethylotrophs, Protaminobacter ruber and Pseudomonas AM1. FEBS Lett. 85:207-210.

25. Schaede, R. 1940. Die Knollchen der adventiven Wasserwurzelnvon Neptunia oleracea und ihre Bakteriensymbiose. Planta31:1-21.

26. Shiba, T. 1984. Utilization of light energy by the strictly aerobicbacterium Erythrobacter sp. OCh 114. J. Gen. Appl. Microbiol.30:239-244.

27. Shiba, T., U. Simidu, and N. Taga. 1979. Distribution of aerobicbacteria which contain bacteriochlorophyll a. Appl. Environ.Microbiol. 38:43-45.

28. Stanier, R. Y., M. Doudoroff, and E. A. Adelberg. 1970. Themicrobial world. Prentice-Hall, Inc., Englewood Cliffs, N.J.

29. Stowers, M. D., and A. R. J. Eaglesham. 1983. A stem-nodulat-ing Rhizobium with physiological characteristics of both fastand slow growers. J. Gen. Microbiol. 129:3651-3655.

30. Takamiya, K., and K. Okamura. 1984. Photochemical activitiesand photosynthetic ATP formation in membrane preparationsfrom a facultative methylotroph, Protaminobacter ruber strainNR-1. Arch. Microbiol. 140:21-26.

31. Thornber, J. P., T. L. Trosper, and S. E. Strouse. 1978.Bacteriochlorophyll in vivo: Relationship of spectral forms tospecific membrane components, p. 133-160. In R. K. Claytonand W. R. Sistrom (ed.), The photosynthetic bacteria. PlenumPublishing Corp., New York.

32. van der Rest, M., and G. Gingras. 1974. An immunological andelectrophoretic study of Rhodospirillum rubrum chromatophorefragments. Arch. Biochem. Biophys. 164:285-292.

33. Vaughn, K. C., and C. D. Elmore. 1985. Ultrastructural charac-terization of nitrogen-fixing stem nodules on Aeschynomeneindica. Cytobios 42:49-62.

34. Woese, C. R. 1987. Bacterial evolution. Microbiol. Rev. 51:221-271.

35. Yatazawa, M., and S. Yoshida. 1979. Stem nodules in Aeschy-nomene indica and their capacity of nitrogen fixation. Physiol.Plant. 45:293-295.

36. Yoch, D. C. 1978. Nitrogen fixation and hydrogen metabolismby photosynthetic bacteria, p. 657-676. In R. K. Clayton andW. R. Sistrom (ed.), The photosynthetic bacteria. Plenum Pub-lishing Corp., New York.

VOL. 56, 1990

on August 5, 2020 by guest

http://aem.asm

.org/D

ownloaded from