Adaptation of the bacterial photosynthetic apparatus to different light intensities

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11 Ray, W. J., Jr and Roscelli, G. A. (1964) J. BioL Chem. 239, 1228-1236

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16 Cheng, P-W. and Carlson, D. M. (1978) Anal. Biochem. 85,533-540 17 Wong, L-J. and Rose, I. A. (1976) J. BioL Chem. 251, 5431-5439 18 Rose, Z. B. and Whalen, R. G. (1973) J. BioL Chem. 248, 1513-1519 19 Rose, Z. B. (1980)Adv. Enzymol. 51,211-253 20 Haggarty, N. W., Dunbar, B. and Fothergill, L. A. (1983) EMBOJ. 2, 1213-1220 21 Han, C-H. and Rose, Z. B. (1979) .L Biol. Chem. 254, 8836-8840

Adaptation of the bacterial photosynthetic apparatus to different light intensifies Gerhart Drews Photosynthetic bacteria respond to variations in light intensity by undergoing a variety of adaptations in their photosynthetic apparatus in order to maintain a sufficient supply of energy and sufficient activity of photosynthetic units. This article reviews current information about how these adaptations are achieved and reguk~d in Rhodopseudomonas capsulata. In photosynthetic organisms most of the incident light energy is absorbed by antenna pigment systems. In purple bacteria these are integral membrane complexes consisting of bacteriochlorophyll a or b, carotenoids and low molecular weight hydrophobic polypeptides (for review see Ref. 1). Most purple bacteria have two antenna complexes which are named by their near infrared bacteriochlorophyll absorption maxima in vivo: B870 and B800-850; lightharvesting bacteriochlorophyll b absorbs at 1020 nm. There are about 20-150 antenna bacteriochlorophyll molecules per photochemical reaction center. In green bacteria bacteriochlorophyll c (d or e) is the predominant antenna bacteriochlorophyll species; it is localized in chlorosomes e and absorbs at 740 nm. The antennae of green bacteria are relatively large, and about 1000 bacteriochlorophyll molecules per reaction center have been determined. The light energy absorbed by antenna pigment molecules creates mobile excited singlet states which migrate by a 'random walk' over the antennae to the reaction center 3, where a charge separation over the membrane is established (for review see Refs 3 and 4). Efficient energy transfer is achieved by strictly oriented and organized pigment molecules in the plane of the membrane. Electrons and protons are transferred from the reaction center via a mobile G. Drews is at the Institut fiir Biologic 11, Mikrobiologie, Schiinzlstrasse 1, D-7800 Freiburg, FRG.

carder and ubiquinone-cytochrome b/c oxidoreductase. An electrochemical proton potential is formed, which drives photophosphorylation, i.e. the formation of ATP from A D P and inorganic phosphate (Pi). Bacterial photosynthesis is only active under anaerobic conditions and is sensitive to an optimum redox potential poised by external and internal redox systems 5. Oxygen is not produced. Changes of oxygen tension in the medium markedly affect the electrochemical proton potential and the rate of photophosphorylation. Additionally, in facultative phototrophic bacteria, oxygen tension regulates the formation of the photosynthetic apparatus. Cells of these bacteria, which have an alternative chemotrophic respiratory metabolism, have no bacteriochlorophyll and no photochemical activity when cultivated in an oxygen saturated culture medium in the dark. The formation of the photosynthetic apparatus is induced strongly when the oxygen partial pressure is reduced to low values of about 70 Pa. The synthesis of the antennae and reaction center complexes is not lightdependent, but light intensity is an external signal which regulates the formation of the photosynthetic apparatus under phototrophic growth conditions. All photosynthetic organisms have developed mechanisms of adaptation to different light intensities. An excess of absorbed light quanta can be dangerous if not channelled to the reaction centers or re-emitted as fluorescence. A shortage of light energy reduces rates of growth and metabolism and diminishes

22 Vanderheiden, B. S. (1970) Biochim. Biophys. Acta 215,242-248 23 Rose, I. A. and Warms, J. V. B. (1974) Biochem. Biophys. Res. Commun. 59, 1333-1340 24 Beitner, R. and Cohen, T. J. (1980) FEBS Len. 115,197-200 25 Wakelam, M. J. O., Emmerich, M. and Pette, D. (1982) Biochem. Z 208, 517-519 26 Bassols, A., Cusso, R. and Carreras, J. (1985) Comp. Biochem. Physiol. 81B, 981-987

the capacity for competition in the ecological niche. Here I will describe the process of light adaptation by membrane differentiation in Rhodopseudomonas capsulata. The photosynthetic apparatus of this facultative phototrophic bacterium is much simpler than that of higher plants; it can be induced and repressed by variations of oxygen partial pressure or light intensity and the process of formation has been studied at the molecular level.

Membrane differentiation after variation of light intensity When exponentially growing cells of R. capsulata are subjected to a sudden decrease in light intensity the growth rate is reduced or arrested because their energy metabolism becomes lightlimited6. The reduction in light intensity, however, initiates immediately an increased synthesis of pigment-protein complexes. Thus, fight is not only a substrate for energy metabolism but also a signal which triggers a process of cell differentiation. After a couple of hours cells are adapted to low light intensity and continue to grow at the same rate as before light intensity was lowered. They are no longer energy-limited. In cells adapted to a low light intensity (low light cells) the area of the intracytoplasmic membrane is more than sixfold that of cells grown under high light intensity (high light cells; Fig. 1, Table I; Ref. 6). The number of photosynthetic units (measured as the number of reaction centers per cell) increases fivefold and the size of the photosynthetic unit (that is the number of antenna bacteriochlorophyll molecules per reaction center) doubles (Table I). Many other photosynthetic organisms adapt to low light intensities by formation of additional and larger photosynthetic units 7. It was, however, a new and surprising result that the rates of photophosphorylation per reaction center showed different saturation kinetics. The K m for light, that is the half-saturating light intensity for photophosphorylation, increased from 5 W m -2 in membrane preparations of low light cells to 36 W m -2 in membrane preparations from

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high light cells the cyclic electron transport is faster and reaction centers have a higher turnover rate* than in membranes from low light cells. Very recent measurements showed a much higher content of ubiquinol-cytochrome b/coxidoreductase and of free ubiquinone per reaction center in high light than in low light cells (A. F. Garcia et al., unpublished). Low light cells are adapted to absorb photons with high efficiency in large antennae at low light intensity in order to funnel enough energy to reaction centers. The incorporation of many pigment-protein complexes into the membrane may limit the insertion of sufficient electron- and proton-transfer components, particularly units of the ubiquinone-cytochrome b / c - o x i d o r e d u c tase and ATPase. The photosynthetic units of high light cells are adapted for maximum photophosphorylation rates under conditions of saturating light intensity, high rates of electron transport and optimal cooperation between the photosynthetic units t°. It is not yet known whether the clustering of the antenna subunits is controlled by covalent modification of pigmentbinding proteins. Small conformational changes may influence the array of

Fig. 1. Rhodopseudomonascapsulata. Cross-sections of cells grown under (a) high light and (b) low light intensity. Under low light intensity there was a considerable increase in the number of intracytoplasmic membrane vesicles (chromatophores). Bar = O.1 pro. Electron micrographby J. R. Golecki.

high light cells6,8. The maximum rate of photophosphorylation under saturating light intensities per reaction center (Vr~a×) in membranes from high light cells was about threefold that of low light cells (Table I). Thus, photosynthetic units of low light cells use the incident light energy with higher efficiency but the photosynthetic units from high light cells achieve higher rates of photophos.phorylation at high light intensities. These different activities were not caused by variation in the sizes of the photosynthetic units, because similar results were obtained with a mutant which lacks the B 8 0 0 ~ 5 0 antenna complex and has a photosynthetic unit of constant sizes . Other experiments suggest that other constituents of the photosynthetic apparatus, as well as the pigment complexes, alter their relative molar concentrations during membrane differentiation. In membranes of high light cells the ratio of ATPase to reaction center was nearly eightfold that of membranes from low light cells (Ref. 8; Table I). Since the at/) operon appears to be constitutively expressed 9, a decrease in the rate of reaction center synthesis relative to membrane formation seems to be responsible for the observed increase in the ratio of ATPase to reaction center in membranes of high light cells. To learn more about the rates of electron transport, samples of the two membranes were excited with saturating flashes and the kinetics of reaction center reduction were measured. Twenty per cent of reaction centers in membranes of high light cells return to the reduced state within 50 ms after the flash, while only 10 per cent of reaction centers from low light cells were reduced within 400 ms (H. Reidl, PhD thesis, 1985). In isolated reaction center preparations with bound

cytochrome c2 this reduction takes less than 1 ms. The slow reduction may be caused by a low content of cytochrome c 2 (the electron donor for reaction centers) in membrane vesicles of low light cells. However, the content of cytochrome c 2 in high light membranes was at least twice that in low light membranes. The decay of the electric field over the membrane was measured by following the carotenoid bandshift after short saturating flashes: faster decay rates occurred in membranes from high light cells than those from low light cells (H. Reidl, PhD thesis, 1985). These results support the idea that in membranes from

*The rate of oxidation/reductioncycles.

Table I. Adaptation of Rhodopseudomonascapsulatato low light intensity a

High light

Low light

Cells

nmolBchl(mgcellprotein)-l nmol RC (mgcellprotein)-i b doubtingtime for cellprotein (min) area of intracytoplasmiemembrane (lam2) photophosphorylationb nmolATP (mg cellprotein)-1 min-I

2.14 0.03 190

18.3 0.145 190

0.4

2.5

140

180

Membranes

nmol Bchl(ragmembraneprotein)-l nmolRC (mg membraneprotein)-I sizeof the photosyntheticunit tool Bchl(mol RC)-1

3.4 0.052 65

25.9 0.205 126

Photophosphorylation nmolATP (nmolRC)-l min-l nmolATP (rag membrane protein)-~min-I

220

260

K~nfor fight(W m-2, 870 nm)

36

5

1100

140

58

28

ATPase mol PO43- (mol RC)-I min-1 nmol PO~- (mg membrane protein)-1 min-I

4.3

1.3

aCells grown phototrophically(anaerobic at high tight intensity, 2000 W) at 30°C in a turbidostat were cultivatedfor 6 h at low light intensity (40 W). Membraneswere isolated from high light and low light cells. Abbreviations:Bchl, baeteriochlorophyll;RC, reactioncenter. ~Calculatedfrom resultsobtained with membranesand Bchlcontent of membranesand cells(Ref. 6).

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antenna units and thus the exciton transfer in the antennae.

Condusion

These results, and preliminary inhibitor studies on bacteriochlorophyU synthesis, are consistent with the idea Regulation of the membrane that the synthesis of the pigment-binding differentiation by fight M e m b r a n e differentiation in photo- polypeptides is regulated at the transsynthetic bacteria, which is induced by criptional level 17,19, but an influence of changes of light intensity or oxygen pigments on translation is possible. In tension, is a process of insertion and bacteriochlorophyll-negative mutants, assembly of newly synthesized pigment-binding polypeptides are sucm e m b r a n e constituents and does not cessfully synthesized and incorporated involve degradation of existing com- into the membrane, but are quickly deplexes H-16. Different rates of phos- graded23; this shows that the assembly pholipid incorporation and of assembly of complexes is dependent on the presof specific complexes in the membrane ence of bacteriochlorophyll. Carotendetermine the final state of membrane oids are not necessary for the differentiation. Reduction of light in- assembly of reaction center and B870 tensity leads to an increase of complex, but seem to be essential for the bacteriochiorophyll, carotenoid and pig- formation of the B800-850 complex ~9. ment-binding polypeptides (particularly Bacteriochlorophyll synthesis seems to the B800-850 complex) in the mem- be regulated mainly by change in the brane fraction; clearly a signal chain activity of two or three key enzymes 24. must be triggered, but the sequence of How synthesis of protein and pigment events is as yet unknown (Refs 15 and 16; are coordinated and how bacteriochlorophyll and its precursor pools influTable I). The pools of m R N A s for bacterio- ence translation and assembly of the pigchlorophyll synthesis increase only ment-binding polypeptides, is still to be slightly and those for carotenoid syn- worked out. One speculation is that varithesis not at all when light intensity or ations of light intensity regulate the biosynthesis of the pigment complexes oxygen partial pressure are lowered, indicating that pigment synthesis is regu- via the redox state of enzymes or regulatlated mainly by changes in enzyme ory proteins 7. Very recently it has been observed activities. The m R N A s for the pigmentbinding polypeptides, however, increase that specific promoter sequences of the strongly 17-19. The genes for two reaction reaction center-B870 operon are oblicenter and two light-harvesting B870 gatory for the regulation of gene exprespolypeptides, which are combined in an sion under the influence of variations of operon 20, are cotranscribed2L The oxygen tension (J. T. Beatty et al., maximum rate of translation and in- unpublished). We propose that regulacorporation of the gene products into the tory gene products, in addition to the membrane, and the assembly of reaction described mechanisms, are active in center and light-harvesting complex coordinating the synthesis and assembly B870, occurs immediately after the of the reaction center-light-harvesting maximum rate of transcription of the constituents, as has been shown for other respective genes is attained w. Ten times functional complexes such as nitroas many B870 polypeptides are syn- genase. thesized as reaction center polypeptides, because their m R N A s have a longer Outlook At present, the formation of the pighalf-life, and their chain length is shorter21,22. The time taken for the genes for ment-protein complexes of the bacterial the three polypeptides of the B800-850 photosynthetic apparatus is under study complex to be transcribed and for in three species: Rhodopseudomonas maximum assembly of the complex in capsulata, R. sphaeroides and Chlorothe membrane is about 30 min later than flexus aurantiacus; all seem to respond to the respective processes for reaction changes in oxygen partial pressure or light intensity in a similar fashion. We center and B87019.

expect that the future work on these organisms will contribute to elucidating the molecular mechanisms involved in the formation of complex systems like membrane-bound functional complexes.

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