Archives of

Microbiology

Arch. Microbiol. 108,281-285 (1976)

9 by Springer-Verlag 1976

The Early Formation of the Photosynthetic Apparatus in Rhodospirillum rubrum J. OELZE and W. PAHLKE Lehrstuhl f(ir Mikrobiologie, Institut ffir Biologie II der UniversitS.t Schfinzlestr. 9, D-7800 Freiburg i. Br., Federal Republic of G e r m a n y

Abstract. The time dependent assembly of the photosynthetic apparatus was studied in Rhodospirillum rubrum after transfer of cells growing aerobically in the dark to low aeration. While bacteriochlorophyll (Bchl) cellular levels increase continuously levels of soluble cytochrome c2 do not change significantly. Absorption spectra of membranes isolated at different times after transfer reveal that incorporation of carotenoids lags behind incorporation of Bchl. However, a carotenoid fraction exhibiting spectral properties of spirilloxanthin isomers was isolated apart from membranes. This carotenoid fraction even was present in homogenates from Bchl-free, aerobically grown cells. Incorporation of U-14C-proteinhydroly zate into membrane proteins showed that proteins are mainly formed which are specific for photosynthetic membranes. Although the proportion of reaction center (RC) Bchl per light harvesting (LH) Bchl does not change the proportions of membrane proteins present in RC and LH preparations change initially. But later on the proportions of the different proteins also reach constant values. Concerning proteins characteristic for cytoplasmic membranes a differential incorporation of label can be observed. The data indicate that the photosynthetic apparatus in Rhodospirillum rubrum is assembled through a sequential mechanism. Key words." Formation of membrane proteins Photosynthetic apparatus - Bacteriochlorophyl! Carotenoid fraction - Soluble cytochrome c2 Rhodospirillum rubrum.

Rhodospirillum rubrum is able to grow phototrophically as well as chemotrophically (reviewed by Oelze Abbreviations. Bchl = bacteriochlorophyll, L H = light harvesting, R C - reaction center, R. = Rhodospirillum, R. = Rhodop seudomonas

and Drews, 1972). While phototrophy depends on light and the absence of oxygen chemotrophy depends on the presence of oxygen. Under chemotrophic conditions cells generate energy coupled to respiration. The essential constituents of the photosynthetic apparatus are not formed under aerobiosis because there is no need for light dependent energy fixation. But, upon transfer of chemotrophically grown cells to specific conditions the photosynthetic apparatus is synthesized. Such conditions can be either anaerobiosis in the light or semiaerobiosis in darkness. The latter conditions were applied in previous investigations concerned with early steps of the adaptive formation of the photosynthetic apparatus in Rhodopseudornonas sphaeroides and R. capsulata (Takemoto, 1974; Nieth and Drews, 1975). With both species it could be demonstrated that at the very beginning of adaptation RC-Bchl and membrane proteins typical for RC preparations are formed primarily. After a short initial phase, however, the proportion of LH-Bchl per RC-Bchl increases continuously (Aagaard and Sistrom, 1972). This also applies to the protein moieties associated with the respective Bchl-species. Consequently it was concluded that the photosynthetic apparatus in R. sphaeroides and R. capsulata is assembled in a sequential mode (Takemoto, 1974; Nieth and Drews, 1975). For R. rubrum, on the other hand, an always constant proportion of RC-Bchl was reported (Aagaard and Sistrom, 1972). This leads to the question whether the assembly of the photosynthetic apparatus in R. rubrum proceeds through a multistep or a singlestep mechanism. The data of the present communication favour the first rather than the latter mechanism.

MATERIALS AND METHODS Rhodospirillurn rubrum, strain FR~, was cultivated highly aerated ~n the dark at 30 ~ C. The malate medium R 8 A H used for previous

282

Arch. Microbiol., Vol. 108 (1976)

2.0

~-.. D

3~

g# _~o.

~c

:3"

3 2

L a E "1.0 o

L

m

o

\

c-----" I

I

I

I

I

1

2

3

4

400

INCUBATION TIME [hi

1

~

q

i

i

I

6(10

t

I

I

I

800

I

I A 950

I

f

f

400

WAVELENGTH & [nm]

2

I

B I

600

I

i

). nm

i

i

800

3

Fig.l. Formation of the photosynthetic apparatus in Rhodospirillum rubrum under low aeration in the dark. Cell protein (x x), molar ratio of RC per total Bchl in isolated membrane fractions (A zx); Bchl per cell protein (9 9 cytochrome c2 per soluble protein (e e) Fig. 2. Absorption spectra of membranes isolated from Rhodospirillum rubrum growing under conditions demonstrated in Figure 1. Membranes were isolated at different times after the transfer of cells to low aeration. A t = 0 (Bchl not detectable); B t = 1.5 h (0.34 nmole Bchl/mg cell protein); C t = 3.5 h (2.5 nmole Bchl/mg cell protein); D represents the membrane spectrum of anaerobically light grown cells for comparison Fig. 3. Spectra of a carotenoid fraction isolated from aerobically grown cells of Rhodospirillum rubrum in a non-membrane-bound form. A spectrum in Tris-HCl-buffer (pH 7.6); B spectrum of the pigment after transfer to acetone/methanol (7 : 2, v/v)

investigations (Drews, 1965) was modified by addition of 0.5 casamino acids and 0.05 % yeast extract (Difco). For pulse label experiments the concentrations of casamino acids and yeast extract were reduced to 0.13% and 0.013% respectively. Cells were harvested from the logarithmic phase of growth by centrifugation. The sedimented material was resuspended with fresh culture medium to a concentration of 0.7 mg cellular protein per ml. The resuspended culture was transferred to a cylindrical culture vessel (11.5 cm diameter) and highly aerated by a constant stream of air and by vigorous stirring. 30 min later the air stream was turned off and in the case of pulse label experiments U-14C protein hydrolyzate (0.6 gCi/ml, Amersham-Buchler) was added. Within 5 rain of time semiaerobic conditions were obtained. Samples taken at different times (see Results) were poured on crunched ice prior to centrifugation. All the following steps were performed at 4 ~C. For measurements of light induced absorbance changes of RC-Bchl cells were resuspended in 0.1 M glycyl-glycine buffer (pH 7.5) containing 2 mM MgC12, 0.5 mM Na-succinate, and 10 ~ sucrose. For all the other experiments cells were resuspended in 0.02 M Tris-HCl-buffer (pH 7.6) supplemented with 3 mM EDTA. Cells were homogenized by two passages through a French pressure cell at 1100 atm. Material sedimenting between 64000• and l l 3 0 0 0 x g for 1 h was washed once with the appropriate buffer and used as enriched membrane fraction. A fraction containing carotenoid floating in Tris-HCl-buffer above the enriched membrane was isolated especially from Bchl-free, fully aerated cells. This fraction was collected by aspiration and precipitated with (NH4)2SO4 between 3 0 - 50 % saturation. The carotenoid was extracted with acetone, transferred to ether and washed several times with water. After this the pigment was transferred to acetone/

methanol (7/2, v/v). Protein was determined by the method of Lowry et al. (1951). Bchl concentrations were calculated using the molar extinction coefficient in methanol given by Cohen-Bazire and Sistrom (1966). RC-Bchl was measured based on light induced absorbance changes at 865 nm according to Aagaard and Sistrom (1972). Molar extinction coefficients for cytochronre c2 were published by Bartsch (1963). Spectral measurements were done with a Cary 14 R spectrophotometer. Solubiiization of membranes and electrophoresis of the resulting proteins were performed by the method of Laemmli (1970) modified as described before (Oelze et al., 1975). To determine the positions of radioactive protein bands the Coomassie-stained gel slabs were dried and exposed in the cold to Osray M 3 film (Agfa-Gevaert). After development the autoradiograms were used to localize exactly the positions of radioactively labeled protein bands within the dried gets. These bands as well as the remaining gel were cut into pieces of equal size, rehydrated over night and treated with NCS tissue solubilizer for liquid scintillation counting (Amersham, Searle Cooperation) according to the instructions given by the manufacturer.

RESULTS

AND

DISCUSSION

T r a n s f e r o f a d e n s e c u l t u r e o f Rhodospirillum rubrum t o l o w a e r a t i o n l e a d s t o c e s s a t i o n o f g r o w t h ( F i g . 1). N e v e r t h e l e s s B c h l is s y n t h e s i z e d a n d its c o n t e n t s i n c r e a s e o n a c e l l u l a r level. I n a g r e e m e n t w i t h d a t a r e p o r t e d f o r R. rubrum b y A a g a a r d a n d S i s t r o m ( 1 9 7 2 )

J. Oelze and W. Pahlke: PhotosyntheticApparatus in R. rubrum and contraryto results obtained with Rhodopseudomonas sphaeroides and R. capsulata the proportion of RC-Bchl per LH-Bchl does not show any significant variation during the early phase of adaptation (Fig. 1). The concentration of soluble cytochrome c2, which represents a constituent of the photochemical electrontransfer system, exhibits only slight if any changes on a protein basis. These data indicate that RC-Bchl and LH-Bchl are formed correlated to each other but not to cytochrome c2. Assuming no significant turnover of cytochrome c2 under the conditions applied one might postulate that from the very beginning on there are sufficient amounts of cytochrome c2 to form the photosynthetic apparatus. This is in accordance with previous findings of Jones and Plewis (1974). Membranes were isolated from cells before and at different times after the transfer to low aeration. The absorption spectra of the membranes are shown in Figure 2. Except for cytochromes membranes from aerobically grown cells exhibit no measurable pigment content. Under low aeration Bchl and also carotenoids are incorporated into the membranes. But it becomes clear that carotenoid incorporation lags behind Bchl incorporation. In wild-type strains of phototrophic bacteria growing under normal conditions Bchl synthesis is known to be accompanied by carotenoid synthesis. Yet, through the experiments of the present investigation it was always possible to isolate a carotenoid fraction even from fully aerated, Bchl-free cells. This carotenoid material floated slightly above the membranes, which sedimented after centrifugation of the prepurified cell homogenate (also see "Materials and Methods"). The spectral properties (Fig. 3) of the carotenoid in acetone-methanol resemble those of spirilloxanthin stereoisomers (Polgfir et al., 1944; Van der Rest and Gingras, 1974). The findings that spirilloxanthin is already formed by aerobically growing cells, although the pigment is not an integral part of membranes yet, suggests that the membranes are not in the proper state to incorporate carotenoids under such conditions. Parallel experiments revealed that carotenoids become constituents of the membranes immediately under anaerobiosis in the light. Changes of the membrane structure were investigated on the basis of protein patterns of solubilized membranes after SDS polyacrylamide gel electrophoresis. An example for the differentiation of the membranes under low aeration is given by Figure 4. This figure shows Coomassie-brilliant-blue stained protein patterns (Fig. 4, A, B) and the corresponding autoradiograms (Fig. 4, a, b) of two stages of growth under low aeration. The presence of the photosynthetic apparatus in the membranes can be demon-

283

Fig.4. Proteinpatterns (A, B) of membranesisolatedfromRhodospirillum rubrum : A = 75 min and B = 240 min after the induction of the photosyntheticapparatus by transfer to low aeration. The correspondingautoradiograms(a, b) giveexamplesfor the differential formationof membraneproteins on the basis of U-14Cprotein hydrolyzateincorporation. Membranes were isolated, solubilized, and subjected to SDS-polyacrylamidegel electrophoresis as outlined in the "Materialsand Methods" strated by the distinct protein bands 11-13 (= H, M, L) and 17. Protein bands 11-13 (H, M, L) are constituents of RC preparations whereas protein band 17 can be detected in LH preparations (Oelze and Golecki, 1975). Other proteins (bands 2, 4, 5) are characteristic for cytoplasmic membranes (Oelze et al., 1975). Cell wall contaminants can be confined to protein bands 8 and 9 (Oelze et al., 1975). The autoradiograms (a,b) make clear that at both of the two stages most of the U-14C protein hydrolyzate was incorporated into proteins associated with the photosynthetic apparatus. It should be noted that among the proteins which are characteristic for cytoplasmic membranes those of band 5 incorporated relatively little of the radioactivity.

284

Arch. Microbiol., Vol. 108 (1976) 8o 9-,,

z o

50

z~

~R 20 LIJ IJno

4O

z

30

o

~-_a 20 z~ [~j B-uo

L~Uno

//

o--.

17

10 ~ - - ' ~

0

6O :--r

z~ w+

./

0

m

o~

RC+17

L,O

i

~'~ i

k i

f.jo--___~ 17