Properties of Chromatophore Fractions Isolated from - Journal of ...

2 downloads 48 Views 3MB Size Report
We thank Carl P. Schaffner and William Rouslin for the use of their Cary spectrophotometers, RobertL. Hall of. William Paterson College for helpful discussions, ...
JouRNAL OF BACTERIOLOGY, June 1976, P. 1326-1338 Copyright © 1976 American Society for Microbiology

Vol. 126, No. 3 Printed in USA.

Membranes of Rhodospirillum rubrum: Physicochemical Properties of Chromatophore Fractions Isolated from Osmotically and Mechanically Disrupted Cells MARY LYNNE PERILLE COLLINS AND ROBERT A. NIEDERMAN* Department of Microbiology, Rutgers University, New Brunswick, New Jersey 08903 Received for publication 22 January 1976

Isolation of highly purified membrane fractions from phototrophically grown Rhodospirillum rubrum was achieved by velocity and isopyknic sedimentation under carefully controlled ionic conditions. Bacteriochlorophyll-rich and succinic dehydrogenase-rich chromatophores that were essentially devoid of contamination by non-chromatophore protein were separated from a denser fraction in extracts disrupted in a French pressure cell. Highly purified chromatophores and a nearly photopigment-free envelope fraction were also obtained from cells lysed by treatment with ethylenediaminetetraacetate-lysozyme-Brij 58. After lysis with lysozyme and ethylenediaminetetraacetate alone, about 50% of the total photosynthetic pigment was released in chromatophores similar to those isolated by the above procedures. Chromatophores prepared by each method were found to have very similar near-infrared absorption spectra, overall chemical composition, equilibrium buoyant densities in CsCl, and protein patterns in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein profiles of the dense, outer membrane-rich fractions were different from those of the chromatophores. The release of much ofthe photosynthetic apparatus as discrete chromatophores in osmotically lysed extracts necessitates a reevaluation of the concept that isolated chromatophores arise only from mechanical comminution of a larger membrane structure.

Photosynthetic particles (chromatophores) from the facultatively photoheterotrophic bacterium Rhodospirillum rubrum were first detected by Schachman et al. (26) in sedimentation studies of cell-free extracts with the analytical ultracentrifuge. Particles with a sedimentation coefficient of 190S were identified in extracts prepared from cells grown under phototrophic but not aerobic conditions. In thin sections of phototrophically grown cells, Vatter and Wolfe (34) observed vesicles that they concluded were identical to the isolated chromatophores observed by Schachman et al. (26). Such structures were absent from thin sections of cells cultured under high aeration (34). On the basis of the regions of continuity between the cytoplasmic membrane and chromatophores observed in the electron micrographs of CohenBazire and Kunisawa (5), it was proposed that chromatophores arise from invaginations of the cytoplasmic membrane (3, 5, 31). These invaginations are thought to be modified by the insertion of chromatophore-specific components (21). The sedimentation of virtually all of the photosynthetic pigment from extracts of osmotically

lysed cells under low centrifugal force has also been interpreted as evidence for the existence of continuity between the chromatophores and the cytoplasmic membrane (33). It was suggested that the free chromatophores observed in extracts of cells broken by sonication or passage through the French pressure cell arise from the fragmentation of a larger structure (5, 12, 13, 31). In this communication, the preparation of purified chromatophores and a photopigmentfree, outer membrane-rich envelope fraction from R. rubrum is reported. Chromatophores isolated from extracts of cells ruptured by three procedures are compared. Two of these methods did not include mechanical disruption, yet free chromatophores were isolated from the resulting cell-free extracts. These findings indicate that a portion of these organelles may be either structurally independent or loosely bound to the cell wall-cytoplasmic membrane complex. (This paper was presented in part at the 75th Annual Meeting of the American Society for Microbiology, New York, N.Y., May 1975. It was taken in part from a dissertation submitted

1326

1327

VOL. 126, 1976

CHROMATOPHORES OF R. RUBRUM

by M. L. P. C. to the Graduate School, Rutgers University, in partial fulfillment of the requirements for the degree of Doctor of Philosophy.)

at 40,000 rpm (200,000 x g). Gradients were formed, collected, and analyzed, and particles were recovered as described above. Characterization of isolated membrane fractions. Equilibrium buoyant density was determined in CsCl gradients as described previously (7) except that the gradients were centrifuged for 16 h. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis was performed by the procedure of Weber and Osborn (35). The methods previously described (7) were employed for membrane solubilization and molecular weight determinations of the polypeptide bands. Samples were exhaustively dialyzed against distilled-deionized water prior to dry weight determinations and chemical analysis. Dry weight, protein, bacteriochlorophyll a (BCHL), phosphorus, nucleic acid, and carbohydrate were quantified in the manner described previously (7). Lipid was extracted by the previously described procedure (7). Electron micrographs of negatively stained chromatophore preparations were obtained with a Philips 300 electron microscope (Philips Electronic Instruments, Mount Vernon, N.Y.). Samples were placed on carbon-coated Formvar membranes supported on 200-mesh grids, kindly provided by B. K. Ghosh. Grids were stained for 1 min with an aqueous solution of 1% (wt/vol) phosphotungstic acid, pH 7.0. Electron micrographs were obtained from thin sections of the isolated cell envelope fractions by a previously described procedure (7). Sedimentation coefficients were measured with a Beckman model E ultracentrifuge equipped with ultraviolet optics, photoelectric scanner, and a RTIC temperature control unit. Cell assembly consisted of sapphire windows and double-sector charcoal-filled epon center pieces of 12-mm light path. The experiment was performed at 20 C at 12,000 rpm. Samples were prepared in 5% CsCl in 1 mM Tris-acetate, pH 7.5. Scans were made at 265 nm. All S values were calculated and standardized to s20. according to Schachman (25). Near-infrared absorption spectra of chromatophores were determined on a Cary model 14 spectrophotometer (Varian Instrument Division, Springfield, N.J.) using quartz cells (1-cm light path) with samples containing 4.0 ,ug of BCHL per ml in 10 mM Tris buffer, pH 7.5. Cytochrome difference spectra were determined as described previously (7). Chromatophore and envelope preparations (250 ,g and 200 gg of protein, respectively) were employed for these determinations. An estimate of total cytochrome content was made from the Soret peak, which probably represents a composite of b- and ctype cytochromes (1, 7). For this purpose, the differ-

MATERIALS AND METHODS Cultivation of organism. R. rubrum (strain S1) was grown phototrophically in the medium of Omerod et al. (23) at 32 C at a light intensity of 170 ft-c (1,830 lux). Cells were cultured in 4-liter glass reagent bottles under an atmosphere of 95% N2-5% CO2. Aerobic cells were cultured as previously described (7). Preparation of cell-free extracts. Cells in the late exponential growth phase (optical density at 680 nm [6] of 1.0 measured on a Gilford spectrophotometer in 1-cm cuvettes [Gilford Instruments, Oberlin, Ohio]) were harvested and washed in 10 mM tris(hydroxymethyl)aminomethane (Tris)-hydrochloride buffer, pH 7.5. These cells were either passed through the French pressure cell or lysed by a modification (7) of the ethylenediaminetetraacetate (EDTA)-lysozyme-Brij 58 method of Godson and Sinsheimer (11). Reagents were added at 5-min intervals, and lysis was carried out in an ice bath. In some experiments, Brij was omitted. In each case, cell debris and remaining whole cells were removed by sedimentation at 750 x g for 10 min. The above procedures and the isolation techniques described below were performed at 0 to 4 C. Isolation of membrane fractions. Cell-free extracts were centrifuged for 75 min at 60,000 rpm (254,000 x g) in a Beckman type 60 Ti rotor in a Beckman model L2-65B preparative ultracentrifuge (Beckman Instruments, Palo Alto, Calif.) to sediment subcellular particles. In some experiments, the crude particulate fraction was obtained from EDTA-lysozyme extracts by a 20-min centrifugation at 14,500 x g in a Sorvall RC2-B centrifuge (Ivan Sorvall, Inc., Newtown, Conn.). In either case, the resulting pellet was homogenized in and dialyzed against 10 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) (Calbiochem, La Jolla, Calif.) buffer, pH 7.5, and layered on linear 35 to 55% (wt/wt) sucrose gradients prepared in the same buffer. Gradient preparation has been described (17). These gradients were centrifuged to equilibrium in a Beckman SW27 rotor (7), and fractions were collected as previously described (7, 17). The absorbance of the diluted fractions was determined at 260 and 280 nm on a Gilford spectrophotometer and at 880 nm on a Beckman DU spectrophotometer; the succinic dehydrogenase (SDH) (succinate:phenazine methosulfate oxidoreductase, EC 1.3.99.1) activity of individual gradient fractions was also assayed (7). Appropriate fractions were pooled from these gradients, diluted, and centrifuged for 30 min at 254,000 x g to sediment the membrane preparations. The chromatophore fractions obtained from these isopyknic gradients were resolved from contaminating ribosomes by velocity differences in 5 to 30 or 5 to 60% (wt/wt) sucrose gradients prepared in 1 mM Tris, pH 7.5. They were centrifuged in a Beckman SW40 Ti rotor for 90 min

ence between the absorbances at 427 and 410 nm was

calculated. Radiochemical procedures. Radiolabeled cells were prepared by growth on the medium of Omerod et al. (23) to which gas-labeled L-[3H]phenylalanine (New England Nuclear Corp., Boston, Mass.) was added. The specific radioactivity of the added phenylalanine (25 mg/liter of medium) was 2.5 ,uCi/mg. Radioactivity incorporated into the protein of the

1328

COLLINS AND NIEDERMAN

J. BACTERIOL.

isolated fractions was determined essentially as described by Mans and Novelli (15). Samples for radioactivity measurement were applied to 3-mm filter paper disks. These disks were then treated in sequence with 10% (wt/wt) trichloroacetic acid in an ice bath, 5% (wt/wt) trichloroacetic acid at 90 C, acetone-methanol (7:2, vol/vol), ethanol-ether (1:1, vol/vol) at 45 C, and twice with ether. After drying, the disks were placed in scintillation vials with 10 ml of scintillation fluid that was prepared with 0.8 g of 2,5-diphenyloxazole and 0.2 g ofp-bis[2-(5-phenyloxazolyl)]-benzene (both scintillation grade, New England Nuclear) per liter of toluene (scintanalyzed, Fisher Chemical Co., Springfield, N.J.). Samples were counted on a Beckman LS-230 liquid scintillation counter. Efficiencies were determined by the method of channel's ratio.

RESULTS Although numerous attempts have been made to achieve a separation of the membranous components of phototrophically grown R. rubrum (4, 9, 13, 14, 20), none has resulted in their complete resolution. In the present investigation, the separation of chromatophores from a photopigment-free, outer membranerich fraction of high buoyant density was achieved through isopyknic sucrose density gradient centrifugation (Fig. 1). Extracts were obtained from cells lysed by passage through the French pressure cell or treatment with EDTA and lysozyme, with or without the addi60

I

I PRESS FREMCM

50 40

~30

ESTA-LYSOZYR ME

A £

A A

tion of the nonionic detergent Brij 58. The membrane fractions isolated from extracts prepared by these procedures will be referred to as "French press," "EDTA-lysozyme," or "Brij" membranes, respectively. The Brij dense fraction is consistently more free from BCHL than this fraction isolated from extracts lysed by the other methods. The photosynthetic pigment and the SDH activity closely coincide. In the case of the French press and Brij-lysed extracts, these markers are almost exclusively confined to the chromatophores at the top of the gradient. In the EDTA-lysozyme preparation, BCHL and SDH are divided between the chromatophores and a fraction of intermediate density. Ribosomal contamination of these chromatophore fractions is indicated by the high ratio of absorbance at 260 nm to that at 280 nm. This ribosomal material was removed by separation on the basis of sedimentation velocity in sucrose gradients (Fig. 2). Under these conditions, ribosomes trail behind the photosynthetic membranes, in which the SDH is localized. Only those fractions with a 260/280 nm absorbance ratio near unity were pooled. Cytoplasmic membrane from aerobically grown cells (7) and chromatophores share several characteristics including substantial amounts of both cytochromes (see below) and SDH. In addition, they band in approximately the same region in isopyknic sucrose gradients.

EA ,

it

A

- SRIJ

A

A_.-

AAA"i.&a' A- £

,

6 290

10

05

0.40.3 0

,SUCCINIC OEIMYOROEI4ASE

0.2

FIG. 1. Resolution ofchromatophores from dense fraction by isopyknic centrifugation. Particulate material sedimented at 254,000 x g was layered on a 35 to 55% (wt/wt) sucrose gradient and centrifuged to equilibrium. Sucrose concentrations were determined on a refractometer. Absorbancy ofdiluted fractions was determined at 260, 280, and 880 nm, which is the absorbance maximum ofBCHL in intact membranes. SDH serves as a membrane marker. Sedimentation is to the right. Chromatophores, fraction number 3-7; diffuse dense fraction, 20-28; intermediate fraction in gradients of EDTA-lysozyme extract, 13-17.

CHROMATOPHORES OF R. RUBRUM

VOL. 126, 1976

5

-L_ _1 10 15

1/l 20 I 5

1329

t 1 10 15 FRACTION

FIG. 2. Separation of chromatophores from ribosomes by sedimentation velocity. Pooled chromatophore fractions from isopyknic gradients were centrifuged in sucrose density gradients at 200,000 x g for 90 min. Sedimentation is to the right.

It was therefore necessary to determine whether chromatophores are free from cytoplasmic membrane. For this purpose, a modification (16) of the reconstruction experiment originally devised in Rhodopseudomonas spheroides by Fraker and Kaplan (8) was performed. Cells grown aerobically in the presence of l3Hlphenylalanine were mixed with unlabeled phototrophically grown cells and disrupted in the French press. Chromatophores were purified from the resulting extract by the usual method. The specific radioactivity (counts per minute/milligram of protein) of the resulting chromatophores was 2,640, whereas that of the crude particulate fraction obtained from aerobically grown cells was 47,600. On this basis, it was calculated that these chromatophores were at least 95% free from contamination by protein of non-chromatophore origin. Although these data result from a reconstruction experiment performed only with French press extracts, they also suggest that chromatophores obtained from EDTA-lysozyme and Brij extracts are similarly free from contamination by non-chromatophore protein. The purity of the latter preparations is inferred from the similar protein patterns in gel electrophoresis (see below) and the fact that the specific BCHL

values of Brij and EDTA-lysozyme chromatophores are higher than those for French press chromatophores. If the content of non-chromatophore protein were greater in Brij and EDTAlysozyme chromatophores, a lower specific BCHL would be expected. The purified chromatophore fractions of all three types were analyzed chemically and physically. The specific BCHL values (Table 1) represent the average of three experiments and reflect a consistent pattern. The value for the Brij chromatophores is greater than that for those obtained with EDTA and lysozyme alone. The latter figure is in turn greater than that for French press chromatophores. The specific BCHL data for the dense fractions obtained from the extracts prepared by the French press, EDTA-lysozyme-Brij, and EDTA-lysozyme are, respectively, 7.9, 4.6, and 10.3. The intermediate fraction of the EDTA-lysozyme preparation is characterized by a specific BCHL value of 30.5. The total chemical composition of isolated chromatophores was determined (Table 2). It may be seen that, aside from their BCHL content, the composition of these particles is typical of bacterial membranes (24). The low total nucleic acid indicates that these chromato-

1330

COLLINS AND NIEDERMAN

J. BACTERIOL.

phores are free from contamination by deoxyri- 33,800) are not entirely resolved in this gel bonucleic acid or ribosomes. Therefore, phos- system (19, 22). When these same preparations pholipid content was calculated from the total were subjected to electrophoresis in the alkaphosphorus. In cases where lipid was extracted line-Tris-SDS gel system of Okamura et al. and analyzed for phosphorus, similar values (22), three individual reaction center polypeptides of molecular weight 21,500, 25,000, and were obtained. The SDS-polyacrylamide disc gel electropho- 31,500 were observed (R. L. Hall, personal comresis profiles (Fig. 3) of purified chromato- munication). The envelope fraction gel patterns phores are quite similar to each other and are reveal a prominent band of molecular weight distinct from the polypeptide pattern of the en- 38,900. This major band may correspond to that velope fraction, as well as that of the cytoplas- observed in Escherichia coli by Schnaitman mic membrane fraction from aerobically grown TABLE 2. Chemical composition of isolated cells (7). A slight depletion of one high-molecuchromatophores lar-weight protein (55,600), probably due to partial solubilization in the presence of Brij 58, Composition (% dry weight) may be observed. The reaction center compoConstituent EDTAFrench EDTAnent proteins (molecular weight of 30,300 and pressa

TABLE 1. Specific BCHL content of isolated chromatophores

Protein 55.2 51.0 Phospholipid 15.6 15.3 BCHL 3.2 3.6 Carbohydrate b 5.6 4.4 Total nucleic acid