PhotosynthesisResearch 47: 301-305, 1996. © 1996 Kluwer Academic Publishers. Primed in the Netherlands. Rapid communication
Membrane vesicles from Synechocystis 6803 showing proton and electron transport and high ATP synthase activities M a r i j k e J.C. Scholts, P e p i j n A a r d e w i j n & H e n d r i k a S. Van Walraven* Department of Physiology and Biochemistry of Plants, Institute for Molecular Biological Sciences (IMBW), BioCentrum Amsterdam, Vrije Universiteit, De Boelelaan 1087, 1081 HV Amsterdam, The Netherlands; *Author for correspondence Received 14 November1995;acceptedin revisedform 12 February1996
Key words: ATP synthase, cyanobacteria, electron transport, proton gradient, spheroplasts, Synechocystis 6803
Abstract
A simple procedure for the preparation of well-coupled and stable membrane vesicles from the transformable cyanobacterium Synechocystis 6803 is described with the primary aim of producing vesicles suitable for the study of photosynthetic electron transport and phosphorylation. Spheroplasts were obtained from the cyanobacterium by lysozyme treatment and stored untill prior to measurement, thylakoid vesicles were obtained by osmotic shock. These vesicles showed very high and stable ATP synthesis rates either driven by light or by acid-base transition, and also performed light-induced ATP hydrolysis and linear electron transport. Formation of a proton gradient is studied by aminoacridines.
Abbreviations: 9-AA - 9-aminoacridine; ACMA - 9-amino-6-chloro-2-methoxyacridine; Chl - chlorophyll; A/2+ - proton electrochemical potential difference; F1F0 - ATP synthase; PMS - phenazine methosulfate; PMSF phenylmethylsulfonylfluoride; S- 13 - 5-chloro-3-t-butyl-2'-chloro-4'-nitrosalicylanilide Introduction
Cyanobacteria are prokaryotes with a high degree of structural and functional similarity to chloroplasts conceming their photosynthetic membranes (thylakoids) (Sherman et al. 1987). Chloroplasts from many sources can be isolated and stored easily and are relatively stable. Therefore, chloroplasts are ideal objects to study photosynthetic electron transport and phosphorylation (see e.g. Graber 1994). Thylakoid vesicles from cyanobacteria are more difficult to obtain and are much more labile, although differences between strains can be observed. But especially Synechocysfis 6803 causes large problems in that respect (R.A. Dilley, J. Schumann, H. Strotmann, H. Lill and W. Junge, pers. comm.). This strain has the great advantage over several other strains and chloroplasts that it takes up DNA naturally and therefore transformation is very easy (Porter 1988). This has made Synechocystis 6803
increasingly popular to study structure-function relationships of different aspects of photosynthetic electron transport and phosphorylation by the use of (sitedirected) mutagenesis. Several strains have been prepared e.g. with mutations in the genes for the subunits of photosytems (see Vermaas 1994 for review) and recently also in two subunits of the ATP synthase. A segment containing two cysteines was inserted into the -y-subunit of Synechocystis 6803 (Werner-G~ne et al. 1994). This segment is solely present in chloroplasts and is supposedly involved in redox regulation and activation of the ATP synthase (see Van Walraven et al. 1993). Due to the lack of a suitable vesicle preparation technique it was not possible to characterise the activation requirements of the mutant ATP synthase. Similarly, a functional study on the effect of truncation of the hydrophylic part of the b'-subunit by mutagenesis could not be performed (Lill et al. 1994).
302 This paper describes a simple procedure to prepare well-coupled and stable membrane vesicles from Synechocystis 6803 which satisfies the need for an experimental system that has been lacking to study the increasing number of mutants that have been produced of the naturally transformable strain. Preliminary results obtained with membrane vesicles from the "y-mutant (Werner-Griine et al. 1994) have recently been described in a colloquium paper (Krenn et al. 1995).
imally 100 min is required for 100% digestion, by comparing the release of phycocyanin during lysozyme treatment with the release upon sonication. The duration of the actual lysis upon osmotic shock should be tested as well. 2. The age of the cells is important for effectiveness of the lysozyme treatment: ceils of 5 to 9 days old (OD 1.2-1.6) give maximal vesicle yield but older cells become increasingly resistant to lysozyme. 3. Cells grown at temperatures higher than 35 °C become resistant to lysozyme as well.
Materials and methods
Organism and culture conditions The cyanobacterium Synechocystis PCC 6803 was kindly provided by Prof. Dr H. Strotmann (Inst. ftir Biochemie der Pflanzen, Univ. DUsseldorf) and was semi-continuous cultured at 34-35 °C in 2-1 airlift fermentors in BG- 11 medium (Lubberding and Bot 1984). The cells were diluted at an optical density (at 798 nm) of 0.2. Cells from a one week old culture (optical density ca. 1.4) were usually harvested.
Preparation of membrane vesicles 400 ml of cyanobacteria at an optical density (at 798 nm) of 1.2-1.6 were harvested and incubated in 30 ml of Mannitol medium (500 mM Mannitol, 10 mM Tricine-KOH, 10 mM MgC12, 5 mM NaH2PO4, 2.5 mM K2HPO4, pH 7.8) supplied with 0.2% lysozyme, 1 mM 6-amino-n-caproic acid and 100 #M PMSF for ca. 2 hours at 35 °C. 6-Amino-n-caproic acid is used by Mamedov et al. (1991) as a proteinase inhibitor (N. Murata, personal communication). Samples were tested for phycocyanin release upon addition of water. As soon as phycocyanin release could be observed, after centrifugation (2 min 10,000 g), the spheroplasts were resuspended in Mannitol medium plus 1 mM 6-aminon-caproic acid and 100 #M PMSF at a chlorophyll a concentration of 0.2-0.5 mg/ml. The spheroplasts were kept at 0 °C and every 2 hours fresh PMSF (100 #M) was added. They were prepared fresh and could be used for 5-10 hours. A number of possible factors should be taken into account: 1. The resistence to lysozyme may vary from one culture to another. Therefore during lysozyme treatment samples must be tested for phycocyanin release upon osmotic shock. It appeared that min-
Determination of ATP synthesis and hydrolysis activity Light-induced ATP synthesis or hydrolysis activity was determined by the uptake or release of scalar protons measured with a sensitive pH-electrode (Chance and Nishimura 1967) in a set-up as described by Krab et al. (1993). The reaction medium contained 2 mM TricineNaOH, 5 mM MgC12, 50 mM KCI, 50 mM NaCI, 2.5 mM K2HPO4 atpH 8.0 and was supplied with 1 mM 6amino-n-caproic acid and 100 #M PMSE Corrections were made for the background acidification due to the presence of PMSE All ATP synthesis activities were tested for sensitivity to uncoupling (1 #M S-13) and samples were taken from the reaction vessel in order to check the ATP content by the luciferin-luciferase assay (see Krenn et al. 1993). ATP hydrolysis activity was also determined by NADH consumption in an ATPregenerating system (Bergmeyer 1974) measured at 340-400 nm with the assay medium as described by Stutterheim et al. (1980) and supplied with 1 mM 6amino-n-caproic acid and 100 #M PMSE ATP synthesis driven by acid-base transition was carried out as described by Krenn et al. (1993). All media were supplied with 1 mM 6-amino-n-caproic acid and 100 #M PMSF. All activities were measured at 35 °C.
9-AA and ACMA measurements The measurements were performed at 35 °C using an Oriel 3090 fibre-optic fluorimeter and a thermostated 1.8 ml cuvette. Actinic light was provided at a wavelenght of >645 nm via a 10 mm-diameter light guide (Schott). The used medium was the same as for the pH measurements but was supplied with 0.1 #M valinomycin. 9-AA was added at a concentration of 0.2 #M; excitation was at 405 nm and emission at 454
303 nm. ACMA was added at a concentration of 1.8 #M; excitation was at 405 nm and emission at 483 nm.
Other methods Chlorophyll a concentration was determined according to Arnon et al. (1974). Values of AGp were calculated according to Krab and Van Wezel (1992).
Materials ATP, ADP, NADH, valinomycin, phosphoenolpyruvate and all enzymes were purchased from Boehringer (Mannheim, Germany) and PMS, PMSF and 6-aminon-caproic acid from Sigma (St. Louis, MO, USA). The luciferin-luciferase mixture was obtained from Pharmacia-LKB Biotechnology (Uppsala, Sweden). S- 13 was a gift from Dr P.C. Hamm (Monsanto Co., St. Louis, MO, USA). All other chemicals were of analytical grade. Stock solutions were prepared in ethanol of S-13 (0.2 and 1 mM), valinomycin (0.1 mM), PMSF (100 mM), 9-AA (0.1 mM) and ACMA (0.72 mM) and were stored at - 2 0 °C.
Table 1. Light-driven ATP synthesis-, light-induced ATP hydrolysis- and linear electron transport activities of membrane vesicles from Synechocystis 6803 at full light intensity. ATP synthesis was started by addition of ADP (1.25 raM) and was determined during illumination; ATP hydrolysis was started by addition of ATP (5 mM) and was determined immediately after pre-illumination for 30 see. 25/zM PMS was present and the concentration of chlorophyll a was 10 #g/ml. Linear electron transport to K3Fe(CN), (1 mM) was measured as the release of scalar protons during water splitting in the presence of 1 M glycinebetaine. All activities are given in #mol/(min • mg Chl). Data represent mean + / - SD. Fifteen independent preparations have been tested over a period of more than one year 13.46 4- 4.57 16.30 4- 6.08 29.50 4- 3.8
ATP synthesis activity ATP hydrolysis activity Linear electron transport activity
10
-¢: 0
8
E "0 E
6
lo
4
. . . .
i
. . . .
I
. . . .
I
. . . .
I
. . . .
I
. . . .
]
i
fit.
Results and discussion
Spheroplasts broken in the reaction vessel show very high activities of the ATP synthase: routinely between 10 and 20 #mol/(min.mg Chl) for both lightdriven ATP synthesis and light-induced ATP hydrolysis (Table 1). Cyclic photophosphorylation as determined by pH measurements (and checked with the luciferin-luciferase assay) remains stable during measurement and a AGp of 43-45 kJ/mol could be reached which is in the same range as found for vesicles from other strains and for chloroplasts (Bakels et al. 1993). ATP hydrolysis activity measured in a pH electrode setup is stable for some minutes and slowly decreases due to accumulation of ADP (see Krab et al. 1993). ATP hydrolysis activity was also measured with an ATP regenerating system and than it was stable for at least 15 min (results not shown). The ATP synthase activities (expressed on basis of chlorophyll content) show a variability which can be explained by variable chlorophyll content also observed in other cyanobacteria (see Krab et al. 1993) and in chloroplasts (Junesch and Gr~iber 1987). Stable activities for linear electron transport to K3Fe(CN)6 of ca. 30 #mol/(min.mg Chl) were obtained. The activities of the spheroplasts
I
Membrane vesicles from Synechocystis 6803 showing proton and electron transport and high ATP synthase activities M a r i j k e J.C. Scholts, P e p i j n A a r d e w i j n & H e n d r i k a S. Van Walraven* Department of Physiology and Biochemistry of Plants, Institute for Molecular Biological Sciences (IMBW), BioCentrum Amsterdam, Vrije Universiteit, De Boelelaan 1087, 1081 HV Amsterdam, The Netherlands; *Author for correspondence Received 14 November1995;acceptedin revisedform 12 February1996
Key words: ATP synthase, cyanobacteria, electron transport, proton gradient, spheroplasts, Synechocystis 6803
Abstract
A simple procedure for the preparation of well-coupled and stable membrane vesicles from the transformable cyanobacterium Synechocystis 6803 is described with the primary aim of producing vesicles suitable for the study of photosynthetic electron transport and phosphorylation. Spheroplasts were obtained from the cyanobacterium by lysozyme treatment and stored untill prior to measurement, thylakoid vesicles were obtained by osmotic shock. These vesicles showed very high and stable ATP synthesis rates either driven by light or by acid-base transition, and also performed light-induced ATP hydrolysis and linear electron transport. Formation of a proton gradient is studied by aminoacridines.
Abbreviations: 9-AA - 9-aminoacridine; ACMA - 9-amino-6-chloro-2-methoxyacridine; Chl - chlorophyll; A/2+ - proton electrochemical potential difference; F1F0 - ATP synthase; PMS - phenazine methosulfate; PMSF phenylmethylsulfonylfluoride; S- 13 - 5-chloro-3-t-butyl-2'-chloro-4'-nitrosalicylanilide Introduction
Cyanobacteria are prokaryotes with a high degree of structural and functional similarity to chloroplasts conceming their photosynthetic membranes (thylakoids) (Sherman et al. 1987). Chloroplasts from many sources can be isolated and stored easily and are relatively stable. Therefore, chloroplasts are ideal objects to study photosynthetic electron transport and phosphorylation (see e.g. Graber 1994). Thylakoid vesicles from cyanobacteria are more difficult to obtain and are much more labile, although differences between strains can be observed. But especially Synechocysfis 6803 causes large problems in that respect (R.A. Dilley, J. Schumann, H. Strotmann, H. Lill and W. Junge, pers. comm.). This strain has the great advantage over several other strains and chloroplasts that it takes up DNA naturally and therefore transformation is very easy (Porter 1988). This has made Synechocystis 6803
increasingly popular to study structure-function relationships of different aspects of photosynthetic electron transport and phosphorylation by the use of (sitedirected) mutagenesis. Several strains have been prepared e.g. with mutations in the genes for the subunits of photosytems (see Vermaas 1994 for review) and recently also in two subunits of the ATP synthase. A segment containing two cysteines was inserted into the -y-subunit of Synechocystis 6803 (Werner-G~ne et al. 1994). This segment is solely present in chloroplasts and is supposedly involved in redox regulation and activation of the ATP synthase (see Van Walraven et al. 1993). Due to the lack of a suitable vesicle preparation technique it was not possible to characterise the activation requirements of the mutant ATP synthase. Similarly, a functional study on the effect of truncation of the hydrophylic part of the b'-subunit by mutagenesis could not be performed (Lill et al. 1994).
302 This paper describes a simple procedure to prepare well-coupled and stable membrane vesicles from Synechocystis 6803 which satisfies the need for an experimental system that has been lacking to study the increasing number of mutants that have been produced of the naturally transformable strain. Preliminary results obtained with membrane vesicles from the "y-mutant (Werner-Griine et al. 1994) have recently been described in a colloquium paper (Krenn et al. 1995).
imally 100 min is required for 100% digestion, by comparing the release of phycocyanin during lysozyme treatment with the release upon sonication. The duration of the actual lysis upon osmotic shock should be tested as well. 2. The age of the cells is important for effectiveness of the lysozyme treatment: ceils of 5 to 9 days old (OD 1.2-1.6) give maximal vesicle yield but older cells become increasingly resistant to lysozyme. 3. Cells grown at temperatures higher than 35 °C become resistant to lysozyme as well.
Materials and methods
Organism and culture conditions The cyanobacterium Synechocystis PCC 6803 was kindly provided by Prof. Dr H. Strotmann (Inst. ftir Biochemie der Pflanzen, Univ. DUsseldorf) and was semi-continuous cultured at 34-35 °C in 2-1 airlift fermentors in BG- 11 medium (Lubberding and Bot 1984). The cells were diluted at an optical density (at 798 nm) of 0.2. Cells from a one week old culture (optical density ca. 1.4) were usually harvested.
Preparation of membrane vesicles 400 ml of cyanobacteria at an optical density (at 798 nm) of 1.2-1.6 were harvested and incubated in 30 ml of Mannitol medium (500 mM Mannitol, 10 mM Tricine-KOH, 10 mM MgC12, 5 mM NaH2PO4, 2.5 mM K2HPO4, pH 7.8) supplied with 0.2% lysozyme, 1 mM 6-amino-n-caproic acid and 100 #M PMSF for ca. 2 hours at 35 °C. 6-Amino-n-caproic acid is used by Mamedov et al. (1991) as a proteinase inhibitor (N. Murata, personal communication). Samples were tested for phycocyanin release upon addition of water. As soon as phycocyanin release could be observed, after centrifugation (2 min 10,000 g), the spheroplasts were resuspended in Mannitol medium plus 1 mM 6-aminon-caproic acid and 100 #M PMSF at a chlorophyll a concentration of 0.2-0.5 mg/ml. The spheroplasts were kept at 0 °C and every 2 hours fresh PMSF (100 #M) was added. They were prepared fresh and could be used for 5-10 hours. A number of possible factors should be taken into account: 1. The resistence to lysozyme may vary from one culture to another. Therefore during lysozyme treatment samples must be tested for phycocyanin release upon osmotic shock. It appeared that min-
Determination of ATP synthesis and hydrolysis activity Light-induced ATP synthesis or hydrolysis activity was determined by the uptake or release of scalar protons measured with a sensitive pH-electrode (Chance and Nishimura 1967) in a set-up as described by Krab et al. (1993). The reaction medium contained 2 mM TricineNaOH, 5 mM MgC12, 50 mM KCI, 50 mM NaCI, 2.5 mM K2HPO4 atpH 8.0 and was supplied with 1 mM 6amino-n-caproic acid and 100 #M PMSE Corrections were made for the background acidification due to the presence of PMSE All ATP synthesis activities were tested for sensitivity to uncoupling (1 #M S-13) and samples were taken from the reaction vessel in order to check the ATP content by the luciferin-luciferase assay (see Krenn et al. 1993). ATP hydrolysis activity was also determined by NADH consumption in an ATPregenerating system (Bergmeyer 1974) measured at 340-400 nm with the assay medium as described by Stutterheim et al. (1980) and supplied with 1 mM 6amino-n-caproic acid and 100 #M PMSE ATP synthesis driven by acid-base transition was carried out as described by Krenn et al. (1993). All media were supplied with 1 mM 6-amino-n-caproic acid and 100 #M PMSF. All activities were measured at 35 °C.
9-AA and ACMA measurements The measurements were performed at 35 °C using an Oriel 3090 fibre-optic fluorimeter and a thermostated 1.8 ml cuvette. Actinic light was provided at a wavelenght of >645 nm via a 10 mm-diameter light guide (Schott). The used medium was the same as for the pH measurements but was supplied with 0.1 #M valinomycin. 9-AA was added at a concentration of 0.2 #M; excitation was at 405 nm and emission at 454
303 nm. ACMA was added at a concentration of 1.8 #M; excitation was at 405 nm and emission at 483 nm.
Other methods Chlorophyll a concentration was determined according to Arnon et al. (1974). Values of AGp were calculated according to Krab and Van Wezel (1992).
Materials ATP, ADP, NADH, valinomycin, phosphoenolpyruvate and all enzymes were purchased from Boehringer (Mannheim, Germany) and PMS, PMSF and 6-aminon-caproic acid from Sigma (St. Louis, MO, USA). The luciferin-luciferase mixture was obtained from Pharmacia-LKB Biotechnology (Uppsala, Sweden). S- 13 was a gift from Dr P.C. Hamm (Monsanto Co., St. Louis, MO, USA). All other chemicals were of analytical grade. Stock solutions were prepared in ethanol of S-13 (0.2 and 1 mM), valinomycin (0.1 mM), PMSF (100 mM), 9-AA (0.1 mM) and ACMA (0.72 mM) and were stored at - 2 0 °C.
Table 1. Light-driven ATP synthesis-, light-induced ATP hydrolysis- and linear electron transport activities of membrane vesicles from Synechocystis 6803 at full light intensity. ATP synthesis was started by addition of ADP (1.25 raM) and was determined during illumination; ATP hydrolysis was started by addition of ATP (5 mM) and was determined immediately after pre-illumination for 30 see. 25/zM PMS was present and the concentration of chlorophyll a was 10 #g/ml. Linear electron transport to K3Fe(CN), (1 mM) was measured as the release of scalar protons during water splitting in the presence of 1 M glycinebetaine. All activities are given in #mol/(min • mg Chl). Data represent mean + / - SD. Fifteen independent preparations have been tested over a period of more than one year 13.46 4- 4.57 16.30 4- 6.08 29.50 4- 3.8
ATP synthesis activity ATP hydrolysis activity Linear electron transport activity
10
-¢: 0
8
E "0 E
6
lo
4
. . . .
i
. . . .
I
. . . .
I
. . . .
I
. . . .
I
. . . .
]
i
fit.
Results and discussion
Spheroplasts broken in the reaction vessel show very high activities of the ATP synthase: routinely between 10 and 20 #mol/(min.mg Chl) for both lightdriven ATP synthesis and light-induced ATP hydrolysis (Table 1). Cyclic photophosphorylation as determined by pH measurements (and checked with the luciferin-luciferase assay) remains stable during measurement and a AGp of 43-45 kJ/mol could be reached which is in the same range as found for vesicles from other strains and for chloroplasts (Bakels et al. 1993). ATP hydrolysis activity measured in a pH electrode setup is stable for some minutes and slowly decreases due to accumulation of ADP (see Krab et al. 1993). ATP hydrolysis activity was also measured with an ATP regenerating system and than it was stable for at least 15 min (results not shown). The ATP synthase activities (expressed on basis of chlorophyll content) show a variability which can be explained by variable chlorophyll content also observed in other cyanobacteria (see Krab et al. 1993) and in chloroplasts (Junesch and Gr~iber 1987). Stable activities for linear electron transport to K3Fe(CN)6 of ca. 30 #mol/(min.mg Chl) were obtained. The activities of the spheroplasts
I
