Molecular Microbiology {1992) 6(11), 1507-1513
Transcriptional regulation of the plastocyanin and cytochrome C553 genes from the cyanobacterium Anabaena species PCC 7937 Arnaud Bovy,* Geert de Vrieze, Miles Borrias and Peter Weisbeek Department of tAoiecuiar Ceii Biology. State University of Utrecht. Padualaan 8, 3584 CH Utrecht, The Netherlands.
Summary The effect of copper on the levels of plastocyanin (PC) and cytochrome 0553 (cyt-c)-specific transcripts from Anabaena sp. PCC 7937 was investigated. The addition of copper resulted in a marked increase in PC mRNA levels, and a decrease in cyt c mRNA levels. Thus the functional exchange between PC and cyt c seems to be regulated at the mRNA level. The copperdependent increase in PC and decrease in cyt c mRNA levels was abolished when chloramphenicol was added to the cells. This suggests that de novo synthesis of at least one frans-acttng element is required to regulate PC and cyt c mRNA levels. Both PC and cyt c mRNA stability was found to be unaltered under varying Cu^ * regimes. This leads to the conclusion that expression of both genes is regulated at the level of initiation of transcription.
Introduction Plastocyanin is a soluble, low-molecufar-weight copper protein (10.5-12.0kDa) that functions as a redox carrier in the photosynthetic electron transport chain of organisms performing oxygenic photosynthesis (Boulter etai., 1977). Piastocyanin catalyses electron transfer from the membrane-bound cytochrome £)6/f complex to P-700. the reaction centre of Photosystem I. by reversible oxidation and reduction of its active copper centre {Crofts and Wood, 1978; Ho and Krogmann, 1982). The iron-haem protein cytochrome C553 is the natural analogue of plastocyanin. Some cyanobacterial species have only cytochrome C553 and no plastocyanin. Other cyanobacteria and some eukaryotic algae have the abiiity to express both plastocyanin and cytochrome C553, either separately or
Received 2 December, 1991; revised 31 January, 1992; a c c ^ t e d 20 February, 1992. *For correspondence. Tel. (30) 534066; Fax (30)513655.
together (Bohner and Boger, 1978; Merchant and Bogorad, 1986a; Sandmann, 1986; Wood, 1978). In higher plants only plastocyanin has been found (Wood. 1978). Anabaenasp. PCC 7937 can express both plastocyanin and cytochrome C553. Plastocyanin is made only in cells grown in the presence of copper, whereas cytochrome C553 is produced only in the absence of copper (Sandmann and Boger, 1980). Thus there must be a copper-regulated switch in the expression of the plastocyanin and cytochrome C553 genes. Depletion of endogenous copper also seems to be the reason for the absence of plastocyanin in dense natural blooms of cyanobacteria and in Anabaena stationary-phase cells from laboratory cultures (Ho ef ai. 1979). Plastocyanin and cytochrome 0553 have been isolated from many organisms. In all eukaryotic organisms both proteins are acidic with isoelectric points around pH 4 (Ho and Krogmann, 1982). Among cyanobacteria, the isoelectric point for both plastocyanin and cytochrome C553 varies from that of a basic protein (pi 9.3) in filamentous species to that of an acidic protein (pi 3.8) in unicellular species. In one and the same species, however, plastocyanin and cytochrome C553 always have the same isoelectric point (Ho and Krogmann, 1984, 1982). This targe variety tn isoelectric points is especially interesting from an evolutionary point of view: plastocyanin and cytochrome C553 apparently have evolved In a parallel fashion from basic to acidic proteins. Genes (or cDNAs) coding for an acidic cytochrome C553 have been cloned from the green alga Chlamydomonas reinhardtii (Merchant and Bogorad, 1987a) and the cyanobacterium Synechococcus sp. PCC 7942 (Laudenbach e( a/.. 1990). Genes (or cDNAs) coding for an acidic plastocyanin have been isolated from higher plants, e.g. Arabidopsis thaiiana (Vorst etal.. 1988), from green algae, e.g. Chiamydomonas reinhardtii (Merchant and Bogorad, 1987a), and from the cyanobacterium Synechocystis sp. PCC 6803 (Briggs etai, 1990). in our laboratory the gene encoding plastocyanin (PC) from the filamentous cyanobacterium Anabaena sp. PCC 7937 has been cloned and sequenced (Van der Pas et ai, 1989). This gene codes for a basic plastocyanin of 10.5 kDa. Southern analysis showed that plastocyanin is a single-copy gene. It is transcribed into a monocistronic mRNA of 740 bases. The amount of PC-specific mRNA in
1508 A. Bovy, G. de Vrieze, M. Borrias and P. Weisbeek cells grown under copper limitation is much lower than in cells grown in complete medium. Recently the gene encoding cytochrome C553 from Anabaena sp. PCC 7937 (cyt c) has been isolated in our laboratory (EMBL. GenBank and DDBJ database accession number X63194; Bovy etai, 1992). This gives us the opportunity to study, in detail, the regulatory interaction between these two genes which encode analogous functions. In this paper we describe the transcriptional regulation of the PC and cytochrome C553 genes from Anabaena sp. PCC 7937. It includes the effect of copper on transcript levels and the role of translation and mRNA stability in copper regulation. We also propose a model for the regulatory interaction between PC and cytochrome C553.
14 kD PC O
0
r-
6
r-
0
0
0
0
0
0
0
Cu-concentration (\tM) Rg. 1. Western analysis of PC protein as a function of medium Cu ion concentration. Log-phase Anabaena cells, grown with various concentrations of CUSO4 (0. 0,05. 0,1, 0.15, 0-2, 0,3, 0.4, 0.5. 0.6 and 0-8I,LM) were harvested by centrifugation, boiled in sample buffer and used for SDS-PAGE. After eiectrophoretic transfer to PVDF membrane, the proteins were incubated with an anti-PC antibody, raised against a synthetic peptide consisting of the first 23 amino acids ot mature Anabaena plastocyanm. The antibody reaction was visualized via horseradish-peroxidase-coupled second antibodies. Each lane contained 2 ^g of protein. The position of a 14 kDa marker protain is also indicated-
Results In the absence of copper in the grov^fth medium, no PC protein can be detected in Anabaena and the steady-state levels of the PC transcript are very low. High levels of PC protein and transcript can be found in cells grown in the presence of 1 ^LM CUSO4 (Sandmann. 1986; Van der Plas etai., 1989), The relationship between protein levels and copper concentration was analysed by Western and Northern blotting. Ceils that were grown in the presence of copper concentrations ranging from 0.05-1 |xM CUSO4 were lysed, proteins separated by SDS-polyacrylamide gel electrophoresis (PAGE) and electroblotted on to PVDF membrane. This blot was probed with anti-PC antibody (Fig. 1). A band of 10.5kDa could be detected which corresponds to the size of mature PC. A clear induction of mature PC protein could be detected at a Cu^^ concentration of at least 0.2 jxM CUSO4. At concentrations above 0.2 (iM CUSO4 the PC level rapidly increased until a maximal level was reached at concentrations above 0.7 jxM CUSO4. These results are in full agreement with the results obsenred for Anabaena variabilis by Sandmann and Boger (1980). The effect of Cu concentration on PC and cyt c transcript leveis was investigated by Northem blotting. Therefore RNA was isolated from cells grown in the same cultures that were used for the Western blotting experiment, separated on a formaldehyde agarose gel. and blotted to a nylon filter. A 630bp H/ndlll-C/al restriction fragment, which covers the whole PC-coding region (see the Experimentai procedures), was used to probe the PC mRNA. After autoradiography, a single band of 740 bases was detected in each lane (Fig. 2A). Duplicate filters were probed with an oligonucleotide, derived from the Anabaena PCC 7937 cytochrome C553 DNA sequence (Bovy et ai., 1992). A single band of approximately 450 bases was detected (Fig. 2A). The results clearly show that, with
increasing Cu^"" concentrations, the PC mRNA level increases, whereas the cyt c mRNA level decreases. Thus there seems to be complementary Cu-regulation of PC and cyt c at the mRNA level. Densitometric quantification of the intensity of the PC-specific bands was done for four independent experiments, and quantification of the cyt c bands for one experiment. From the combined results a plot was drawn of the relationship between PC/cyt c transcript levels and copper concentration (Fig. 2B). The data in this panel show that an increase in the copper concentration from 0 0.2 ^iM CUSO4 leads to a 16-fold increase in the PC mRNA level and a 5-fold decrease in the cyt c mRNA level. A further increase in the copper concentration from 0.2-0.6 H-M still leads to an increase in PC mRNA levels and a decrease in cyt c levels, although the effect is much smaller (only 1.2-fold for PC and 1.3-fold for cyt c). The maximal PC expression level is reached at a concentration of 0.6M-M CUSO4. We investigated the kinetics of the induction of PC and cyt c mRNA after a shift from low- to high-copper growth conditions. At several times after the addition of 1 txM CUSO4 to tog-phase cells that were grown in the absence of copper. RNA was isolated and analysed on a Northern blot (Fig. 3A). PC mRNA levels rapidly increased after the addition of Cu^^, whereas cyt c mRNA levels decreased. The PC and cyt-c-speclfic mRNA levels, obtained from two independent experiments, were quantified by densitometry and a plot was drawn of PC and cyt c mRNA levels versus time (Fig. 3B). After the addition of copper, PC transcript levels remained at a low basal level for about 20 min and then rapidly increased, until a maximal level was reached after 90 min (the results of longer incubation times in the presence of copper are not shown). Cyt c mRNA levels remained high during the first 20 min after Cu^* addition and then rapidly decreased. The lag phase of 20
Transcriptionai reguiation of plastocyanin and cyt C553 genes 1509
PC
Cyt-c
o
m o
tn o
o
O
O
1-
lO
•-
^
00
o o o o o o o Cu-concentration (jj M) mRNA lavel (%)
B
•
120-
• •
100;
Jdlll/C/al restriction fragment from pPCV6; Van der Plas et al.. 1989), Duplicate filters were hybricfized with a racfiolabelled cyt-c-specific oligonucleotide. B. The relationship between copper-concentration and PC/cyt c transcript levels. Autoradiograms of four independent PC experiments and 1 cyt c experiment, performed as indicated in (A), were scanned with a densitometer and quantified. The PC and cyt-c-specific transcript levels were expressed as a percentage of the maximal level. From the combined results of these experiments a plot was drawn of relative transcript level versus copper concentration.
min, observed in the responses of both PC and cyt c, could represent combinations of the time needed for the uptake of copper into the cell, the synthesis or modulation of regulatory protein(s}. and the synthesis or degradation of the transcript. To investigate whether protein synthesis is necessary for the induction and repression of PC and cyt c mRNA levels, respectively, a Cu-induction experiment similar to that described above was performed in the presence of the translational inhibitor, chloramphenicol (300p-g ml"'). The results in Fig. 4 show that, when translation is blocked, PC mRNA levels cannot be induced, and cyt c mRNA
Cyt-c —»• 9 O
* O n^
O Q KD A
min
0
10
20
JO
40
SO
60
70
80
90
Tim* (min)
Fig. 3. A, Northem blot ot total RNA isolated at several time points after the addition of 1 tiM CUSO4 to cells grown In the absence of copper. Ttie filters were hybridized with the PC and cyt c probe. Conditions were as described in Fig. 1. B. Densitometric quantification of autoradiograms from two exfwnments, performed as indicated in (A).
1510 A. Bovy, G. de Vrieze, M. Borrias and P. Weisbeei
Transcriptional regulation of the plastocyanin and cytochrome C553 genes from the cyanobacterium Anabaena species PCC 7937 Arnaud Bovy,* Geert de Vrieze, Miles Borrias and Peter Weisbeek Department of tAoiecuiar Ceii Biology. State University of Utrecht. Padualaan 8, 3584 CH Utrecht, The Netherlands.
Summary The effect of copper on the levels of plastocyanin (PC) and cytochrome 0553 (cyt-c)-specific transcripts from Anabaena sp. PCC 7937 was investigated. The addition of copper resulted in a marked increase in PC mRNA levels, and a decrease in cyt c mRNA levels. Thus the functional exchange between PC and cyt c seems to be regulated at the mRNA level. The copperdependent increase in PC and decrease in cyt c mRNA levels was abolished when chloramphenicol was added to the cells. This suggests that de novo synthesis of at least one frans-acttng element is required to regulate PC and cyt c mRNA levels. Both PC and cyt c mRNA stability was found to be unaltered under varying Cu^ * regimes. This leads to the conclusion that expression of both genes is regulated at the level of initiation of transcription.
Introduction Plastocyanin is a soluble, low-molecufar-weight copper protein (10.5-12.0kDa) that functions as a redox carrier in the photosynthetic electron transport chain of organisms performing oxygenic photosynthesis (Boulter etai., 1977). Piastocyanin catalyses electron transfer from the membrane-bound cytochrome £)6/f complex to P-700. the reaction centre of Photosystem I. by reversible oxidation and reduction of its active copper centre {Crofts and Wood, 1978; Ho and Krogmann, 1982). The iron-haem protein cytochrome C553 is the natural analogue of plastocyanin. Some cyanobacterial species have only cytochrome C553 and no plastocyanin. Other cyanobacteria and some eukaryotic algae have the abiiity to express both plastocyanin and cytochrome C553, either separately or
Received 2 December, 1991; revised 31 January, 1992; a c c ^ t e d 20 February, 1992. *For correspondence. Tel. (30) 534066; Fax (30)513655.
together (Bohner and Boger, 1978; Merchant and Bogorad, 1986a; Sandmann, 1986; Wood, 1978). In higher plants only plastocyanin has been found (Wood. 1978). Anabaenasp. PCC 7937 can express both plastocyanin and cytochrome C553. Plastocyanin is made only in cells grown in the presence of copper, whereas cytochrome C553 is produced only in the absence of copper (Sandmann and Boger, 1980). Thus there must be a copper-regulated switch in the expression of the plastocyanin and cytochrome C553 genes. Depletion of endogenous copper also seems to be the reason for the absence of plastocyanin in dense natural blooms of cyanobacteria and in Anabaena stationary-phase cells from laboratory cultures (Ho ef ai. 1979). Plastocyanin and cytochrome 0553 have been isolated from many organisms. In all eukaryotic organisms both proteins are acidic with isoelectric points around pH 4 (Ho and Krogmann, 1982). Among cyanobacteria, the isoelectric point for both plastocyanin and cytochrome C553 varies from that of a basic protein (pi 9.3) in filamentous species to that of an acidic protein (pi 3.8) in unicellular species. In one and the same species, however, plastocyanin and cytochrome C553 always have the same isoelectric point (Ho and Krogmann, 1984, 1982). This targe variety tn isoelectric points is especially interesting from an evolutionary point of view: plastocyanin and cytochrome C553 apparently have evolved In a parallel fashion from basic to acidic proteins. Genes (or cDNAs) coding for an acidic cytochrome C553 have been cloned from the green alga Chlamydomonas reinhardtii (Merchant and Bogorad, 1987a) and the cyanobacterium Synechococcus sp. PCC 7942 (Laudenbach e( a/.. 1990). Genes (or cDNAs) coding for an acidic plastocyanin have been isolated from higher plants, e.g. Arabidopsis thaiiana (Vorst etal.. 1988), from green algae, e.g. Chiamydomonas reinhardtii (Merchant and Bogorad, 1987a), and from the cyanobacterium Synechocystis sp. PCC 6803 (Briggs etai, 1990). in our laboratory the gene encoding plastocyanin (PC) from the filamentous cyanobacterium Anabaena sp. PCC 7937 has been cloned and sequenced (Van der Pas et ai, 1989). This gene codes for a basic plastocyanin of 10.5 kDa. Southern analysis showed that plastocyanin is a single-copy gene. It is transcribed into a monocistronic mRNA of 740 bases. The amount of PC-specific mRNA in
1508 A. Bovy, G. de Vrieze, M. Borrias and P. Weisbeek cells grown under copper limitation is much lower than in cells grown in complete medium. Recently the gene encoding cytochrome C553 from Anabaena sp. PCC 7937 (cyt c) has been isolated in our laboratory (EMBL. GenBank and DDBJ database accession number X63194; Bovy etai, 1992). This gives us the opportunity to study, in detail, the regulatory interaction between these two genes which encode analogous functions. In this paper we describe the transcriptional regulation of the PC and cytochrome C553 genes from Anabaena sp. PCC 7937. It includes the effect of copper on transcript levels and the role of translation and mRNA stability in copper regulation. We also propose a model for the regulatory interaction between PC and cytochrome C553.
14 kD PC O
0
r-
6
r-
0
0
0
0
0
0
0
Cu-concentration (\tM) Rg. 1. Western analysis of PC protein as a function of medium Cu ion concentration. Log-phase Anabaena cells, grown with various concentrations of CUSO4 (0. 0,05. 0,1, 0.15, 0-2, 0,3, 0.4, 0.5. 0.6 and 0-8I,LM) were harvested by centrifugation, boiled in sample buffer and used for SDS-PAGE. After eiectrophoretic transfer to PVDF membrane, the proteins were incubated with an anti-PC antibody, raised against a synthetic peptide consisting of the first 23 amino acids ot mature Anabaena plastocyanm. The antibody reaction was visualized via horseradish-peroxidase-coupled second antibodies. Each lane contained 2 ^g of protein. The position of a 14 kDa marker protain is also indicated-
Results In the absence of copper in the grov^fth medium, no PC protein can be detected in Anabaena and the steady-state levels of the PC transcript are very low. High levels of PC protein and transcript can be found in cells grown in the presence of 1 ^LM CUSO4 (Sandmann. 1986; Van der Plas etai., 1989), The relationship between protein levels and copper concentration was analysed by Western and Northern blotting. Ceils that were grown in the presence of copper concentrations ranging from 0.05-1 |xM CUSO4 were lysed, proteins separated by SDS-polyacrylamide gel electrophoresis (PAGE) and electroblotted on to PVDF membrane. This blot was probed with anti-PC antibody (Fig. 1). A band of 10.5kDa could be detected which corresponds to the size of mature PC. A clear induction of mature PC protein could be detected at a Cu^^ concentration of at least 0.2 jxM CUSO4. At concentrations above 0.2 (iM CUSO4 the PC level rapidly increased until a maximal level was reached at concentrations above 0.7 jxM CUSO4. These results are in full agreement with the results obsenred for Anabaena variabilis by Sandmann and Boger (1980). The effect of Cu concentration on PC and cyt c transcript leveis was investigated by Northem blotting. Therefore RNA was isolated from cells grown in the same cultures that were used for the Western blotting experiment, separated on a formaldehyde agarose gel. and blotted to a nylon filter. A 630bp H/ndlll-C/al restriction fragment, which covers the whole PC-coding region (see the Experimentai procedures), was used to probe the PC mRNA. After autoradiography, a single band of 740 bases was detected in each lane (Fig. 2A). Duplicate filters were probed with an oligonucleotide, derived from the Anabaena PCC 7937 cytochrome C553 DNA sequence (Bovy et ai., 1992). A single band of approximately 450 bases was detected (Fig. 2A). The results clearly show that, with
increasing Cu^"" concentrations, the PC mRNA level increases, whereas the cyt c mRNA level decreases. Thus there seems to be complementary Cu-regulation of PC and cyt c at the mRNA level. Densitometric quantification of the intensity of the PC-specific bands was done for four independent experiments, and quantification of the cyt c bands for one experiment. From the combined results a plot was drawn of the relationship between PC/cyt c transcript levels and copper concentration (Fig. 2B). The data in this panel show that an increase in the copper concentration from 0 0.2 ^iM CUSO4 leads to a 16-fold increase in the PC mRNA level and a 5-fold decrease in the cyt c mRNA level. A further increase in the copper concentration from 0.2-0.6 H-M still leads to an increase in PC mRNA levels and a decrease in cyt c levels, although the effect is much smaller (only 1.2-fold for PC and 1.3-fold for cyt c). The maximal PC expression level is reached at a concentration of 0.6M-M CUSO4. We investigated the kinetics of the induction of PC and cyt c mRNA after a shift from low- to high-copper growth conditions. At several times after the addition of 1 txM CUSO4 to tog-phase cells that were grown in the absence of copper. RNA was isolated and analysed on a Northern blot (Fig. 3A). PC mRNA levels rapidly increased after the addition of Cu^^, whereas cyt c mRNA levels decreased. The PC and cyt-c-speclfic mRNA levels, obtained from two independent experiments, were quantified by densitometry and a plot was drawn of PC and cyt c mRNA levels versus time (Fig. 3B). After the addition of copper, PC transcript levels remained at a low basal level for about 20 min and then rapidly increased, until a maximal level was reached after 90 min (the results of longer incubation times in the presence of copper are not shown). Cyt c mRNA levels remained high during the first 20 min after Cu^* addition and then rapidly decreased. The lag phase of 20
Transcriptionai reguiation of plastocyanin and cyt C553 genes 1509
PC
Cyt-c
o
m o
tn o
o
O
O
1-
lO
•-
^
00
o o o o o o o Cu-concentration (jj M) mRNA lavel (%)
B
•
120-
• •
100;
Jdlll/C/al restriction fragment from pPCV6; Van der Plas et al.. 1989), Duplicate filters were hybricfized with a racfiolabelled cyt-c-specific oligonucleotide. B. The relationship between copper-concentration and PC/cyt c transcript levels. Autoradiograms of four independent PC experiments and 1 cyt c experiment, performed as indicated in (A), were scanned with a densitometer and quantified. The PC and cyt-c-specific transcript levels were expressed as a percentage of the maximal level. From the combined results of these experiments a plot was drawn of relative transcript level versus copper concentration.
min, observed in the responses of both PC and cyt c, could represent combinations of the time needed for the uptake of copper into the cell, the synthesis or modulation of regulatory protein(s}. and the synthesis or degradation of the transcript. To investigate whether protein synthesis is necessary for the induction and repression of PC and cyt c mRNA levels, respectively, a Cu-induction experiment similar to that described above was performed in the presence of the translational inhibitor, chloramphenicol (300p-g ml"'). The results in Fig. 4 show that, when translation is blocked, PC mRNA levels cannot be induced, and cyt c mRNA
Cyt-c —»• 9 O
* O n^
O Q KD A
min
0
10
20
JO
40
SO
60
70
80
90
Tim* (min)
Fig. 3. A, Northem blot ot total RNA isolated at several time points after the addition of 1 tiM CUSO4 to cells grown In the absence of copper. Ttie filters were hybridized with the PC and cyt c probe. Conditions were as described in Fig. 1. B. Densitometric quantification of autoradiograms from two exfwnments, performed as indicated in (A).
1510 A. Bovy, G. de Vrieze, M. Borrias and P. Weisbeei
