Plant Molecular Biology 20:481-491, 1992. © 1992 Kluwer Academic Publishers. Printed in Belgium.
Cloning and sequencing of the Synechococcus sp. PCC 7002
481
petBD operon from the cyanobacterium
Susan N. Brand, Xiaolin Tan and William R. Widger*
Department of Biochemical and Biophysical Sciences, University of Houston, Houston, TX 77204-5500, USA (* authorfor correspondence) Received 5 November 1991; accepted in revised form 3 June 1992
Key words:photosynthesis, cytochrome b6, gene regulation, genome mapping Abstract
The genes encoding the photosynthetic cytochrome b6 (petB) and subunit 4 (petD) have been cloned and sequenced from the unicellular, photoheterotrophic, transformable cyanobacterium Synechococcus sp. PCC 7002, formerly designated Agmenellum quadruplicatum. The gene arrangement was found to be similar to that reported in the cyanobacterium Nostoc PCC 7906. The DNA and derived protein sequences were compared to chloroplast and the other cyanobacterial sequences. By pulsed-field electrophoresis, the petBD operon and the petCA operon, encoding the Rieske iron-sulfur protein and cytochrome f, were found to be located on separate, unlinked, Not I-digested DNA fragments. The petBD operon was found on the third largest Not I fragment (NC-325) while the petCA operon was found on the second largest Not I fragment (NB-370). These results suggest the two operons are not in proximity. The 1.35 kb transcript was shown to be light-regulated. Transcripts from cells grown under constant illumination showed a decrease in petB transcript levels to undetectable levels within 2 h after the cells were placed in the dark. Upon reillumination, transcript levels rose to three-fold over that seen initially under constant illumination.
Introduction
The cytochrome bcf complex transfers electrons from reduced plastoquinone to the soluble electron carrier, plastocyanin, in plant chloroplasts and some cyanobacteria and to cytochrome c553 in most algae and most cyanobacteria. As a direct result of the electron transfer, protons are vectorally transferred to the lumenal side of the
thylakoid membrane creating a A/~H+ which drives the synthesis of ATP. A functionally similar complex, the cytochrome bcl complex, is found in the inner membranes of mitochondria and in the chromatophores of purple bacteria such as Rhodobacterspheroides [9, 10]. The initial electron transfer step in cytochrome b/bcl is the reduction of heme bp from a ubisemiquinone generated from the initial one electron transfer of a
The nucleotide sequence data reported ~11 appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number X63049.
482 fully reduced ubiquinol to the iron-sulfur protein [11]. The transfer of the electron on heme bp across the membrane to the heme bn and on to an oxidized quinone (the Q cycle) allows extra protons to be cycled back across the membrane thus increasing the proton/electron ratio. Recently [16] differences in the electron transfer mechanisms between the mitochondrial bcl complexes and the chloroplast cytochrome b6f complexes have been noted suggesting a modified mechanism of electron transfer in the b6f complex [ 8 ]. The apparent lack of intra-heme electron transfer in the cytochrome b6f complex [16] and a proton/electron ratio close to one [8, 25] in steady-state light indicate a lack of a protonpumping Q cycle. The amino acid sequence differences among the cytochrome b/bcl and cytochrome b6 peptides [33] suggest the electron transfer mechanisms may also be altered. The lack of a strong n-side inhibitor for cytochrome b6 [8] gives support to a mechanism of electron transfer differing from the cytochrome b/bcl. Although antimycin A was shown to inhibit PSI cyclic electron transport, linear electron transport remains unaffected [27]. Studies on inhibitor-resistant (Inh r) mutants of mitochondrial and purple bacterial cytochrome b/bcl (for review see [33] and trypsin digestion studies of the spinach cytochrome b6 [31] have led to the prediction that the cytochrome b6 contains four transmembrane helices, not five as previously predicted [34]. Atrazine- and turbutrynresistant mutants visualized by X-ray crystal structure analysis were found to affect amino acid residues in the Rhodobacterviridis reaction center at the quinone-binding pocket [26]. By analogy, the Inh r mutants found in the cytochrome b/bcl have led to predictions of amino acid residues involved in the reduced quinone-binding site. A similar system for analysis of mutants in chloroplast-derived cytochrome b6 have not been forthcoming. The cyanobacterium Synechococcus sp. PCC 7002 is a unicellular, naturally transformable, photoheterotropic prokaryote that possesses an oxygenic photosynthetic electron transport chain similar to that found in higher-plant chlo-
roplasts. Cloning and sequencing of the petCA and petBD operons were undertaken to take advantage of these properties and to construct a system in which site-directed mutations could be used to help elucidate amino acid residues involved in the electron and proton transport. This manuscript reports on the cloning and D N A sequencing of the petBD operon of Synechococcus sp PCC 7002. Transcript level of the petBD operon were shown to be dependent on light and the proximity of this operon to the petCA operon was shown to be at least 445 kb distant. Methods and materials
Growth and maintenance of PCC 7002 Synechococcus sp. PCC 7002 obtained from the ATCC collection was maintained and cultured under photoautotrophic conditions using an artificial seawater medium [30]. Water-saturated air augmented with carbon dioxide was bubbled into 300 ml culture tubes (42 cm x 3.5 cm diameter) exposed to direct and continuous fluorescent light from two Sylvania cool white 30 W fluorescent bulbs. Tubes were inoculated with 5 ml of cyanobacteria cultured in 50 ml flasks grown from single colonies isolated on agar plates. Cells were harvested at late exponential phase, 1-5 x 10s cells per ml and used to prepare genomic DNA. Agar plates for maintaining the cyanobacteria were prepared using the artificial seawater media plus 1~o Oxiod bacteriological agar 1 from Oxiod Limited, Basingstoke, Hampshire, England. Lambda dash genomic DNA library Synechococcus sp PCC 7002 genomic D N A was isolated as previously described [32] and was used to construct a lambda Dash (Stratagene, La Jolla, CA) phage clone library from partially digested Sau3AI genomic DNA. Gridded library construction 1539 individual plaques were suspended in 1 ml each of SM medium in 1.5 ml tubes. Individual
483 clones were amplifiied in microtiter plates (81 per plate) in 0.3 ml of host cells by adding 15 #1 of lysate from individual phage to each well and incubated at 37 °C overnight. The amplified clones were transferred, gridded, on 150 mm culture dishes containing top agar with host cells using a 96 prong transfer tool. Plaques 3-6 mm in diameter were allowed to form and were lifted onto nylon filters (Magnagraph; MSI, Westburo, MA), three filters per plate. Reference notches were cut in each filter for easy identification and orientation.
Southern transfers Genomic DNA, cut with appropriate restriction enzymes, was electrophoresed along with size markers on 0.7~o agarose gels and transferred to nylon membranes in 4 x SSC as described [2] using a Vacublot system. Transfers were complete in 90 min and after rinsing in 2 x SSC, the filters were prehybridized in 50~o formamide, 1 M sodium chloride, 0.2~o SDS, 0.05 M Tris-HC1 pH 8.0, 1 x Denhardt's solution, and 1 m M EDTA containing 0.5~o non-fat dry milk as a blocking agent (HA buffer).
Hybridization Hybridization conditions were the same for the Southern-blotted filters or the gridded-library filters. H A buffer (25 ml) was placed in a roller drum hybridization chamber containing the prehybridized filter. Radioactive probe D N A was added after denaturation at 95 °C for 5 min. Hybridization was carried out for 24-48 h at room temperature after which the filters were washed in 2 x SSC, 0.5~o SDS, twice at room temperature and once at 55 °C using a Schleichher & Schuell disk wisk filter bath. Probes were labeled using the random hexamer method with the D N A polymerase Klenow fragment [ 14].
SubcIoning D N A restriction fragments, isolated in lowmelting agar from restriction-digested lambda
phage, were ligated [2] into the appropriate restriction sites of the pUC19 vector or in M13 mpl8 or mpl9. After transformation and confirmation of the fragment insert size by alkalinelysis minipreps, single-stranded D N A was isolated and used for sequencing.
DNA sequencing Sequencing was performed as described by S anger [24] using [e-thio-35S] dATP and S equenase (U. S. Biochemical, Cleveland, O H ) according to the instructions of the manufacturer. Oligonucleotide primers were synthesized using an Applied Biosystems Biosearch model 8600 D N A synthesizer.
Transcript isolation and northern analysis Total R N A was isolated by a method developed for Physarum polycephalum [23] and modified for use with Serratia marcescens (G. Shipley, pers. comm.) as follows. Four ml aliquots of Synechococcus PCC 7002 cells, OD at 550 nm of 1.2-1.5, exposed various times in the dark or illuminated after dark treatment was treated in DEPC-treated oven-baked 15 ml Corex tubes containing 1 ml of 5 x NES lysis buffer (0.5 M sodium acetate, 0.25 M E D T A and 5 ~o SDS) at 95 ° C for 10 min. The solubilized cells were extracted with preheated phenol at 65 °C equilibrated in 1 x NES and cooled to 5 ° C in ice before being centrifuged at 11 000 x g for 10 min. The aqueous layer was reextracted with a 1:1 (v/v)phenol plus Sevag solution and centrifuged as above. The R N A was precipitated overnight at 4 °C by the addition of 0.25 volumes of 10 M lithium chloride (2 M final LiC1 concentration). After centrifugation the R N A pellet was rinsed in 2 M LiC1 and redissolved in 1 x NES and reprecipitated by the addition of 2.5 volumes of ethanol. The R N A pellet was rinsed in 76~o ethanol and dried under vacuum. The R N A was dissolved in DEPCtreated water and treated with RNase-free DNase, RQ1 (Promega, Madison, WI) prior to electrophoresis and northern analysis. The 3.1 kb Hind III fragment from p A Q P R 2
484 containing the cpcBA C operon [ 12] and the 1.6 kb Bam HI-HindIII fragment from pUHWRW1 [32] containing the petCA operon were used as hybridization probes.
Formaldehyde RNA gel electrophoresis RNA formaldehyde agarose gels containing 2 0 m M MOPS, 5 mM sodium acetate, l m M EDTA pH 7.0 with 1.85 ~o formaldehyde [2] was used for separation of formamide/formaldehyde denatured RNA. The ethidium bromide-stained gels were photographed using a 35 mm camera with Tmax 100 film (Kodak, Rochester, NY) with the appropriate filters. The RNA was transferred to nylon filters using 10 x SSC on a Vacublot vacuum apparatus.
from Dr T. Kallas) [ 18] was used as a hybridization probe to screen Southern transferred genomic D N A digested with Eco RI, Barn HI, Hind III, Xba I and BglII (Fig. 1A) and double digests (Fig. 1B) of the same enzymes. A single hybridizing band was seen for all digestions strongly suggesting a single copy of the cytochrome b6 is present in the genome. The Bgl IIHind III double digest produced the smallest fragment size (2.9 kb). The lambda Dash library was screened using the Nostoc PCC 7906 cytochrome b 6 probe and five positive clones were identified, AG13-8,
Pulsed-fieM gel electrophoresis Pulsed-field electrophoresis was done using an in-house built C H E F system [7] assembled with directions obtained from R. Davis and G. Volrath (pers. comm.). Switch times were controlled by a PC computer using the parallel port connected to an optically isolated digital relay. Up to ten switch times and durations can be programmed. Genomic D N A was isolated in agarose blocks as described [29]. Separation of Not I digested D N A fragments was done using 180 V at 4 °C in 0.5 x TBE buffer at various switch times. Separation of the larger fragments 440-130 kp was done using switch times of 40 and 20 s for 18 h duration each while smaller fragments were separated using switch times between 5 and 15 s (unpublished results).
Results
Identification and subcloning of the petBD operon The 600 bp internal fragment from the clone PN.6b6-4.1 containing the petB gene from the cyanobacterium Nostoe PCC 7906 (a kind gift
Fig. 1. Synechococcus PCC 7002 genomic DNA digested with
various restriction enzymes, Southern transferred, and probed with the Nostoc PCC 7906 cytochrome b6 fragment. Genomic DNA digested singly (panel A) with Bam HI (B), Bgl II (Bg), Eeo RI (E), Hind III (H) and Xba I (X) and doubly (panel B) with BglII-Hind II (Bg-H), BglII-Xba I (Bg-X), Eco RIHind IIl (E-H), Eco RI-Xba I (E-X), and Hind III-Xba I (H-
X).
485 in the Nostoc PCC 7906 sequence. Shine-Delgarno sequences are present before both genes. The petB gene is 666 bp long encoding a 222 amino acid cytochrome b6 peptide which is seven amino acids longer at the N-terminus than the Nostoc PCC 7906 peptide. PetD is 480 bp in length encoding a 160 amino acid subunit 4 peptide. The comparison of the amino acid sequence differences among the two cyanobacteria, Synechocoeeus PCC 7002 and Nostoc 7906 [18] and spinach chloroplast cytochrome b 6 subunit 4 [ 17 ], Fig. 4 shows a 16.4~o difference between Nostoc PCC 7906 and spinach, 16.8 ~o difference between Synechococcus PCC 7002 and spinach and 13.5~o difference between Synechococcus PCC 7002 and Nostoc PCC 7906. Subunit 4 shows 19.4~o, 28.0~o, and 24.4~o while the combined sequences show 17.6~o, 19~o and 18.1~o differences for Nostoc PCC 7906 and spinach, Synechoeoecus PCC 7002 and spinach, and Synechococcus PCC 7002 and Nostoc PCC 7906 respectively. This suggests that the evolutionary distance between the Synechococcus PCC 7002
AG13-32, AG17-18, AG17-19, and AG17-40. The phage AG17-40 was amplified and the isolated D N A was digested with the above restriction enzymes and showed hybridization patterns predicted from the genomic Southerns. The 2.9 kb BglII-Hind III fragment was excised and ligated into the Barn HI-Hind III ends ofpUC19 yielding p U H S N B 1. This fragment was also ligated into the same ends of M13mp18 and M13mp19. The sequence was determined on both strands with overlapping determinations as shown on Fig. 2. The first start codon ofthepetBD operon was located 1464 bp from the start of the sequence from the Bgl II end and the stop codon of petD was located at position 2671. The Bgl IIHind III fragment was 2858 bp long. A large open reading frame was found on the opposite strand starting at 1233 and extending beyond the Bgl II site. Figure 3 shows the D N A sequence starting 163 bases upstream from the start codon of the petB gene. A gap of 55 bp is located between the stop codon of the petB gene and the start of the petD gene and there is no inverted repeat as seen
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Fig. 2. The strategies for the sequencing of the petBD operon. The 2.85 kb Bgl II-Hind III fragment was digested with Sau3A1 or Hae III and fragments were sequenced by subcloning into M13 mp18 to obtain single-stranded DNA. Overlaps and opposite strand data were obtained from synthesized oligonucleotide primers. The unidentified open reading frame (URF) coding on the opposite strand continues beyond the Bgl II site. Search of the GenBank sequence data has not been fruitful in determining the function of the protein encoded on this URF.
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Fig. 3. The nucleotide and calculated amino acid sequence of the petBD operon of Synechococcus PCC 7002. The nucleotide sequence starts at -163 upstream from the ATG start of the petB gene. The petB sequence, 666 bp is followed by a gap of 55 bp separating the 480 bp petD gene.
and Nostoc PCC 7906 sequences are similar to the distances from either cyanobacteria to spinach chloroplast cytochrome b6-subunit 4. However, the paucity of sequence data from Nostoc PCC 7906 forbids any substantial determinations and these relationships are not readily resolvable. The same relationship is seen for the petCA gene products [32]. The codon usage for the Synechococcus PCC 7002 petBD operon mirrors that which has been reported for the Nostoc PCC 7906 petBD and other cyanobacteria [ 18, 19]. The absence of ATA-Ile, AGG-Arg, AGA-Arg and one occurrence of TCG-Ser and CGA-Arg is seen while GGT-Gly, CAA-Gln, CAC-His, and ACC-Thr are more frequently used. The codon usage for the petBD operon of Synechococcus PCC 7002 matches the overall codon usage seen in this cyanobacterium determined from 11600 bp of coding sequences derived from GenBank [5] using the codon frequency program in the U W G C G package [13].
The light-dependent transcript concentrationsfor cytochrome b 6 The transcript levels for the petB gene were measured as a function of time in the dark and from the onset of illumination. Cells continuously illuminated with light described under growth conditions were dark adapted in the absence of bubbled CO2. Aliquots, removed as indicated in Fig. 5, were analyzed for m R N A levels by isolating the m R N A and separating the transcripts on R N A gels. The northern transferred R N A was analyzed by hybridization with a radiolabeled probe containing just the petB reading frame or the cpcBAC operon [ 12]. After three hours in the dark, the cells were illuminated as before and aliquots were again removed and the transcripts analyzed. The autoradiograph depicting the temporal response of the petB and the cpcBAC transcript to light/dark conditions was scanned. The area under the curve was plotted as a function of time (Fig. 5). As seen the transcript levels under
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c o n s t a n t illumination diminished rapidly to undetectable levels within 120 min. W h e n the illumination w a s restored to the cells, the transcript level increased rapidly and reached a m a x i m u m level, three fold over steady state, within 8 0 100 min and then slowly declined. This increase in transcript levels in r e s p o n s e to light has been seen for the petCA o p e r o n as well. Several other p h o t o s y s t e m II transcripts h a v e been s h o w n as being light inducible in Synechocystis 6803 [21] and Anacystis nidulans R 2 [6] as well as the other
SynechococcusPCC 7002
are shown as asterisks and numbered to
Synechococcus P C C 7 0 0 2 p h y c o b i l i s o m e o p e r o n apcABC (unpublished data). Probes for these transcripts were kindly D r D o n a l d Bryant.
supplied
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us
by
Distance separating the p e t C A and the p e t B D operons T h e minimal distance separating the petBD and petCA o p e r o n s w a s estimated to be at least 445 kb
488 I
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Cloning and sequencing of the Synechococcus sp. PCC 7002
481
petBD operon from the cyanobacterium
Susan N. Brand, Xiaolin Tan and William R. Widger*
Department of Biochemical and Biophysical Sciences, University of Houston, Houston, TX 77204-5500, USA (* authorfor correspondence) Received 5 November 1991; accepted in revised form 3 June 1992
Key words:photosynthesis, cytochrome b6, gene regulation, genome mapping Abstract
The genes encoding the photosynthetic cytochrome b6 (petB) and subunit 4 (petD) have been cloned and sequenced from the unicellular, photoheterotrophic, transformable cyanobacterium Synechococcus sp. PCC 7002, formerly designated Agmenellum quadruplicatum. The gene arrangement was found to be similar to that reported in the cyanobacterium Nostoc PCC 7906. The DNA and derived protein sequences were compared to chloroplast and the other cyanobacterial sequences. By pulsed-field electrophoresis, the petBD operon and the petCA operon, encoding the Rieske iron-sulfur protein and cytochrome f, were found to be located on separate, unlinked, Not I-digested DNA fragments. The petBD operon was found on the third largest Not I fragment (NC-325) while the petCA operon was found on the second largest Not I fragment (NB-370). These results suggest the two operons are not in proximity. The 1.35 kb transcript was shown to be light-regulated. Transcripts from cells grown under constant illumination showed a decrease in petB transcript levels to undetectable levels within 2 h after the cells were placed in the dark. Upon reillumination, transcript levels rose to three-fold over that seen initially under constant illumination.
Introduction
The cytochrome bcf complex transfers electrons from reduced plastoquinone to the soluble electron carrier, plastocyanin, in plant chloroplasts and some cyanobacteria and to cytochrome c553 in most algae and most cyanobacteria. As a direct result of the electron transfer, protons are vectorally transferred to the lumenal side of the
thylakoid membrane creating a A/~H+ which drives the synthesis of ATP. A functionally similar complex, the cytochrome bcl complex, is found in the inner membranes of mitochondria and in the chromatophores of purple bacteria such as Rhodobacterspheroides [9, 10]. The initial electron transfer step in cytochrome b/bcl is the reduction of heme bp from a ubisemiquinone generated from the initial one electron transfer of a
The nucleotide sequence data reported ~11 appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number X63049.
482 fully reduced ubiquinol to the iron-sulfur protein [11]. The transfer of the electron on heme bp across the membrane to the heme bn and on to an oxidized quinone (the Q cycle) allows extra protons to be cycled back across the membrane thus increasing the proton/electron ratio. Recently [16] differences in the electron transfer mechanisms between the mitochondrial bcl complexes and the chloroplast cytochrome b6f complexes have been noted suggesting a modified mechanism of electron transfer in the b6f complex [ 8 ]. The apparent lack of intra-heme electron transfer in the cytochrome b6f complex [16] and a proton/electron ratio close to one [8, 25] in steady-state light indicate a lack of a protonpumping Q cycle. The amino acid sequence differences among the cytochrome b/bcl and cytochrome b6 peptides [33] suggest the electron transfer mechanisms may also be altered. The lack of a strong n-side inhibitor for cytochrome b6 [8] gives support to a mechanism of electron transfer differing from the cytochrome b/bcl. Although antimycin A was shown to inhibit PSI cyclic electron transport, linear electron transport remains unaffected [27]. Studies on inhibitor-resistant (Inh r) mutants of mitochondrial and purple bacterial cytochrome b/bcl (for review see [33] and trypsin digestion studies of the spinach cytochrome b6 [31] have led to the prediction that the cytochrome b6 contains four transmembrane helices, not five as previously predicted [34]. Atrazine- and turbutrynresistant mutants visualized by X-ray crystal structure analysis were found to affect amino acid residues in the Rhodobacterviridis reaction center at the quinone-binding pocket [26]. By analogy, the Inh r mutants found in the cytochrome b/bcl have led to predictions of amino acid residues involved in the reduced quinone-binding site. A similar system for analysis of mutants in chloroplast-derived cytochrome b6 have not been forthcoming. The cyanobacterium Synechococcus sp. PCC 7002 is a unicellular, naturally transformable, photoheterotropic prokaryote that possesses an oxygenic photosynthetic electron transport chain similar to that found in higher-plant chlo-
roplasts. Cloning and sequencing of the petCA and petBD operons were undertaken to take advantage of these properties and to construct a system in which site-directed mutations could be used to help elucidate amino acid residues involved in the electron and proton transport. This manuscript reports on the cloning and D N A sequencing of the petBD operon of Synechococcus sp PCC 7002. Transcript level of the petBD operon were shown to be dependent on light and the proximity of this operon to the petCA operon was shown to be at least 445 kb distant. Methods and materials
Growth and maintenance of PCC 7002 Synechococcus sp. PCC 7002 obtained from the ATCC collection was maintained and cultured under photoautotrophic conditions using an artificial seawater medium [30]. Water-saturated air augmented with carbon dioxide was bubbled into 300 ml culture tubes (42 cm x 3.5 cm diameter) exposed to direct and continuous fluorescent light from two Sylvania cool white 30 W fluorescent bulbs. Tubes were inoculated with 5 ml of cyanobacteria cultured in 50 ml flasks grown from single colonies isolated on agar plates. Cells were harvested at late exponential phase, 1-5 x 10s cells per ml and used to prepare genomic DNA. Agar plates for maintaining the cyanobacteria were prepared using the artificial seawater media plus 1~o Oxiod bacteriological agar 1 from Oxiod Limited, Basingstoke, Hampshire, England. Lambda dash genomic DNA library Synechococcus sp PCC 7002 genomic D N A was isolated as previously described [32] and was used to construct a lambda Dash (Stratagene, La Jolla, CA) phage clone library from partially digested Sau3AI genomic DNA. Gridded library construction 1539 individual plaques were suspended in 1 ml each of SM medium in 1.5 ml tubes. Individual
483 clones were amplifiied in microtiter plates (81 per plate) in 0.3 ml of host cells by adding 15 #1 of lysate from individual phage to each well and incubated at 37 °C overnight. The amplified clones were transferred, gridded, on 150 mm culture dishes containing top agar with host cells using a 96 prong transfer tool. Plaques 3-6 mm in diameter were allowed to form and were lifted onto nylon filters (Magnagraph; MSI, Westburo, MA), three filters per plate. Reference notches were cut in each filter for easy identification and orientation.
Southern transfers Genomic DNA, cut with appropriate restriction enzymes, was electrophoresed along with size markers on 0.7~o agarose gels and transferred to nylon membranes in 4 x SSC as described [2] using a Vacublot system. Transfers were complete in 90 min and after rinsing in 2 x SSC, the filters were prehybridized in 50~o formamide, 1 M sodium chloride, 0.2~o SDS, 0.05 M Tris-HC1 pH 8.0, 1 x Denhardt's solution, and 1 m M EDTA containing 0.5~o non-fat dry milk as a blocking agent (HA buffer).
Hybridization Hybridization conditions were the same for the Southern-blotted filters or the gridded-library filters. H A buffer (25 ml) was placed in a roller drum hybridization chamber containing the prehybridized filter. Radioactive probe D N A was added after denaturation at 95 °C for 5 min. Hybridization was carried out for 24-48 h at room temperature after which the filters were washed in 2 x SSC, 0.5~o SDS, twice at room temperature and once at 55 °C using a Schleichher & Schuell disk wisk filter bath. Probes were labeled using the random hexamer method with the D N A polymerase Klenow fragment [ 14].
SubcIoning D N A restriction fragments, isolated in lowmelting agar from restriction-digested lambda
phage, were ligated [2] into the appropriate restriction sites of the pUC19 vector or in M13 mpl8 or mpl9. After transformation and confirmation of the fragment insert size by alkalinelysis minipreps, single-stranded D N A was isolated and used for sequencing.
DNA sequencing Sequencing was performed as described by S anger [24] using [e-thio-35S] dATP and S equenase (U. S. Biochemical, Cleveland, O H ) according to the instructions of the manufacturer. Oligonucleotide primers were synthesized using an Applied Biosystems Biosearch model 8600 D N A synthesizer.
Transcript isolation and northern analysis Total R N A was isolated by a method developed for Physarum polycephalum [23] and modified for use with Serratia marcescens (G. Shipley, pers. comm.) as follows. Four ml aliquots of Synechococcus PCC 7002 cells, OD at 550 nm of 1.2-1.5, exposed various times in the dark or illuminated after dark treatment was treated in DEPC-treated oven-baked 15 ml Corex tubes containing 1 ml of 5 x NES lysis buffer (0.5 M sodium acetate, 0.25 M E D T A and 5 ~o SDS) at 95 ° C for 10 min. The solubilized cells were extracted with preheated phenol at 65 °C equilibrated in 1 x NES and cooled to 5 ° C in ice before being centrifuged at 11 000 x g for 10 min. The aqueous layer was reextracted with a 1:1 (v/v)phenol plus Sevag solution and centrifuged as above. The R N A was precipitated overnight at 4 °C by the addition of 0.25 volumes of 10 M lithium chloride (2 M final LiC1 concentration). After centrifugation the R N A pellet was rinsed in 2 M LiC1 and redissolved in 1 x NES and reprecipitated by the addition of 2.5 volumes of ethanol. The R N A pellet was rinsed in 76~o ethanol and dried under vacuum. The R N A was dissolved in DEPCtreated water and treated with RNase-free DNase, RQ1 (Promega, Madison, WI) prior to electrophoresis and northern analysis. The 3.1 kb Hind III fragment from p A Q P R 2
484 containing the cpcBA C operon [ 12] and the 1.6 kb Bam HI-HindIII fragment from pUHWRW1 [32] containing the petCA operon were used as hybridization probes.
Formaldehyde RNA gel electrophoresis RNA formaldehyde agarose gels containing 2 0 m M MOPS, 5 mM sodium acetate, l m M EDTA pH 7.0 with 1.85 ~o formaldehyde [2] was used for separation of formamide/formaldehyde denatured RNA. The ethidium bromide-stained gels were photographed using a 35 mm camera with Tmax 100 film (Kodak, Rochester, NY) with the appropriate filters. The RNA was transferred to nylon filters using 10 x SSC on a Vacublot vacuum apparatus.
from Dr T. Kallas) [ 18] was used as a hybridization probe to screen Southern transferred genomic D N A digested with Eco RI, Barn HI, Hind III, Xba I and BglII (Fig. 1A) and double digests (Fig. 1B) of the same enzymes. A single hybridizing band was seen for all digestions strongly suggesting a single copy of the cytochrome b6 is present in the genome. The Bgl IIHind III double digest produced the smallest fragment size (2.9 kb). The lambda Dash library was screened using the Nostoc PCC 7906 cytochrome b 6 probe and five positive clones were identified, AG13-8,
Pulsed-fieM gel electrophoresis Pulsed-field electrophoresis was done using an in-house built C H E F system [7] assembled with directions obtained from R. Davis and G. Volrath (pers. comm.). Switch times were controlled by a PC computer using the parallel port connected to an optically isolated digital relay. Up to ten switch times and durations can be programmed. Genomic D N A was isolated in agarose blocks as described [29]. Separation of Not I digested D N A fragments was done using 180 V at 4 °C in 0.5 x TBE buffer at various switch times. Separation of the larger fragments 440-130 kp was done using switch times of 40 and 20 s for 18 h duration each while smaller fragments were separated using switch times between 5 and 15 s (unpublished results).
Results
Identification and subcloning of the petBD operon The 600 bp internal fragment from the clone PN.6b6-4.1 containing the petB gene from the cyanobacterium Nostoe PCC 7906 (a kind gift
Fig. 1. Synechococcus PCC 7002 genomic DNA digested with
various restriction enzymes, Southern transferred, and probed with the Nostoc PCC 7906 cytochrome b6 fragment. Genomic DNA digested singly (panel A) with Bam HI (B), Bgl II (Bg), Eeo RI (E), Hind III (H) and Xba I (X) and doubly (panel B) with BglII-Hind II (Bg-H), BglII-Xba I (Bg-X), Eco RIHind IIl (E-H), Eco RI-Xba I (E-X), and Hind III-Xba I (H-
X).
485 in the Nostoc PCC 7906 sequence. Shine-Delgarno sequences are present before both genes. The petB gene is 666 bp long encoding a 222 amino acid cytochrome b6 peptide which is seven amino acids longer at the N-terminus than the Nostoc PCC 7906 peptide. PetD is 480 bp in length encoding a 160 amino acid subunit 4 peptide. The comparison of the amino acid sequence differences among the two cyanobacteria, Synechocoeeus PCC 7002 and Nostoc 7906 [18] and spinach chloroplast cytochrome b 6 subunit 4 [ 17 ], Fig. 4 shows a 16.4~o difference between Nostoc PCC 7906 and spinach, 16.8 ~o difference between Synechococcus PCC 7002 and spinach and 13.5~o difference between Synechococcus PCC 7002 and Nostoc PCC 7906. Subunit 4 shows 19.4~o, 28.0~o, and 24.4~o while the combined sequences show 17.6~o, 19~o and 18.1~o differences for Nostoc PCC 7906 and spinach, Synechoeoecus PCC 7002 and spinach, and Synechococcus PCC 7002 and Nostoc PCC 7906 respectively. This suggests that the evolutionary distance between the Synechococcus PCC 7002
AG13-32, AG17-18, AG17-19, and AG17-40. The phage AG17-40 was amplified and the isolated D N A was digested with the above restriction enzymes and showed hybridization patterns predicted from the genomic Southerns. The 2.9 kb BglII-Hind III fragment was excised and ligated into the Barn HI-Hind III ends ofpUC19 yielding p U H S N B 1. This fragment was also ligated into the same ends of M13mp18 and M13mp19. The sequence was determined on both strands with overlapping determinations as shown on Fig. 2. The first start codon ofthepetBD operon was located 1464 bp from the start of the sequence from the Bgl II end and the stop codon of petD was located at position 2671. The Bgl IIHind III fragment was 2858 bp long. A large open reading frame was found on the opposite strand starting at 1233 and extending beyond the Bgl II site. Figure 3 shows the D N A sequence starting 163 bases upstream from the start codon of the petB gene. A gap of 55 bp is located between the stop codon of the petB gene and the start of the petD gene and there is no inverted repeat as seen
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Fig. 2. The strategies for the sequencing of the petBD operon. The 2.85 kb Bgl II-Hind III fragment was digested with Sau3A1 or Hae III and fragments were sequenced by subcloning into M13 mp18 to obtain single-stranded DNA. Overlaps and opposite strand data were obtained from synthesized oligonucleotide primers. The unidentified open reading frame (URF) coding on the opposite strand continues beyond the Bgl II site. Search of the GenBank sequence data has not been fruitful in determining the function of the protein encoded on this URF.
486 -163
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137 46
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337 112
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T C A C C G G T G G C T T T A A A C G T C C C C G T G A G C T G A C T T C - G A T T A C T G G G G T C A T C A T G G C GA C G A T C A C C G T T T C C T T C G G T G T A A C T G G T T A C T C C T T G C C L T G G F K R P R E L T W I T G V I M A T I T V S F G V T G Y S L P
437 146
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537 179
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637 212
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737 5
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A A A A A G C C G G A T C T T A G C G A T C C A A A A C T C C ~ G G C A A A A C T G G C T C A A A A C A T G G G A C A C A A T T A C T A T G G T G A G C C G G C T T G G C C C A A T GA C A T T C T A T K K P D L S D P K L R A K L A Q N M G H N Y Y G E P A W P N D I L
837 37
T T A C C T T C C C C A T C T G T A T T G C G G G A A C C A T C G G T T T A A T T A C C G G C T T G G C G A T T C T C G A T C C A G C G A T G A T C G G T G A A c C G G GT A A T C C T T T C G C A A C T F P I C I A G T I G L I T G L A I L D P A M I G E P G N P F A T
937 72
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A C • C C T G G A A A T T T T G • C G G A G T G G T A C C T C T A C • C C G T C T T C C A G A T C C T C C G G G T A • T G C C C A A C A A A C T G C T G G G T A T T G C T T G T C A A G G G GC G A T T P L E I L P E W Y L Y P V F Q I L R V L P N K L L G I A C Q G A I
1037 105
10 38 106
CCGTTGGGTTTGATGATGGTGCCTTT~ATTGAAAGCGTCAACAAATTCCAAAACCCCTTCCGTCGTCCGGTGG~GATGGCTGTTTTCCTCTTTC-GGACTG P L G L M M V P F I E S V N K F Q N P F R R P V A M A V F L F G T
1137 138
1138 139
CGGTCACTCTCTGGCTCGGTG CGGGTGCTTGTTTCCCTATTGATGAGTCTTTGACTTTGGGCCTGTTCTAAAACCCACGCCTAATTTTTTGGACTAATTT A V T L W L G A G A C F P I D E S L T L G L F *
1237 161
123 8
TAY"~"i'I'ICATTTCTAAACAGATTGGC G A T C G C C C C C T ~ G C T C C G G C T A A A C G A T G G C G A T C G C C T T T T T T G T T A G G A T G G T G A A C A C C T G A C T T C T A A
838 38
1338
F
AGGAAGTCAAGATCCCCATTTTATTTTTCATTTCTAAACAGATTGGCGATCGCCCCCT
1337 1396
Fig. 3. The nucleotide and calculated amino acid sequence of the petBD operon of Synechococcus PCC 7002. The nucleotide sequence starts at -163 upstream from the ATG start of the petB gene. The petB sequence, 666 bp is followed by a gap of 55 bp separating the 480 bp petD gene.
and Nostoc PCC 7906 sequences are similar to the distances from either cyanobacteria to spinach chloroplast cytochrome b6-subunit 4. However, the paucity of sequence data from Nostoc PCC 7906 forbids any substantial determinations and these relationships are not readily resolvable. The same relationship is seen for the petCA gene products [32]. The codon usage for the Synechococcus PCC 7002 petBD operon mirrors that which has been reported for the Nostoc PCC 7906 petBD and other cyanobacteria [ 18, 19]. The absence of ATA-Ile, AGG-Arg, AGA-Arg and one occurrence of TCG-Ser and CGA-Arg is seen while GGT-Gly, CAA-Gln, CAC-His, and ACC-Thr are more frequently used. The codon usage for the petBD operon of Synechococcus PCC 7002 matches the overall codon usage seen in this cyanobacterium determined from 11600 bp of coding sequences derived from GenBank [5] using the codon frequency program in the U W G C G package [13].
The light-dependent transcript concentrationsfor cytochrome b 6 The transcript levels for the petB gene were measured as a function of time in the dark and from the onset of illumination. Cells continuously illuminated with light described under growth conditions were dark adapted in the absence of bubbled CO2. Aliquots, removed as indicated in Fig. 5, were analyzed for m R N A levels by isolating the m R N A and separating the transcripts on R N A gels. The northern transferred R N A was analyzed by hybridization with a radiolabeled probe containing just the petB reading frame or the cpcBAC operon [ 12]. After three hours in the dark, the cells were illuminated as before and aliquots were again removed and the transcripts analyzed. The autoradiograph depicting the temporal response of the petB and the cpcBAC transcript to light/dark conditions was scanned. The area under the curve was plotted as a function of time (Fig. 5). As seen the transcript levels under
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Fig. 4. The amino acid sequences from SynechococcusPCC 7002 (AGB6S4), Nostoc PCC 7906 [ 18] (Nostoc), and spinach [17] derived from the D N A sequences. Amino acids common to the spinach sequence.
c o n s t a n t illumination diminished rapidly to undetectable levels within 120 min. W h e n the illumination w a s restored to the cells, the transcript level increased rapidly and reached a m a x i m u m level, three fold over steady state, within 8 0 100 min and then slowly declined. This increase in transcript levels in r e s p o n s e to light has been seen for the petCA o p e r o n as well. Several other p h o t o s y s t e m II transcripts h a v e been s h o w n as being light inducible in Synechocystis 6803 [21] and Anacystis nidulans R 2 [6] as well as the other
SynechococcusPCC 7002
are shown as asterisks and numbered to
Synechococcus P C C 7 0 0 2 p h y c o b i l i s o m e o p e r o n apcABC (unpublished data). Probes for these transcripts were kindly D r D o n a l d Bryant.
supplied
to
us
by
Distance separating the p e t C A and the p e t B D operons T h e minimal distance separating the petBD and petCA o p e r o n s w a s estimated to be at least 445 kb
488 I
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