Construction and Characterization of Cyanobacterial Mutants Lacking ...

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1991 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 266, No. 20, Issue of July 15,pp. 13370-13376, 1991 Printed in U.S.A .

Construction and Characterization of Cyanobacterial Mutants Lacking the Manganese-stabilizingPolypeptide of Photosystem 11” (Received for publication, December 21, 1990)

Judith B. Philbrickt, BruceA. Diners, and Barbara A. Zilinskastll From the $Department of Biochemistry and Microbiology, Cook College, Rutgers, The State University of New Jersey, New Brunswick, New Jersey08903 and §CR&D Department, Experimental Station, E. I. du Pont de Nemours and Company, Wilmington, Delaware 19880-0173

Mutants of the cyanobacterium Synechocystis sp. thylakoid vesicles and photosystem I1 (PS 11)’ preparations Pasteur Culture Collection (PCC) 6803 that specifi- that retain the ability to evolve oxygen has made possible a cally lack the extrinsic 33-kDa manganese-stabilizingwide variety of in vitro studies, including those wherein the polypeptide of the photosystem I1 oxygen-evolving three extrinsic polypeptides of the oxygen-evolving complex (OEC) can be reversibly removed and the effects of their complex have been constructed by two independent removal on PS I1 activity observed. The results from such methods. Cartridge mutagenesis was used toinsertionally inactivate the psbO gene of one mutant andcom- experiments support amodel for the function of these proteins pletely delete thepsbO gene of the other mutant. Thesein which the 16- and 24-kDa extrinsic proteins mediate C1and Ca2+binding to PS I1 (4, 5), while the 33-kDa extrinsic mutantshave no detectablemanganese-stabilizing protein, often referred to as themanganese stabilizing protein polypeptide, but theydo accumulate steady-state levels (MSP), interacts more closely with the manganese cluster, of the intrinsic photosystem I1 polypeptides D l , D2, protecting the manganese binding site from exogenousreducand CP-43 that are comparable to wild-type,as deter- tants, and possibly stabilizes the conformation of PS I1 that mined by immunoblot analysis. Measurement of the is required for S-state transitions and oxygen evolution (6evolution of the relative quantum yields of chlorophyll 8). fluorescencefollowing actinic flash excitation indiRemoval of MSP from PS I1 preparations by washing with cates that though the concentration of reaction centers 1-1.5 M CaC12 results in a loss of 02-evolvingactivity which in mutantcells is comparable to that of wild-type cells, can be significantly reconstituted by allowing the rebinding approximately 40% of these centers harbora fluores- of the protein (6, 9, 10). Under these conditions, the mancence-quenching species other than P680+. The mu- ganese itself is not lost from the membranes, suggesting that tants are capable of photoautotrophicgrowth at a MSP does not provide any ligands that are essential for the slower rate than that of wild-type. Under conditions binding of the manganese cluster to PS11. However, evidence of Ca2+ depletion where wild-type growth is unaf- that some MSP may remain bound to PS I1 preparations fected, the mutants are unable to grow at all. The after such treatments has been raised (11, 12). The only data manganese-stabilizing protein, therefore, enhances the published to dateregarding the effects of an absence of MSP binding of Caz+or protects the reaction centerat low in an intactsystem (as opposed to isolated PS I1 preparations) Ca2+ concentrations. The mutants evolve oxygen at come from the work of Mayfield et al. (13). These authors approximately 70% of the wild-type rate, but are com- describe a mutant of the green alga Chlamydomonas reinhardpletely photoinactivated by high light intensities. Our tii which contains a large insertion in the gene whichencodes results indicate that the manganese-stabilizing poly- MSP, referred to by various laboratories as psbl (13), oeel peptide is not absolutely required for photosystem I1 (14), wowl (15), or psbO (16). In this mutant, the absence of MSP results in a lowered steady-state accumulation of the assembly or function in cyanobacteria, but its absence PS I1 reaction center complex (approximately 10% of wilddoes leadto anenhanced sensitivity to photoinhibition. type) and a more rapid turnover of its polypeptide components. We have sought to augment the existing data concerning MSP by genetically engineering mutants in the naturally transformable, facultatively photoheterotrophic cyanobacterium Synechocystis sp. Pasteur Culture Collection (PCC) 6803. The past decade has seen tremendous advances inour This unicellular cyanobacterium has become a widely utilized knowledge of the components and stepsinvolved in photosynthetic oxygen evolution (for recent reviews, see Refs. 1-3). The abbreviations used are: PS 11, photosystem 11; OEC, oxygenThe development of methods for the isolation of inverted evolvingcomplex; MSP,manganese-stabilizing polypeptide; PCC, * Supported by National Science Foundation Grant DCB-8820168. This is New Jersey Agricultural Experiment Station Publication D01104-2-90, supported in part by state funds and by the United StatesHatch Act. Thecosts of publication of thisarticle were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 To whom correspondence should be addressed. Tel.: 908-9329563; Fax: 908-932-8965.

Pasteur Culture Collection; SDS, sodium dodecyl sulfate; kan, kanamycin; strep, streptomycin; spec, spectinomycin; kb, kilobase pair(s); bp, base pair(s); DCMU,3-(3,4-dichlorophenyl)-l,l-dimethylurea;Q~, the primary quinone acceptor; P680, the primary electron donor of PS 11; F , relative quantum yield of chlorophyll fluorescence; F,, lowest relative quantum yield of chlorophyll fluorescence (QA oxidized); F,,,,,, maximum quantum yield of chlorophyll fluorescence (QA reduced); F,, variable fluorescence = F - F,; HEPES, 4-(2-hydroxyethyl)-lpiperazineethanesulfonic acid SI and Sz, the states of the OEC in which 1 and 2 oxidizing equivalents, respectively, have been stored.

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CyanobacterialMSP- Mutants system for the investigation of PS I1 function via cloning and mutagenesis of the various polypeptide components of the complex (17-22). We report here the construction of two mutants which lack MSP (one by the insertional inactivation by deletion of psbO), and theresults of of psb0, and the other various analyses of their photosynthetic capabilities. Previously (23), we reported the cloning, nucleotide sequence, and mutational analysis of psbO from Synechocystis sp. PCC 6803. Our observations at that time indicated that inactivation of psbO led to instability of PS I1 and the complete loss of oxygen-evolving ability, in agreement with the conclusions from studies of Chlamydomonas MSP- mutants (13). These earlier results, however,were clouded by the subsequent detection of a secondary mutation. As the results presented here show, our initial conclusions were incorrect. Compared to wild-type cells, mutants with inactivated psbO genes display only a partial loss of oxygen-evolving activity, but appear to have an enhanced susceptibility to photoinactivation. EXPERIMENTALPROCEDURES

MiscellaneousCloning Procedures-Isolation of restriction fragments from agarose gels wasdone using GeneCleanTM(BIO-101 Inc., La Jolla, CA). [32P]dATPlabeling of restriction fragments for probes in Southern and Northern blot analyses was done using the randomprimed DNA-labelling kit from U. S. Biochemicals. DNA Isolation and Southern Hybridization Analysis-Isolation of genomicDNA for Southern analyses was done by the method of Rogers and Bendich (24), using pelleted cells from cultures in late exponential to early stationary phase. Southern hybridization was performed overnight at 65 "C in 5 X SSC (1 X SSC: 15 mM sodium citrate at pH 7.0,0.15 M NaCl), 0.1% SDS, 0.02% bovine serum albumin, 0.02% Ficoll, 50 pg/ml sheared and denatured calf thymus DNA. Filters were then washed at 65 "C in several changes of 0.1 X SSC, 0.2% SDS before exposure to film. RNA Isolation and Northern Hybridization Analysis-RNAwas isolated from Synechocystis PCC 6803 wild-type and mutant cells grown under photoheterotrophic conditions to an O D ~ Xof,1.0. ~~ Cells were harvested by centrifugation at 8,000 X g for 5 min, washed once in 0.15 M NaC1/5 mM EDTA, and quick frozen in liquid nitrogen. The frozen cells were pulverized to a fine powder under liquid nitrogen, and the RNAwas isolated using guanidinium isothiocyanate according to theprotocol of Chomczynski and Sacchi (25). RNA was denatured with formamide at 65 "C, and 10 pg of RNA per lane was electrophoresed in a 1.2% agarose gel containing formaldehyde. The size-separated RNA was then transferred to nylon membranes (Duralon, Stratagene) and hybridized under high stringency conditions (as described above for Southern blot analysis) to 32P-labeledSymchocystis psbA,D, and 0 gene-internal probes. Filters were washed with 0.1 X SSPE (1 X SSPE: 180 mM NaCl, 10 mM sodium phosphate at pH 7.4, 1 mM EDTA), 0.5% SDS a t 60 "C and exposed to x-ray film for 1-3 days until exposures of each filter were of approximately the same intensity. Western Blot Analysis-Thylakoid membranes were isolated from late log phase cultures of Synechocystis wild-type and MSP deletion and insertion mutant strains grown in the presence of 5 mM glucose and 25 pg/ml kanamycin where appropriate. The membranes were isolated according to the protocol described by Burnap et al. (26), although cells were disrupted with a Bead-heater (Biospec Products, Bartlesville, OK) with 0.1-mm glass heads. Chlorophyll concentrations were determined by spectrophotometric analysis of methanolic extracts using extinction coefficients reported by MacKinney (27). The membranes were denatured in SDSaccording to conditions as described previously (28), and polypeptides were separated by electrophoresis in polyacrylamide gradient gels (11-15%), using a gel system developed by Thornber (28) to minimize aggregation and improve resolution of membrane polypeptides. Immunoblotting was done as described earlier (23). Monospecific polyclonal antibodies raised against spinach Dl, D2, and CP-43 were generously provided by Dr. W. Vermaas (Department of Botany and the Center for the Study of Early Events in Photosynthesis,Arizona State University, Tempe, AZ). Antibodies raised against spinachMSP were as described (23). Growth of Cyanobacteria-The glucose-tolerant strain of Synecho-

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cystis PCC 6803 was grown in liquid BG-11 medium (29) at 30"C under constant illumination (approximately 35 peinsteins m-* s-') with shaking (200 rpm) in a PsychrothermTM Controlled Environment Incubator. Antibiotics were added as appropriate to the final concentrations 25 pg of kanamycin/ml, 5 pg of streptomycin/ml, and 20 pg of spectinomycin/ml. For photoheterotrophic growth, glucosewas added to 5 mM. Construction of Cyanobacterial Mutants-Mutants lacking functionalpsbo genes were constructed intwo ways.One mutant, referred to as SK, bears an insertionally inactivatedpsbo, and was constructed by ligating a 1.3-kb BamHI fragment (obtained from the vector pUC4K, Pharmacia LKB Biotechnology Inc.) containing the Tn903 kanamycin resistance gene into theunique BamHI site inthe coding region of psbO (see Fig. 1).The other, referred to asSA, is a deletion mutant and was constructed by deleting the entire coding region of psbO from the StuI site 49 bp upstream of the ATG initiation codon to the XbaI site approximately 275 bp downstream of the TAA stop codon. The cohesive ends of the XbaI site were filled in using Klenow fragment so that a 1.2-kb SmaI fragment (obtained from the vector pUC4-KIXX, Pharmacia) containing the Tn5 kanamycin resistance gene could be ligated into theresultant blunt-ended gap (Fig. 1).The cyanobacterial genomic DNAused in these constructionswas carried as a 2.8-kb insert in pUC13, with approximately 0.8 and 1 kb of cyanobacterial flanking sequences 5' and 3', respectively, of the psbO coding region. The construct used to complement SA hack to wild-type was made by ligating a 2-kb EcoRI fragment containing a gene encoding streptomycin/spectinomycin resistance (obtained from pRL453 (30)) into the XbaI site downstream ofpsbO afterfilling in all ends with Klenow (Fig. 1).The complemented cyanobacterial mutant is referred to as

ss.

Transformation of Cyanobacteria-Plasmid DNA isolated by alkaline lysis was used to transform cyanobacterial cells as described (23). Transformants were selected on BG-11 agar plates supplemented with 0.3% sodium thiosulfate, 5 mM glucose, 10 p M DCMU and the appropriate antibiotic (25 pg/ml kanamycin or 2.5 pg/ml streptomycin). (Note: some strep' transformants were later found not to contain the strep/spec' marker, having most likely arisen from spontaneous mutation of the cyanobacterial genome; from that point on, spectinomycin at 20 pg/ml was used to select for the presence of the strep/ spec' marker in both Synechocystis and Escherichia coli). Individual antibiotic resistant colonies were serially streak-purifed at least three times to ensure segregation of homozygous mutants. Measurement of Oxygen Euolution-The oxygen-evolvingactivity of whole cells was measured a t 26 "C in aYellow Springs Instruments model 53 oxygen monitor fitted with a Clark-type electrode. Actinic light was supplied by a projection lamp and was filtered through a 1%CuS04solution. Precise adjustment of light intensity was achieved through the use of neutral density filters. The assay medium wasBG11 supplemented with 1.5 mM NaHC03,to which an aliquot of concentrated cells was added to a final chlorophyll concentration of 5 pgof chlorophyll per ml. The concentration of chlorophyll in methanol extracts of the concentrated cells was calculated using the extinction coefficients of MacKinney (27). Measurement of Flash-induced Fluorescence and Charge Recombination Kinetics-The relative quantum yield of chlorophyll fluorescence is ameasure of the redox state of the primary quinone electron acceptor QA of P S 11. In Synechocystis6803,where there is little energy transfer between centers, the concentration of QA- is proportional to F-F,. When QA is oxidized,the quantum yield of fluorescence except is minimal (Fo). When QA is fully reduced, it is maximal (FmaX), when a quencher of fluorescence is also present. The time-dependent variation of the quantum yield of chlorophyll fluorescence, following a saturating flash, was monitored in the fluorescence mode of a flashdetection spectrophotometer modeled after an instrument described by Joliot et al. (31). The actinic flash (EG&G FX199, 2 ps at halfheight) was filtered by an infrared reflecting filter (MTO Athervex TA2), aCorning 4-96, and a Kodak Wratten 34. The evolution of the relative fluorescence yield was measured by a series of probe flashes (EG&G FX199) a t 422 nm, where each flash excited no more than -0.2% of the centers. The sample photodiode was protected by an infrared reflecting filter (as above), a Schott KV470, a Schott KV550, and a Corning 2-64 which transmitted fluorescence >660 nm. RESULTS

Construction of psbO Mutants-Mutants lacking functional psbO genes were constructed in two different ways, as de-

Mutants Cyanobacterial MSP-

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scribed under "Experimental Procedures." The psbO gene of B C A the mutant referred to as SK has been insertionally inactiw-ss WT SK SA WT SK SA SS vated with the Tn903 kanamycin resistance gene cassette, while that of the mutant referred to as S A has been deleted kb entirely andreplaced with the Tn5 kanamycin resistance gene cassette (Fig. 1). T o demonstrate that the mutant phenotype of SA wasindeed due to a lesioninvolving only pshO, we attempted andwere successful inthe complementationof this mutant back to awild-type phenotype by transforming it with 4.8 m 0 4.5 a plasmid bearing a wild-type copy ofpshO linked to a marker 4.2 4.2 0 geneencoding streptomycin/spectinomycin resistance. The construct used to returnpsbO coding sequences to the mutant SA, resulting in the complemented mutantreferred to as SS, is also shown in Fig. 1. 0 2.0 The structure andhomozygosity of the cyanobacterial mu.,, 1.8 1.8 0 1.6 tants were confirmed by Southern analysis; results are shown in Fig. 2. Fig. 2A depicts the resultsof probing HindIII digests of DNA from wild-type ( WT) and the various mutants with the "?P-labeled 318-bp HindII/RstEII fragment from within ka n psbO Wac the psbO coding region (see Fig. 1). The 4.8-kb HindIII fragFIG. 2. Southern analysis of genomic DNA from wild-type ment containing psbO in wild-type DNA is absent in SK, sp. PCC 6803. and psbO mutant strains of Synerhocyslin replaced by 4.2- and 1.8-kb fragments that result from intro- Genomic DNA from wild-type 1 W 7 ' )and mutant strains SK iinserducing into the sequence a HindIII site in the kan' cassette tionally inactivated), SA (deleted), and SS (complemented) was tii(see Fig. 1). No hyhridization to thepshO probe is detectable gestedwith HindlII, electrophoresed on a 0.Y; aEarose gel. transin the lane containing SA DNA, confirming the absence of ferred to a nylon memhrane,and prohedwith [ -P]dA'l'P-laheIed f r a m e n t s . Prohes: pshO. 318-hp HindIl/HstEIl internalfragment; psbOin this mutant. However,in DNA from the complekan, a comhination of 1.2-kh Smnl fragment from pU('4-KINN mented mutant SS, the probe does recognize a 4.5-kb fragcontaining the Tn5 kan'gene and 2-kh HarnHI fragment from pl:CJment expected from the return of psbO linked to the spec' KSAC containing the TnSO.? kan' gene; s p r , 2-kt) b,'coHI fragment marker (see Fig. 1). from pRL453 containing the strep/spec' gene. Similar lanes were probed with "'P-labeled kan' cassettes (Fig. 2 R ) or the strep/spec' cassette (Fig. 2C). No hybridizaA E C 0 tion was detected in lanes of W T DNA in either case, while WT S A Y ( WT 56 5 1 WT SA sa W S A Y the kan probe did hybridize to the 4.2- and 1.8-kb fragments koa in SK DNA and a 1.6-kb fragment in SA DNA, all of which are expected to contain kan' cassette sequences. The spec 97.4ea 2probe hybridized to a 2-kb fragment as predicted from the map of SS (see Fig. 1). 450-

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