Genetic Manipulation of the Cyanobacterium - Plant Physiology

Plant Physiol. (1994) 104: 349-354

Genetic Manipulation of the Cyanobacterium Synechocystis sp. PCC 6803'

Development of Strains Lacking Photosystem I for the Analysis of Mutations in Photosystem I I

Lawrence B. SmartZ*, Neil R. Bowlby, Shawn 1. Anderson3, ldah Sithole4, and Lee Mclntosh

Michigan State University-Department of Energy Plant Research Laboratory, Michigan State University, East Lansing, Michigan 48824-1 31 2

We have taken a genetic approach to eliminating the presence of photosystem I (PSI) in site-directed mutants of photosystem I I (PSII) in the cyanobacterium Synechocystis sp. PCC 6803. By selecting under light-activated heterotrophic conditions, we have inactivated the psaA-psaS operon encoding the PSI reaction center proteins in cells containing deletions of the three psbA genes. We have also introduced deletions into both copies of psbD in a strain containing a mutation that inactivates psaA (ADK9). These strains, designated Dl-/PSI- and DZ-/PSI-, may serve as recipient strains for the incorporation of site-directed mutations in either psbAZ or psbD1. The characterization of these cells, which lack both PSI and PSII, is described.

The study of structure and function relationships in the PSII reaction center has been aided in recent years by the resolution of the three-dimensional structure of the bacterial reaction center (Allen et al., 1987; Deisenhofer and Michel, 1989). The conservation of overall structural and functional components has enabled accurate predictions of the corresponding architecture of PSII (Michel and Deisenhofer, 1988). However, the nonoxygen-evolving bacterial reaction center provides few clues about the organization of the OEC of PSII or the mechanism of water oxidation. In contrast, a wealth of biochemical, biophysical, and physiological data have been collected describing the enzymic and chemical properties of the four Mn atoms that form the active site of the OEC (Diner and Joliot, 1977; Ghanotakis and Yocum, 1990). This Mn cluster serves to accumulate oxidizing equiv-

Supported by National Science Foundation Postdoctoral Fellow- ship in Plant Biology DMB-9148737 (to N.R.B.), by Michigan State University Research Excellence Funds (to L.M.), and by Department of Energy grant DE-FG02-90-ER 20021 (to L.M.).

* Present address: Department of Vegetable Crops, Mann Labo- ratory, University of Califomia, Davis, CA 95616.

Present address: National Science Foundation Center for Biolog- ical Timing, Department of Biology, University of Virginia, Char- lottesville, VA 22901. ' Present address: Biochemistry Department, University of Zim-

babwe, Mount Pleasant, Harare, Zimbabwe. * Corresponding author; fax 1-916-752-4554. -

349

alents needed for the oxidation of water in a linear series of S-states driven by four single photon events (Kok et al., 1970; Forbush et al., 1971). By using molecular genetics, precise determination of the protein scaffold that coordinates this unique and crucial structure may be possible.

Structure and function studies on the photosystems has been accelerated by the use of the transformable unicellular cyanobacterium Synechocystis sp. PCC 6803 (Debus et al., 1988a; Vermaas et al., 1988b). This strain is naturally com- petent and has an active homologous recombination system, making it very amenable to molecular genetic manipulation (Williams, 1988). Synechocystis 6803 may also be grown pho- toheterotrophically, in the presence of DCMU and Glc (Wil- liams, 1988). These conditions eliminate the selective advantage conferred by the presence of wild-type PSII and have allowed for the segregation of mutations in PSII reaction center genes (Debus et al., 1988a; Vermaas et al., 1988b; Metz et al., 1989; Nixon and Diner, 1992).

Synechocystis 6803 lacks a trait found in chloroplast thylakoid membranes that allows for easy purification of PSII, lateral heterogeneity of PSI and PSII (Andersson and Ander- son, 1980). In chloroplasts, PSII is concentrated in the stacked membranes and PSI is confined to the unstacked regions (Andersson and Anderson, 1980). Current techniques for the purification of PSII from Synechocystis 6803 rely on selective detergent solubilization, Suc density centrifugation, and/or a series of chromatographic separations (Bumap et al., 1989; Rogner et al., 1990; Noren et al., 1991), techniques that can compromise OEC activity, especially in mutants (N. Bowlby, unpublished results). The present status of PSII purification has served well for certain types of analysis of PSII mutants (Debus et al., 1988a; Nixon and Diner, 1992), and detection of the EPR multiline spectrum from the S1 state of the Mn cluster has been reported (Noren et al., 1991). However, a method yielding highly purified and active PSII with a min-

Abbreviations D1-, D1 (PsbA) deficient; D2-, D2 (PsbD) deficient; EPR, electron paramagnetic resonance; LAHG, light-activated heterotrophic growth; OEC, oxygen-evolving complex; PSI-, PSI deficient; PSII-, PSII deficient; WT-LAHG, LAHG-grown wild type; WT- MIXO, mixotrophically grown wild type.

Dow

nloaded from https://academ

ic.oup.com/plphys/article/104/2/349/6068042 by guest on 21 D

ecember 2021

350 Smart et al. Plant Physiol. Vol. 104, 1994

imum of biochemical manipulation would be highly benefi- cial for the analysis of mutants in the OEC.

To facilitate such studies, we have embarked on a genetic approach to the elimination of PSI, a detrimental contaminant of PSII preparations, in Synechocystis 6803. Because there is no effective inhibitor of PSI activity, cells must be grown heterotrophically to eliminate the selective advantage conferred by wild-type PSI. Synechocystis 6803 may be grown in complete darkness using Glc as a carbon source, requiring only a brief (5 min) pulse of light every 24 h (Anderson and McIntosh, 1991). By selecting under these LAHG conditions (Anderson and McIntosh, 199 I), we have successfully inactivated the genes encoding the reaction center proteins of PSI, psuA and psaB (Smart et al., 1991; Smart and McIntosh, 1993). These mutants were devoid of any detectable PSI complex but still accumulated functional PSII at wild-type levels (Smart et al., 1991; Smart and McIntosh, 1993).

Here we describe the development of recipient strains for the mutagenesis of the PSII reaction center genes psbA and psbD. These strains, Dl-/PSI- and DZ-/PSI-, have mutations in psuA that prevent assembly of PSI. To avoid problems associated with the segregation of a mixture of wild-type and mutant psbA or psbD sequences, the regions of the chromosome targeted for mutagenesis have been deleted. In Synech- ocystis 6803, psbA is present in three copies (Jansson et al., 1987); therefore, deletions were introduced into a11 three copies by replacing those regions of the chromosome with three different drug-resistance cassettes (1. Sithole, N.R. Bowlby, J. Sinclair, G.T. Babcock, L. McIntosh, unpublished data). Likewise, psbD is present in two copies (Williams and Chisholm, 1987), and deletions were introduced into both of them. The rationales for attempting particular mutations in the psbA or psbD genes will be discussed elsewhere (I. Sithole, N.R. Bowlby, J. Sinclair, G.T. Babcock, L. McIntosh, unpublished data). Characterization of the strains Dl-/PSI- and DZ-/PSI- also gives us insight into the biogenesis of PSII and aspects of cyanobacterial physiology.

MATERIALS A N D METHODS

Materials

Chemicals and antibiotics used were of high purity and were obtained from Sigma or Research Organics (Cleveland, OH). Restriction and other enzymes were purchased from New England Biolabs (Beverly, MA). Radioactive isotope ([a-32P]dATP) was purchased from Amersham, nitrocellulose membrane from Schleicher & Schuell, and Bacto-Agar from Difco (Detroit, MI).

Strains and Crowth Conditions

A11 studies were performed using a Glc-tolerant strain of the cyanobacterium Synechocystis sp. PCC 6803 (Williams, 1988). The mutant ADK9, an insertional inactivation mutant of psaA, was previously described (Smart et al., 1991). The mutant 3ApsbA, with a11 three copies of psbA deleted, will be described elsewhere (I. Sithole, N.R. Bowlby, J. Sinclair, G.T. Babcock, L. McIntosh, unpublished data). Cells were grown in liquid BG-11 medium with 5 mM Glc or on solid BG-11 with 5 mM Glc and 1.5% purified Bacto-Agar under LAHG

conditions, as previously described (Anderson and McIntosh, 1991). Transformation of Synechocystis 6803 was performed essentially as described (Williams, 1988). For analysis of mutants, cells were grown in carboys containing 15 L of medium, were harvested using a continuous flow rotor (DuPont Sorvall, Wilmington, DE), and were frozen at -7OOC in BG-11 with 15% (v/v) glycerol. When appropriate, media were supplemented with antibiotics in the following concentrations: chloramphenicol, 1 O mg/L; erythromycin, 5 mg/L; spectinomycin, 20 mg/L; kanamycin, 5 mg/L; and gentamycin, 1 mg/L.

Nucleic Acids

A11 nucleic acid manipulations were performed using standard procedures (Sambrook et al., 1989) unless otherwise stated. DNA was isolated from Synechocystis 6803 using a modification of the procedure described by Golden et al. (1987) for the isolation of RNA from Synechocystis 6803. The aqueous phase obtained after vortexing the cells with glass beads, buffer, phenol-chloroform, and detergent,, was extracted twice with chloroform, then purified over a cesium chloride gradient with ethidium bromide by centrifiigation in a Beckman vTi50 rotor at 45,000 rpm for 18 h. The ethidium- bromide-stained DNA band was recovered, extracted with isopropanol to remove ethidium bromide, and precipitated using ethanol. Hybridization conditions and preparation of radiolabeled probes were previously described (Smart and McIntosh, 1991). The probe for psbD2 was a 0.5-kb KpnI/ SmaI fraginent from plasmid pRD655 (Debus et ai., 1988a). The psbDI probe was a 3.5-kb HindIII fragment from plasmid pRD1219 (Debus et al., 1988a). The psaA probe was a 1.8-kb KpnI fragment from plasmid pLS18 (Smart and McIntosh, 1991).

Chl and Protein Analysis

Chl was extracted from whole cells or from membranes using methanol and was quantified using the extinction coef- ficients of Lichtenthaler (1987). Cells were broken using a bead beater (Biospec Products, Bartelsville, OK), and membranes were prepared essentially as previously described (Smart et al., 1991). Membrane proteins were prepared for electrophoresis as described by Wynn et al. (1989) and were separated by 10% SDS-PAGE using the buffers of Laemmli (1970). Proteins were transferred to nitrocellulose and immunoblotting was performed as described by Tovvbin et al. (1979) and Smart et al. (1991), respectively. Antibodies raised to the PsaA/B proteins from Synechococcus have been described (Henry et al., 1990), as have antibodies raised to a portion of the D1 protein from Amaranthus hybridus (Ohad et al., 1985). Antibodies raised to the D2 polypeptide from spinach were previously described (Vermaas et al., 1988a).

EPR Spectroscopy

Room temperature EPR spectroscopy was performed using a Bruker ERZOOD spectrometer as previously described (Smart et al., 1991). Spectrometer conditions were: power, 20 mW; modulation amplitude, 0.4 mT; time constant, 200 ms; and

Dow



ecember 2021

Strains for Photosystem II Mutagenesis that Lack Photosystem I 351

sweep time, 200 s. Illumination was provided by a high-intensity microscope lamp. Dark-adaptation was for at least10 min.

RESULTS

Plasmid Constructs and Transformations

We used different strategies to generate the two recipientstrains for mutagenesis of psbA and psbD. In the case of theD2 recipient, we started with a PSr mutant, ADK9 (Smartet al., 1991), and sequentially introduced deletions into bothcopies of psbD. To generate the Dl recipient, we inactivatedthe psaA gene in a strain, SApsM, that had deletions in allthree copies of psbA (I. Sithole, N.R. Bowlby, J. Sinclair, G.T.Babcock, L. Mclntosh, unpublished data).

The first step in constructing the D2 recipient was thedeletion of the psbD2 gene. The strain ADK9 was transformedwith the plasmid pRD655Cm" (Debus et al., 1988a), in whichmost of the psbD2 gene had been replaced by a gene encodingresistance to chloramphenicol. Colonies resistant to kana-mycin and chloramphenicol were selected and streak purifiedfrom single colonies to accomplish gene segregation. DNAwas isolated from one of these colonies, ADK9Apsi>D2, andwas subjected to Southern analysis using a probe recognizingthe 3' flanking region of the psbDl gene that does notrecognize psbDl (Fig. 1A). Because these cells were found tobe fully segregated for the deletion of psbD2 (Fig. 1A), theywere then transformed with the plasmid pSLA1219:GmR (Fig.2A). This plasmid was a modification of pRD1219 (Debus etal., 1988a). In pSLA1219:GmR most of the psbDl gene hadbeen replaced by a 2.0-kb BamHl fragment, originally puri-fied from the plasmid pRZ1107, which contains a geneencoding resistance to gentamycin (Yin et al., 1988). The GmR

B1 2 4 5 6

kb

2.6-

1.4-

3.2-

1.8-

T.6-

3.8- I

1.6-

0.4-

Figure 1. Autoradiographs of Southern blots of genomic DNA fromwild-type and mutant cells. Lanes are: 1, 3, 5, wild type; 2,ADK9ApsbD2; 4, D2-/PSI"; 6, Dr/PSI"; 5 Mg DNA/lane. A, psbD2probe, DNA digested with H/ncll; B, psbDl probe, DNA digestedwith BstEII; C, psaA probe, DNA digested with Kpn\. Autoradi-ographs were exposed for different lengths of time.

psbC

T T HE

pSLA1219:Om

(B) flm" (B)

B psaA psaB

N K BK K

1kb

PLS1813G

Figure 2. Partial restriction maps of plasmids used for mutagenesisand of portions of the Synec/iocyst/s 6803 chromosome. A, Map ofthe psbDl -psbC region and of the insert region from the plasmidpSLA1219:GmR. B, Map of the psaA-psaB operon and of the insertregion from the plasmid pLS1813G. Thick lines represent regionsof the Synechocyst/s 6803 chromosome. Hatched boxes representcoding regions. The cross-hatched boxes represent the gentamycinresistance gene cassette. Crosses depict possible sites of homolo-gous recombination. Restriction sites in parentheses were de-stroyed in the cloning process. Restriction sites are represented by:H, H/ndlll; T, BstEII; E, fcoRI; B, BamHl; N, Ncol; K, Kpnl.

gene was also subcloned into the BamHl site of pUC119(Vieira and Messing, 1987), yielding pUC119-gen. Coloniesresistant to chloramphenicol, kanamycin, and gentamycinwere selected and streak purified from single colonies toallow for segregation. DNA was isolated from one of thesecolonies, and Southern analysis using a probe for psbDlrevealed complete segregation of the deletion (Fig. IB). Inaddition to wild-type bands (3.8, 1.6, and 0.4 kb), the psbDlprobe cross-hybridized to higher molecular mass bands rep-resenting psbDl (Fig. IB, lane 3). None of the wild-typepsbDl or psbD2 bands were detectable in the mutant strain,which was designated D2"/PSI~ (Fig. IB, lane 4).

The strain 3ApsM was generated by targeted deletion ofpsbA2 in a strain with psbAl and psbA3 deleted (Debus et al.,1988b), resulting in a strain with genes encoding resistanceto chloramphenicol, spectinomycin, and erythromycin in thechromosome (I. Sithole, N.R. Bowlby, J. Sinclair, G.T. Bab-cock, L. Mclntosh, unpublished data). To inactivate PSI ge-netically in this strain, we constructed a plasmid, pLS1813G,with a portion of the psaA gene replaced by a BamHl-Kpnlfragment from the plasmid pUC119-gen encoding resistanceto gentamycin (Fig. 2B). The strain 3Apsfe<4 was transformedwith pLS1813G, and colonies resistant to chloramphenicol,erythromycin, spectinomycin, and gentamycin were selectedand allowed to segregate by streak purification from singlecolonies. DNA was isolated from one of the resistant coloniesand was subjected to Southern analysis to verify completesegregation of the psaA mutation (Fig. 1C). This strain wasdesignated Dl"/PSr.

Dow



ecember 2021


Table 1. Chl determinations of whole cells and thylakoid membranesWT-MIXO' WT-LAHG'

Whole cells Ug Chl mL-' A™'1) 3.66 ± 0.63 0.81Membranes (^g Chl mg~' protein) 43.1 ± 7.2 9.9

' WT, Wild type. Data taken from Smart et al. (1991).

±0.31±0.4bN.D.,

or/Psr D2-/Psr0.31 ±0.06 0.15

5.5 + 0.1 3.2

Not determined.

± N.D.6

±0.3

Characterization of D1~/PSI~ and D2~/PSI~

Analysis of the strains Dl"/PSr and D2~/PSI~ includedChl determination, immunoblotting, and EPR spectroscopy.Chl determinations performed on whole cells revealed verylow levels of Chl per cell in strain D2~/PSI~, relative to wildtype, grown either mixotrophically (in the light with Glc) orunder LAHG conditions (Table I). The Chl levels in strainDr/PSr were also lower than in WT-MIXO or WT-LAHG(Table I) but greater than in D2~/PSI~. The ratio of Chl toprotein in isolated membranes followed the same trend (TableI). Both strains, Dl~/PSr and D2-/PSI", grew at near wild-type rates under LAHG conditions but did not grow incontinuous light above approximately 3 /nmol m~2 s"1 (datanot shown). Also, both cell types exhibited the turquoise-blue color seen previously in cells lacking PSI (Smart et al.,1991; Smart and Mclntosh, 1993).

To determine if any PSI or PSII reaction center proteinswere accumulating in the recipient strains, membrane pro-teins were subjected to immunoblotting. Antibodies raised tothe PsaA/B polypeptides did not detect the PSI reactioncenter proteins in membranes from either Dl~/PSr or D2~/PSI" (Fig. 3A). Likewise, antibodies raised against Dl did notdetect that protein in membranes from either of the tworecipient strains but did display apparent nonspecific cross-reaction to a polypeptide of approximately 33 kD (Fig. 3B).However, when antibodies raised to the D2 polypeptide wereused, a reduced amount of D2 protein was detected in

A1 2 3

B1 2 3

c1 2 3

kDa

95.5-

65.0-943.0-

36.0-

29.0-

18.4-

Figure 3. Immunoblots of membrane proteins isolated from wildtype, Dr/PSr, and D2~/PSr. Lanes are: 1, wild type; 2, Dr/PSr;

3, D2"/PSI"; 150 Mg protein/lane. Antibodies used for the threepanels recognized the following proteins: A, PsaA/B; B, the Dlpolypeptide; C, the D2 polypeptide. Molecular mass standardswere prestained proteins purchased from Diversified Biotech (New-ton Centre, MA).

membranes from the strain D1~/PSI~ (Fig. 3C). No D2 wasdetected in membranes from the strain D2~/PSr (Fig. 3C).

EPR spectroscopy is a sensitive and quantitative methodfor detecting redox components in the two photosystems. Wecollected room-temperature EPR spectra from membranes ofboth recipient strains, conditions under which one wouldexpect to detect both signal I (from P70o+ in PSI) and signal II(from Tyr YD

+ in PSII). The spectra from both recipient strainsinclude a prominent feature centered at g = 2.00425 with alinewidth (1 mT) and a lineshape (Fig. 4) that clearly indicatethat it does not arise from Tyr YD

+ (g = 2.0045, linewidth =2 mT) (Barry and Babcock, 1987). This feature is seen underillumination and in the dark and is of approximately equalintensity in samples from the two strains, but is much lessintense than one would expect from signal II in membranesof WT-LAHG (Fig. 4). There are no light-induced signals inthe spectra from either recipient strain (Fig. 4).

DISCUSSION

The purification of active PSII is essential for the analysisof site-directed mutations targeted at the OEC. The processof purification of PSII from Synechocystis 6803 is complicatedby the lack of lateral segregation of PSI from PSII that isfound in chloroplasts (Andersson and Anderson, 1980). Wehave taken a genetic approach to eliminate PSI from mutantswith site-directed changes in PSII by generating recipientstrains in which PSI has been genetically inactivated and inwhich deletions have been introduced into the targeted PSIIreaction center genes. By deleting the regions targeted formutagenesis, problems associated with segregating a mixtureof wild-type and mutant sequences are avoided. These strains(Dr/PSr and D2~/PSr) may be transformed with a plasmid

or/PS

— IlluminatedDark-Adapted

330 332 334 338H(mT)

338 340

Figure 4. Room-temperature EPR spectra of thylakoids from D1~/PSI~ and D2"/PSI~. The solid lines are the spectra obtained underconstant illumination. The dotted lines are the spectra collected inthe dark after illumination. Spectra were normalized to approxi-mately equal Chl concentrations.

Dow



ecember 2021

Strains for Photosystem II Mutagenesis that Lack Photosystem I 353

containing DNA that overlaps the deletion and includes the desired mutation, with an adjoining drug-resistance gene for selection. The mutation will be readily integrated into the genome by homologous recombination (Williams, 1988). This is the first published use of the gentamycin-resistance gene (Yin et al., 1988) for selection of mutations in a cyanobacterium. This gene adds another option for manipulation of the cyanobacterial chromosome.

We have shown previously that cells with mutations that inactivate the PSI reaction center genes exhibit normal assembly of stable PSII complex (Smart et al., 1991; Smart and McIntosh, 1993). However, these strains are extremely light- sensitive, and, therefore, must be grown in very dim light (approximately 3 pmol m-' s-') or under LAHG conditions. The genetic inactivation of PSII does not appear to have relieved that light sensitivity, suggesting that there is some other light-induced component creating toxic elements or that respiration cannot synthesize ATP in light above approximately 3 pmol m-* s-'. Our previous analysis of isolated membranes from strains ADK9 or BDK8 yielded EPR spectra with PSII signals of wild-type intensity and essentially free of other contaminating signals (Smart et al., 1991; Smart and McIntosh, 1993). Thus, we are quite confident that the inactivation of PSI does not significantly alter the stable assembly of PSII in Synechocystis 6803.

The strains Dl-/PSI- and D2-/PSI- give us some insight into the biogenesis of PSII. Genetic inactivation of the two copies of psbD, which encode the D2 polypeptide, also pre- vented accumulation of the D1 polypeptide. However, targeted deletion of the three copies of psbA, which encode D1, caused only partia1 reduction of the accumulation of D2. The D2 protein that did accumulate did not assemble into functional reaction centers, since we detected no signal I1 in the EPR spectra of D2-/PSI- membranes and no oxygen-evolving activity in the presence of 2,6-dichloro-p-benzoquinone (data not shown), an artificial electron acceptor from PSII. Our data suggest that a lack of PSI assembly does not alter the pattem of PSII biogenesis that has been observed in prior mutagenesis experiments using strains containing PSI. When both copies of psbD were inactivated, the D1 polypeptide failed to accumulate (Vermaas et al., 1988a; Yu and Vermaas, 1990). In addition, this strain failed to accumulate some of the other PSII proteins (Vermaas et al., 1988a). In a mutant with the three copies of p s b A inactivated, CP43, Cyt bS59, and the 33-kD protein accumulated to levels slightly lower than wild type, and the accumulation of D2 and CP47 was greatly reduced (Jansson et al., 1987; Nilsson et al., 1990). Our results reaffirm previous observations that both D1 and D2 are crucial for stable assembly and accumulation of PSII complex.

Assays of Chl accumulation per cell serve well in estimating the accumulation of PSI or PSII reaction center proteins (Smart et al., 1991; Smart and McIntosh, 1993) and may be used for initial characterization of mutants. This would be expected, since cyanobacteria do not contain peripheral Chl- binding antennae proteins. The Chl content in the Dl-/PSI- cells is reduced relative to ADK9 or BDK8, cells lacking only PSI (Smart et al., 1991; Smart and McIntosh, 1993). The Chl content of D2-/PSI- is about half that of Dl-/PSI-. The higher Chl levels in Dl-/PSI- may reflect the accumulation of low levels of D2 and possibly higher accumulation of

CP43 and/or CP47. We did not examine the accumulation of CP43 or CP47 in these strains. However, the Chl levels in both Dl-/PSI- and D2-/PSI- seem higher than one might expect for cells that lack both PSI and PSII. This may represent free Chl that is trapped in the membranes or Chl in some stage of synthesis or degradation, which may or may not be bound to a photosynthetic protein.

The EPR spectra from Dl-/PSI- and D2-/PSI- contained neither signal I, from P~,,o+, nor signal 11, from Tyr YD+ in D2. The prominent feature, common to spectra from both strains, was not light induced. One possible explanation is that this feature may have arisen from a semiquinone in the Cyt bs/f complex (G.T. Babcock, personal communication).

The strains Dl-/PSI- and DZ-/PSI- represent the first mutants in a photosynthetic organism with both photosystems genetically inactivated. In addition to serving in the process of site-directed mutation of PSII genes, these strains may be particularly well suited for other studies. In cyanobacteria, the Cyt b6/f chain is shared between photosynthesis and respiration (Scherer, 1990). The coordinate regulation of electron flow between these pathways is poorly understood. These strains may be appropriate for the study of respiration in cyanobacteria, since the interaction with the photosystems has been eliminated. The membranes from D2-/PSI- are essentially free of photosynthetic proteins, so they may serve as excellent starting material for the purification of the Cyt b6/f complex.

ACKNOWLEDCMENTS

We wish to thank G.T. Babcock (Department of Chemistry, Mich- igan State University) for the use of his spectroscopy facilities and for helpful discussions. The PsaA/B antibodies were the kind gift of J. Guikema (Kansas State University, Manhattan, KS). The D2 antibodies were the kind gift of W. Vermaas (Arizona State University, Tempe, AZ). The plasmid pRZ1107, containing the gentamycinresistance gene, was the kind gift of C.P. Wolk (M.S.U-D.O.E. Plant Research Laboratory, Michigan State University.)

Received September 2, 1993; accepted September 27, 1993. Copyright Clearance Center: 0032-0889/94/104/0349/06.

LITERATURE ClTED

AllenJP, Feher G, Yeates TO, Komiya H, Rees DC (1987) Structure of the reaction center from Rhodobacter sphaeroides R-26: the cofactors. Proc Natl Acad Sci USA 8 4 5730-5734

Anderson SL, McIntosh L (1991) Light-activated heterotrophic growth of the cyanobacterium Synechocystis sp. strain PCC 6803: a blue-light-requiring process. J Bacterioll73 2761-2767

Andersson B, Anderson JM (1980) Lateral heterogeneity in the distribution of chlorophyll-protein complexes of the thylakoid membranes of spinach chloroplasts. Biochim Biophys Acta 593

Barry BA, Babcock GT (1987) Tyrosine radicals are involved in the photosynthetic oxygen-evolving system. Proc Natl Acad Sci USA

Burnap R, Koike H, Sotiropoulou G, Sherman LA, Inoue Y (1989) Oxygen evolving membranes and particles from the transformable cyanobacterium Synechocystis sp. PCC6803. Photosynth Res 2 2

Debus RJ, Barry BA, Babcock GT, McIntosh L (1988a) Site-directed mutagenesis identifies a tyrosine radical involved in the photosynthetic oxygen-evolving system. Proc Natl Acad Sci USA 8 5

427-440

8 4 7099-7103

123-130

427-430

Dow



ecember 2021


Debus RJ, Barry BA, Sithole I, Babcock GT, McIntosh L (1988b) Directed mutagenesis indicates that the donor to P+680 in photosystem I1 is tyrosine-161 of the D1 polypeptide. Biochemistry 27:

Deisenhofer J, Michel H (1989) The photosynthetic reaction center from the purple bacterium Rhodopseudomonas viridis (Nobel lec- ture). Angew Chem Int Ed Engl28: 829-847

Diner BA, Joliot P (1977) Oxygen evolution and manganese. In A Trebst, M Avron, eds, Photosynthesis I: Photosynthetic Electron Transport and Photophosphorylation. Encyclopedia of Plant Phys- iology. Springer-Verlag, New York, pp 187-205

Forbush B, Kok B, McGloin M (1971) Cooperation of charges in photosynthetic O2 evolution. 11. Damping of flash yield osdlation, deactivation. Photochem Photobiol14 307-321

Ghanotakis DF, Yocum CF (1990) Photosystem I1 and the oxygen- evolving complex. Annu Rev Plant Physiol Plant Mo1 Biol 41:

Golden SS, Brusslan J, Haselkorn R (1987) Genetic engineering of the cyanobacterial chromosome. Methods Enzymoll53 215-231

Henry RL, Li M, Guikema JA (1990) A comparison of the PS1 polypeptide organization of soybean and the cyanobacterium, Anacystis nidulans. In M Baltscheffsky, ed, Current Research in Photosynthesis, Vol 11. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp 567-570

Jansson C, Debus RJ, Osiewacz HD, Gurevitz M, McIntosh L (1987) Construction of an obligate photoheterotrophic mutant of the cyanobacterium Synechocystis 6803. Plant Physiol 8 5

Kok B, Forbush B, McGloin M (1970) Cooperation of charges in photosynthetic 0 2 evolution. 1. A linear four step mechanism. Photochem Photobiol 11: 457-475

Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685

Lichtenthaler HK (1987) Chlorophylls and carotenoids: pigments of photosynthetic biomembranes. Methods Enzymol148 350-382

Metz JG, Nixon PJ, Roegner M, Brudvig GW, Diner BA (1989) Directed alteration of the D1 polypeptide of photosystem 11: evi- dente that tyrosine-161 is the redox component, Z, connecting the oxygen-evolving complex to the primary electron donor, P680. Biochemistry 28: 6960-6969

Michel H, Deisenhofer J (1988) Relevance of the photosynthetic reaction center from purple bacteria to the structure of photosystem 11. Biochemistry 27: 1-7

Nilsson F, Andersson B, Jansson C (1990) Photosystem I1 charac- teristics of a constructed Synechocystis 6803 mutant lacking synthesis of the D1 polypeptide. Plant Mo1 Biol 1 4 1051-1054

Nixon PJ, Diner BA (1992) Aspartate 170 of the photosystem I1 reaction center polypeptide D1 is involved in the assembly of the oxygen-evolving manganese cluster. Biochemistry 31: 942-948

Noren GH, Boerner RJ, Barry BA (1991) EPR characterization of an oxygen-evolving photosystem I1 preparation from the transfom-

9071-9074

255-276

1021-1025

able cyanobacterium Synechocystis 6803. Biochemistry 30

Ohad I, Kyle DJ, Hirschberg J (1985) Light-dependent degradation of the Qe-protein in isolated pea thylakoids. EMBO J 4 1655-1659

R-er M, Nixon PJ, Diner BA (1990) Purification and characterization of photosystem I and photosystem I1 core complexes from wild-type and phycocyanin-deficient strains of the cyanobacterium Synechocystis PCC 6803. J Biol Chem 265 6189-6196

Sambrook J, Fritsch EF, Maniatis T (1989) Molecular Cloning: A Laboratory Manual, Ed 2. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

Scherer S (1990) Do photosynthetic and respiratory electron transport chains share redox proteins? Trends Biochem Sci 15: 458-462

Smart LB, h d e r s o n SL, McIntosh L (1991) Targeted genetic inactivation of the photosystem I reaction center in the cyanobacterium Synechocystis sp. PCC 6803. EMBO J 1 0 3289-3296

Smart LB, McIntosh L (1991) Expression of photosynthesis genes in the cyanobacterium Synechocystis sp. PCC 6803: psaA-psaB and psbA transcripts accumulate in dark-grown cells. Plant Mo1 Biol

Smart LB, McIntosh L (1993) Genetic inactivation of the psaB gene in Synechocystis sp. PCC 6803 disrupts assembly of photosystem I. Plant Mo1 Biol 21: 177-180

Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedures and some applications. Proc Natl Acad Sci USA 76

Vermaas WFJ, Ikeuchi M, Inoue Y (1988a) Protein composition of the photosystem I1 core complex in genetically engineered mutants of the cyanobacterium Synechocystis sp. PCC 6803. Photosynth Res 17: 97-113

Vermaas WFJ, Rutherford AW, Hansson O (1988b) Site-directed mutagenesis in photosystem I1 of the cyanobacterium Synechocystis sp. PCC 6803: donor D is a tyrosine residue in the D2 protein. Proc Natl Acad Sci USA 85: 8477-8481

Vieira J, Messing J (1987) Production of single-stranded plasmid DNA. Methods Enzymoll53 3-11

Williams JGK (1988) Construction of specific mutations in photosystem I1 photosynthetic reaction center by genetic engineering methods in Synechocystis 6803. Methods Enzymol167: 766-778

Williams JGK, Chisholm DA (1987) Nucleotide sequences of both psbD genes from the cyanobacterium Synechocystis 6803. Zn J Biggens, ed, Progress in Photosynthesis Research, Vol IV. Martinus Nijhoff Publishers, Dordrecht, The Netherlands, pp 809-812

Wynn RM, Omaha J, Malkin R (1989) Structural and functional properties of the cyanobacterial photosystem I complex. Biochem- istry 2 8 5554-5560

Yin JCP, Krebs MP, Reznikoff WS (1988) Effect of dam methylation on Tn5 transposition. J Mo1 Bioll99 35-45

Yu J, Vermaas WFJ (1990) Transcript levels and synthesis of photosystem I1 components in cyanobacterial mutants with inactivated photosystem I1 genes. Plant Cell2 315-322

3943-3950

17: 959-971

4350-4354

Dow



ecember 2021

Documents

Genetic Manipulation of the Cyanobacterium - Plant Physiology