Synechocystis Sp PCC 6803 and Phycobilisome Function Strains

The Plant Cell, Vol. 5, 1853-1863, December 1993 O 1993 American Society of Plant Physiologists

Synechocystis sp PCC 6803 Strains Lacking Photosystem I and Phycobilisome Function

Gaozhong Shen,”’ Sammy Boussiba,aib and Wim F. J. Vermaasa a Department of Botany and Center for the Study of Early Events in Photosynthesis, Arizona State University, Tempe, Arizona 85287-1601 The Jacob Blaustein lnstitute for Desert Research, Ben-Gurion University of the Negev, Sede-Boker 84990, ISiael

To design an in vivo system allowing detailed analysis of photosystem II (PSII) complexes without significant interference from other pigment complexes, part of the psaAB operon coding for the core proteins of photosystem I (PSI) and part of the apcE gene coding for the anchor protein linking the phycobilisometo the thylakoid membrane were deleted from the genome of the cyanobacterium Synechocystis sp strain PCC 6803. Upon transformation and segregation at low light intensity (5 pE m+ sec-l), a PSI deletion strain was obtained that is light tolerant and grows reasonably well under photoheterotrophic conditions at 5 pE m-2 sec-l (doubling time -28 hr). Subsequent inactivation of apcE by an erythromycin resistance marker led to reduction of the phycobilin-to-chlorophyllratio and to a further decrease in light sensitivity. The resulting PSI-IesslapcE- strain grew photoheterotrophicallyat normal light intensity (50 pE m-2 se&) with a doubling time of 18 hr. Deletion of apcE in the wild type resulted in slow photoautotrophic growth. The remaining phycobilins in apcE- strains were inactive in transferring light energy to PSII. Cells of both the PSI-less and PSI-IesslapcE- strains had an approximately sixfold enrichment of PSll on a chlorophyll basis and were as active in oxygen evolution (on a per PSll basis) as the wild type at saturating light intensity. Both PSI-less strains described here are highly appropriate both for detailed PSll studies and as background strains to analyze site- and region-directed PSll mutants in vivo.

INTRODUCTION

During recent years, the cyanobacterium Synechocystis sp interpretation of experimental data. Thus, we set out to inves- strain PCC 6803 has been used widely and productively in tigate the possibility of developing strains lacking PSI and/or functional and structural analyses of photosystem II (PSII). A depleted in phycobilisome components; such strains would main reason for this is that Synechocystis 6803 is eminently provide a highly suitable background into which PSll muta- suitable for genetic modification of PSII: this cyanobacterium tions can be introduced. is a spontaneouslytransformable facultative (photo)heterotroph The core of the PSI complex consists of the PSI-A and PSI-B showing homologous recombination and can survive in the proteins (encoded by psaA and psaB, respectively) that to- absence of photosynthetic activity in the presence of glucose gether harbor the reaction center and -100 antenna chlorophyll (reviewed by Shestakov and Reaston, 1987; Williams, 1988). molecules (Golbeck, 1992). In cyanobacteria and higher plants, A large number of directed mutations have been made in PSll the psaA and psaB genes are adjacent and are cotranscribed. of this cyanobacterium (reviewed by Nixon et al., 1992; Pakrasi In cyanobacteria, targeted deletion of PSI was found to corre- and Vermaas, 1992). However, detailed functional and biochem- late with an extreme light sensitivity of the resulting mutants; ical characterization of PSll mutants generally is complex this is observed both in Anabaena variabilis ATCC 29413 because the PSlllPSl reaction center ratio is unfavorable in (Mannan et al., 1991; Toelge et al., 1991) and Synechocystis this cyanobacterium (Fujita and Murakami, 1988). Even though 6803 (Smart et al., 1991; Smart and Mclntosh, 1993). Thus, severa1 useful PSll preparation procedures are available for mutants lacking PSI were grown in darkness (as can be done wild-type Synechocystis 6803 (Burnap et al., 1989; Noren et for Anabaena 29413); strains such as Synechocystis 6803, al., 1991; Kirilovsky et al., 1992; Nilsson et al., 1992), prepara- which cannot be propagated in complete darkness, were tion of oxygen-evolvingPSll particles from a number of mutants propagated by light-activatedheterotrophic growth (LAHG). Un- has been unsuccessful, possibly due to a destabilized oxygen- der LAHG conditions, cells are grown in the presence of evolving complex in these mutants. Apart from PSI, the pres- glucose in darkness except for 5 min of light every 24 hr ente of phycobilisome components may also complicate the (Anderson and Mclntosh, 1991). By selection under LAHG conditions, complete genetic segregation after directed inactivation ofpsaA (Smart et al., 1991) orpsa6 (Smart and Mclntosh, 1993) To whom correspondence should be addressed. was obtained in Synechocystis 6803. The resulting mutants 1854 The Plant Cell

were reported not to grow in light and could be propagated predicted to result in a 2.0-kb apcE fragment. The PCR- only under LAHG conditions. However, as will be pointed out amplified Synechocystis 6803 DNA was cloned in pUC118 and in more detail in the Discussion section, it is relatively incon- sequenced to verify that it was highly homologous to apcE venient and possibly physiologically artifactual to grow under sequences from other organisms. The PCR-generated Syn- LAHG conditions. Therefore, the developmentof light-tolerant echocystis 6803 DNA sequence was 64 to 69% identical with PSI-less strains would be highly advantageous. apcEsequences from other cyanobacteria, but was much less The elimination of phycobilisomes could also be useful to homologous to any other gene in the data base. Thus, we have simplify PSll studies. The phycobilisome is a large phycobilin assigned this sequence to be part of the Synechocystis 6803 binding protein complex that serves as a primary light-harvest- apcf sequence. ing antenna for PSll in cyanobacteria and red algae (Bryant, A 940-bp Smal-Smal fragment within this Synechocystis 1991). The large anchor protein LCM,encoded by the apcf 6803 apcf gene was deleted and replaced by an erythromy- gene, appears to be a central component in attachment of the cin resistance cassette (Elhai and Wolk, 1988). The plasmid phycobilisome to the thylakoid membrane and in the transfer construct (pEE25) is shown in Figure 1. This plasmid was used of excitation energy from phycobilins to chlorophyll in the thy- to transform wild-type Synechocystis 6803. Transformants were lakoid (Gantt, 1988). Upon inactivation or deletion of theapcE selected by screening for erythromycin resistance and were gene in the cyanobacterium Synechococcus sp strain PCC subcultured to allow segregation to occur (a single Synechocys- 7002, the resulting mutant is still photoautotrophic(even though tis 6803 cell contains multiple genome copies) and thus to it grows slower than the wild type under photoautotrophic con- obtain a homogeneous genotype. As shown in Figure 2, the ditions because of a decreased PSll antenna size), and no strain is homozygous at apcf locus and is designated as intact phycobilisomes can be isolated (Bryant, 1988; Bryant apcE-. et al., 1990). Thus, it could be expected that inactivation of PCR-mediated amplification of part of the psaAB operon, apcf in Synechocystis 6803 would lead to a very much looser which is followed by creation of a plasmid that can be used attachment of phycobilisome components to thylakoids so that to replace part of thepsaA8 operon by a chloramphenicol re- the remaining phycobilisome components can be washed off sistance marker in Synechocystis 6803, has been described easily. These properties would be highly desirable for PSll by Boussiba and Vermaas (1992). The resulting plasmid car- studies in relatively intact systems, such as thylakoids. rying a deletion in the psaAB operon was used to transform In this study, we show that a PSI-less strain of Synechocys- tis 6803 can be developed by segregation in dim light. Upon deletion of apcf, this strain can grow under the same conditions as the wild type and with a similar doubling time. These strains are highly appropriate backgrounds into which PSll mutations can be introduced.

,Xba I apcE // RV

RESU LTS

Molecular Cloning and Construction of apcE- and PSI-less Strains

To generate an apcE- strain, at least part of apcE needed to be cloned from Synechocysfis 6803 so that a plasmid could be constructed carrying an antibiotic resistance marker flanked by Synechocystis 6803 apcE regions. For this purpose, a 2.0- kb apcE gene fragment from Synechocysfis 6803 was amplified using the polymerase chain reaction (PCR) with two primers designed to recognize conserved regions of apcf. Figure 1. Plasmid Map of the Construct Used To Generate the apcE- These conserved regions were identified by comparison of Strain of Synechocystis sp Strain PCC 6803. apcf sequences from Synechococcus sp strain PCC 6301 A 0.9-kb Smal-Smal fragment of the Synechocystis apcE gene was (Capuano et al., 1991), Synechococcus sp strain PCC 7002 deleted and replaced by a DNA fragment conferring erythromycin re- (Bryant, 1991), Calofhrix sp strain PCC 7601 (Houmard et al., sistance. Ampr, ampicillin resistance gene; Em', erythromycin 1990), and Cyanophoraparadoxa (Bryant, 1988). The primers resistance cartridge; M131G, the intergenic region of wild-type bacte- used were 5'-TATGCTATCGTAGCTGGGGATCCCAACATC-3' riophage M13 for initiation and termination of bacteriophage M13 DNA and 5'-CGTTCATA AG GTACCGTATCTTCACCA A A-3' an d were synthesis and for packaging of DNA into bacteriophage particles. PSI-les apcE-d san Synechocystis Strains 1855

seer shows 1.A Figurnn i thi, e3 s transformant strain lackn sa intact psaAB operon and is homozygous at the psaAB locus. The absence of PSI in this strain was confirmed by protein gel blotting using antibodies raised against PSI-A and PSI-B from spinach (data not shown) and by fluorescence emission wild type measurements at 77 K (as discussed later). To combine in one strain the lack of PSI with the deletion of apcE, the PSI-less strain was used as the host for transformation with the apcE deletion plasmid construct pEE25.

apcF a Rc5 agc'o c _ Q _ . Q I_Q — t_. t O L- Q ZX* QX^X 2^ Emr 1 kb . l _ 1 1 1l l L I JI , B wild type psaA psaB wild type apcE' PSI-less/apcE' I 1 prohn 123123123 _ kb 1 8 •c 1 c £- 8- Q Z 5-1 £ | 1 1 ?, 1 1 1 —t Z 1 6- H J V PSI-less / 1 5- L\\\\XV\\.N 4- Cmr 1 kb

3- B wild type PSI-less PSI-less/apcP 2- kb 123123123 1.6- 8- 6- 5- 4-

Figure 2. DMA Gel Blot Analysis of the Wild Type and apcE~ Strains. (A) Restriction maps of the apcE gene fragment from Synechocystis wile 680th n dapc£i n i 3typd ~an e strains. Emr, erythromycin 2- resistance. 1.6- (B) DNA gel blot of wild type, apcE~, and PSI-less/apcf- probed with a 32P-labeled intragenic 1.9-kb Clal-Kpnl apcE fragment froA mDN . the differen witt straincu hs Hindllswa l (lane Cla, s1) l d (lanean , s2) 1 - EcoRV and Hpal (lanes 3).

0.5-

wild-type Synectocysf/s 6803. Transformants were propagated Figure 3. DNA Ge, Btot Ma^s of the WNdTyp e and ps,.,essstrain s at low light intensity (5 nE m~2 sec'1) on plates containing 5 mM of glucose and 25 ng/mL of chloramphenicol. As trans- ) Restrictio(A n psaABmape th f so operon from Synechocystis 6803 in the wild type and PSI-less strains. Cm', chloramphenicol formants deplete werI PS e dn expectei relativele b do t y blue resistance. dudepletioa eo t chlorophyln i l while retaining phycobilisomes, (B) DNA gel blot of the wild type, PSI-less, and PSI-less/apc£- probed bluish colonies were selecte r restreaksdfo . After several with a 32P-labeled intragenic 2.3-kb Ncol-Hindlll fragment. DNA from restreaks turquoisa , e strain resulted relativelthas twa y light the different strains was cut with Oral (lanes 1), Kpnl (lanes 2), and tolerant and could grow well in continuous light at 5 uE rrr2 Ncol (lanes 3). 1856 The Plant Cell

Erythromycin-resistant colonies were selected, and segrega- in this organism leads to a loss of most phycobilins from the tion was allowed to occur while the transformants were cell and thus to a yellow-green color of the culture. Absence propagated at low light intensity (5 pE m-2 sec-I). As shown of PSlA and PSI-6 leads to a loss of the PSI core complex in Figures 26 and 36, results of DNA gel blotting indicated and thus of most of the chlorophyll in the cell, while the leve1 that the appropriate deletions and insertions indeed had oc- of phycobilisomes is not much affected. The increase in the curred in the genome of the PSI-IesslapcE- strain and that phycobilisome-to-chlorophyll ratio results in a turquoise blue the appropriate wild-type genes no longer could be detected. color of the PSI-less culture. The PSI-IesslapcE- strain dis- Also, after amplification of the EcoRV-Kpnl fragment of apcE plays a light yellow-green color because of the loss of most in the apcE- and PSI-IesslapcE- strains, and of the Ndel-Sphl phycobilins and the PSI core complex. fragment of psaAB in the PSI-less and PSI-IesslapcE- strains by PCR, the size of the fragments obtained was as expected from the size of the deletion constructs, and no trace of the Absorption Spectra wild-type fragment sizes could be observed (data not shown). These results indicated that these strains are homozygous, For a more quantitative analysis of the spectral features of these that only one copy per genome exists both for the psaAB op- strains, absorption spectra of intact cells were measured. The eron and for the apcE gene in the wild type, and that thispsaAB results are presented in Figure 4. The apcE- strain showed andlor apcE copy has been inactivated (by partia1 deletion) a large reduction in the 620-nm absorption maximum (originat- in the corresponding strains. ing from phycobilins) as compared to the wild type. The PSI-less strain was decreased in its 680- and 440-nm absorption peaks, reflecting a depletion in chlorophyll a due to the Reason for Light Tolerance in the PSI-less Strain loss of the PSI core proteins. The phycobilin peak was virtually unaffected. The PSI-IesslapcE- strain showed reduction The fact that we have been able to generate fully segregated in both the phycobilin and chlorophyll absorption peaks. The PSI-less strains under dim light conditions seems at variance relative amount of chlorophyll remaining in the PSI-less strains with the observation that PSI-less cells grown under LAHG was difficult to estimate precisely because of the underlying conditions are extremely light sensitive (Smart et al., 1991; phycobilin absorption, but seemed at least fourfold lower than Smart and Mclntosh, 1993). There are two possible explana- the amount present in the wild type. In strains lacking apcE, tions for this apparent discrepancy: (1) a secondary mutation the amount of phycobilins (as approximated by 620 nm ab- has occurred, making the PSI-less strain light tolerant; or (2) sorption) was decreased significantly (by a factor of -2). These a transition of PSI-less cells from LAHG to light conditions (and results imply that absence of apcE (and thus impairment of vice versa) may require a lengthy adaptation period during phycobilisome assembly and attachment) by itself does not which cell growth is inhibited. To distinguish between these possibilities, a liquid culture of the light-tolerant PSI-less strain, resulting from segregation under dim light, was transferred to LAHG conditions and propagated under these conditions for -4 weeks (four subcultures). Then, both this strain and a

PSI-less strain that always had been propagated under LAHG 0.08O.’ ~ conditions and had never been in continuous dim light were plated out and exposed to continuous dim light (5 pE m-2 se&). In both cases, a lag of -25 days was ObServed, after which some colonies started to grow. Eventually, the number of surviving colonies was very similar for both types of PSI- less cells. This implies that the reason PSI-less Synecbocys- tis 6803 is light sensitive when grown under LAHG conditions is because it needs to undergo a lengthy adaptation before it can propagate under dim light conditions. There is no evi- dente for the occurrence of a secondary (frequent) mutation 400 500 600 700 800 that makes our PSI-less Synechocystis 6803 strain light Wavelength (nm) tolerant. Figure 4. Absorption Spectra of the Wild Type and the apcE- and/or PSI-less Strains. Phenotype Absorption spectra were measured using whole cells of the wild type (-), and of apcE- (- - -), PSI-less (-.-.), and PSI-IesslapcE- The apcE-, PSI-less, and PSI-IesslapcE- strains are easily (. . . . .) strains. Spectra were corrected for scattering by putting tissue distinguished from each other and from the wild type by the paper in the reference beam. Chlorophyll absorbs at -440 and 680 nm, color of the strains. As will be discussed later, the lack of apcE and phycobilins at -620 nm. PSI-less and apcE- Synechocystis Strains 1857

0.5 -0.2

-0.4 O E -0.6 4 6 -0.5 2 -0.8 O O 8 m 3 -1 -1 -1 -1.2

-1.5 -1.4 O 20 40 60 80 100 120 O 20 40 60 80 100 120 Time (hcurs) lime (hours)

0.8 I'I'I'I'I' 0.4

0.4 O E oO k - m -0.4 O O O $ -0.4 0) -1 3 -0.8 -0.8

-1.2 -1.2 O 20 40 60 80 1w120 O 20 40 60 80 100 120 Tims (hours) Time (hours)

Figure 5. Growth Curves of Wild Type and Mutant Synechocystis 6803 Strains. Time-dependent growth of the wild type (O), the apcf- strain (A),the PSI-less strain (O),and the PSI-IesslapcE- strain (x) propagated under the following conditions. (A) Photoautotrophic conditions at 50 pE m-* sec-l. (B) Photoautotrophic conditions at 5 pE m-'sec-l. (C) Photomixotrophic conditions at 50 pE sec-l. (D) Photomixotrophic conditions at 5 pE m-' sec-l.

affect the amount of chlorophyll in the cell, whereas the ab- from a strain with decreased PSll antenna size, under pho- sence of the PSI core complex by itself does not change the tomixotrophic conditions the apcE- strain grew at a rate leve1 of phycobilisome pigments. similar to that of the wild type. The PSI-less strain did not grow photoautotrophically and, as shown in Figure 5C, it did not even grow appreciably under Growth Rates photoheterotrophic conditions at higher light intensity (50 pE m-2 sec-l). However, it grew reasonably well at lower light in- In Figure 5, the growth of the wild type and mutant strains un- tensity (5 pE m-2 sec-l) with a doubling time of 28 hr (Figure der photoautotrophic and photomixotrophic growth conditions 5D). In contrast, a PSI-less strain resulting from segregation is presented. As shown in Figure 5A, the apcE- strain was under LAHG conditions that has not been adapted to continu- photoautotrophic, but grew slowly (doubling time of 38 hr) in ous dim light (Boussiba and Vermaas, 1992) did not grow at the absence of glucose at 50 pE m-2 sec-I. Photoautotrophic 5 pE m-2 sec-l (data not shown). The light sensitivity of the growth of this mutant was even slower (doubling time 5 to 6 PSI-less strain adapted to LAHG conditions is in agreement days) at lower light intensity (Figure 5B), which is correlative with data obtained by Smart et al. (1991). evidence for a smaller PSll antenna size. As may be expected After inactivation of phycobilisome function in the PSI-less 1858 The Plant Cell

strain, the resulting PSI-IesslapcE- strain could be propa- was similar to that in the wild type (770). As shown in Figure gated well in continuous light at 50 pE m-* sec-I: the light 66, the PSI-less and PSI-IesslapcE- strains had a chlorophyll- intensity used for the wild type (Figure 5C). This implies that to-PSII ratio of 130 and 110, respectively, and had approximately a PSI-less strain has been generated that can grow reason- six times more PSll on a chlorophyll basis than the wild type. ably fast (with a doubling time of 18 hr) under normal light This suggests that in wild-type Synechocystis 6803,80 to 85% conditions. The significantly decreased light sensitivity of the of all chlorophyll is associated with the PSI core complex. This PSI-less strain upon decreasing the PSll antenna size implies corresponds reasonably well to the relative decrease in chlo- PSll activity is the main reason for light sensitivity in the PSI- rophyll concentration seen in PSI-less strains (see Figure 3) less strain. and to other estimates regarding the relative amounts of chlorophyll associated with PSll and PSI (Fujita and Murakami, 1988). The diuron dissociation constants were similar in the PSll Quantitation wild type and these strains, indicating that dissociation of phycobilisomes and loss of the PSI reaction centers have no To measure the relative amount of PSll in the wild type and effect on the conformation of the herbicide binding niche in the other strains, herbicide binding analysis was used to de- PSII. termine PSII-to-chlorophyll ratios in intact cells. Diuron is a PSII-directed herbicide with one high-affinity binding site per physiologically functional PSll unit, and from the number of Fluorescence Spectra diuron binding sites on a chlorophyll basis, the in vivo chlorophyll-to-PSII ratio can be calculated. As illustrated in Fig- A convenient way to confirm that the introduced mutations have ure 6A, the chlorophyll-to-PSII ratio in the apcf- strain (740) the desired effects on pigment-protein complexes is by fluorescence emission measurements at 77 K. Spectra obtained by I excitation of chlorophyll at 440 nm are presented in Figure 7A. The PSI-less and PSI-lesslapcf- strains yielded peaks at 685 C and 695 nm, which are characteristic for PSII, whereas the üs 725-nm fluorescence emission peak, originating from PSI, was -0 C absent. This confirms that chlorophyll associated with the PSI 3 reaction center core is missing in the PSI-less and PSI- nO IesslapcE- strains. As presented in Figure 76, upon 590-nm excitation (excit- ing phycobiliproteins), cells of the wild type showed three fluorescence emission maxima when measured in 60% glycerol, which tends to functionally uncouple phycobilisomes from thylakoid components. The peaks at 645 and 665 nm orig- O 400 1200 1600 inate from phycobilisome components (phycocyanin and allophycocyanin, respectively). The 685-nm peak originates 3, i from allophycocyanin 6 and PSII. This 685-nm peak was absent in the apcf- and PSI-lesslapcf- strains. Also, the phy- c O cocyanin (645 nm) and allophycocyanin (665 nm) emission .-5 peaks were blue shifted in the apcf- and PSI-IesslapcE- U strains, which may be the result of a lack of proper assembly Uc =I O of the remaining components of the phycobilisome complex. n Interestingly, at 590-nm excitation, fluorescence emission shoulders were observed at 725 nm, even in strains lacking PSI. It is likely that the 725-nm emission shoulder in this case originated f rom p hycobil ins. o) E To monitor the effect of glycerol on energy transfer from the

n phycobilisometo PSII, the low-temperaturefluorescence emis- 1 I I I I O 400 800 1200 1600 sion spectra upon 590-nm excitation were also measured in 25 mM of Hepes-NaOH, pH 7. The results are shown in Figure l/(free diuron) (l/pM) 7C. In the apcE- strains, only a single peak (at 660 nm) was Figure 6. Double-Reciproca1Plot of 14C-Diuron Binding to lntact observed in the absence of glycerol. Strikingly, in contrast to Cells. the wild type, no 685-nm peak and a barely discernible 725- nm shoulder were shown for the apcE- strains. This indicates (A) The wild type (O)and the apcE- strain (A). (e) The PSI-less (O) and the PSI-IesslapcE- (x) strains. the absence of efficient energy transfer from phycobilisomes Chl, chlorophyll. to pigments in the thylakoid membrane in the absence of apcE. PSI-less and apcE- Synechocystis Strains 1859

1.2 It appears that in the apcE- strains the remaining phycobilins are either free or loosely associated with the thylakoid mem- 1 brane: upon isolation of thylakoids from these strains, virtually all phycobilins were separated from the thylakoid fraction upon 0.8 centrifugation of broken cells (data not shown). In the wild type

oO under the same conditions, a sizeable portion of the phycobili- c 0.6 somes remained in the thylakoid fraction. In the absence of v)E PSI and glycerol, the major emission maximum was at 685 nm er 0.4 O and may represent PSII. In contrast to the observations made 3 ii in the presence of glycerol, fluorescence emission originating 0.2 from phycobilisome components was low, indicating efficient O energy transfer from phycobilisomes to PSII. Energy transfer 600 625 650 675 700 725 750 between phycobilisomes and PSll in the PSI-less strain appeared to be more efficient than in the wild type at low temperature in the absence of glycerol because there was a 1.2 ~ much higher relative fluorescence emission at 665 nm in the wild type than in the PSI-less strain. This apparent difference in energy transfer efficiency may be related to a difference in energy transfer states. Dark-adaptedwild-type cells are in state 2 (Fork and Satoh, 1983), with inefficient energy transfer between the phycobilisome and PSll (Mullineaux et al., 1990). In contrast, dark-adapted PSI-less cells appear to be in state 1.

Electron Transport

To determine whether the remaining phycobilins in the apcf- and PSI-lesslapcf- strains still were able to harvest any light energy for PSII, oxygen evolution was measured upon excitation with light from different regions of the spectrum. As illustrated in Table 1, upon 600 nm illumination (transmission maximum at 600 nm, band width 50 nm), which preferentially excites phycobilins, little oxygen evolution could be measured in the apcf- and PSI-lesslapcf- strains, in spite of the fact that a reasonable amount of phycobilins remained spectrally detectable in the apcf- strains, as indicated in Figure 4. The low rate of oxygen evolution detected can be explained by light absorption from chlorophylls because the transmission band width of the filter used is rather broad (50 nm). This indicates 600 625 650 675 700 725 750 that light absorbed by remaining phycobilins cannot be used Wavelength (nm) efficiently (if at all) to excite PSll in the apcf- strains, confirm- ing the results obtained by 77 K fluorescence emission Figure 7. FluorescenceEmission Spectra Detected at 77 K Using In- (Figure 7). tact Cells. Upon excitation at wavelengths greater than 665 nm (mainly (A) Emission spectra from the wild type (-) and from the PSI-less absorbed by chlorophyll), the oxygen evolution rates of PSI- (---) and the PSI-IesslapcE- (. . . . .) strains. Excitation was at 440 less (compared to the wild type) and PSI-lesslapcf- (com- nm (chlorophyll excitation). Spectra were normalized to 1.0 at 685 nm pared to the apcE- strain) were five to six times higher on a for the PSI-less and PSI-IesslapcE- strains and at 725 nm for the wild per chlorophyll basis, which is compatible with the results of me. PSll quantitation. On a per cell basis, the apcE-, PSI-less, (B) Emission spectra from the wild type (-) and from the apcE- and PSI-lesslapcf- strains showed oxygen evolution rates (---) and PSI-lesslapcf- (. . . . .) strains. Excitation was at 590 nm (phycobilisome excitation), and cells were suspended in 60% glycerol similar to those of the wild type (Table 1). in 25 mM of Hepes-NaOH, pH 7,2 min before freezing. Spectra were normalized to 1.0 at the 640- to 650-nm peak. (C) Emission spectra from the wild type (-) and from the apcE- was added. Spectra were normalized to 1.0 at the 665-nm peak for (---), PSI-less (-.-.-.), and PSI-IesslapcE- (. . . . .) strains. Ex- the wild type, the 660-nm peak for the apcE- and PSI-IesdapcE- citation was at 590 nm, but in contrast to the situation in (E),no glycerol strains, and the 685-nm peak for the PSI-less strain. 1860 The Plant Cell

and W. Vermaas, unpublished data), and this would pose prob- Table 1. Rates of Oxygen Evolution in Cells upon Excitation at Various Wavelengthsa lems for routine application of strains grown under such conditions. Excitation Therefore, it was of interest to develop a Synechocystis 6803 Wavelength pmol O2 pmol O2 strain lacking PSI but retaining the capacity to propagate in Strain (nm) (mg Chl hr)-l (OD7m L hr)-l light. By carrying out transformation and subsequent segre- Wild type 600b 300 1150 gation at low light intensity (5 pE m-* sec-I), we have ob- >665c 330 950 tained a genetically homozygous strain carrying a deletion of apcE- 600 40 120 part of thepsaA6 operon and growing satisfactorily at low light >665 290 870 intensity in the presence of glucose. This PSI-less strain con- PSI-less 600 2460 1200 tained no 725-nm fluorescence emission component that is >665 21 80 1110 characteristic for PSI (Figure 7A), lost most of its chlorophyll PSI-lessl 600 190 150 aDcE- >665 1970 1290 (Figure 4), and did not show any PSI-mediated electron flow (data not shown). Because most, if not all, of the chlorophylls a Data shown are the average of four experiments and reproducible associated with the PSI core complex are associated with the within of each value reported. 20% gene products of psaA and psa6 (Bryant, 1992) and because Light intensity, pE rr2sec-l. 1800 there is no light-harvesting chlorophyll I complex in cyanobac- C Light intensity, 2400 pE m-2 sec-l. Chl, chlorophyll. teria, it is likely that inactivation of the psaA6 operon in Synechocystis 6803 leads to a loss of all pigments associated with PSI. Light Saturation In the PSI-less as well as the PSI-IesslapcE- strains, diuron binding assays yielded a ratio of one diuron binding siíe As noted above, the size of the PSll antenna in apcE- strains (one PSll reaction center) per 110 to 130 chlorophylls. This ra- appears to be decreased significantly. To quantitate this, oxy- tio was two- to threefold higher than could be expected from gen evolution was measured at different light intensities. As a simple addition of the number of chlorophylls associatedwith shown in Figure 8, the apcE- and PSI-lesslapcE- strains CP43 and CP47 (15 to 25 each; reviewed by Vermaas and needed approximately a fourfold higher light intensity to ob- Ikeuchi, 1991) and with D1 and D2 (approximately six total; tain a similar degree of saturation as compared to the wild type. Montoya et al., 1991). It is likely that chlorophyll binding pro- The light saturation characteristics of the PSI-less strain resem- teins other than CP43 and CP47 and the PSll and PSI reaction bled those of the wild type at low light intensity; however, the PSI-less strain appeared more prone to photoinhibition at higher light intensity. The results obtained with the apcE- strains are compatible with the notion that the apcE- and PSI- lesslapcf- strains have a small antenna size for PSll and that light energy absorbed by phycobilins in these strains is not transferred efficiently (if at all) to PSII.

DlSCUSSlON

Deletion of the PSI Reaction Center Core

The unicellular cyanobacteriumSynechocystis 6803 is a highly O 11111,1,1,1, convenient system for directed mutagenesis of the PSll com- O 2000 4000 6000 8000 10000 12000 14000 plex, and a PSI-less strain would be a considerable asset for detailed analysis of PSll mutants. Previous works on targeted Light lntensity (pE.m-?s-’) inactivation of the PSI reaction center in Synechocystis 6803 Figure 8. Light Saturationof Steady State Oxygen Evolution in lntact suggested that PSI-less mutants could be obtained only un- Cells. der LAHG conditions (Smart et al., 1991). However, because Light saturation curves were measured for cells from the wild type (O) light influences expression of PSll genes (Mullet, 1988) and and the apcE- (A),the PSI-less (O), and the PSI-lesslapcf- (x) is required for photoactivation (Tamura and Cheniae, 1988), strains. The data presented are the average of three measurements properties of PSll in cells grown under LAHG conditions are for each data point. For the wild type, 100% oxygen evolution represents not necessarily identical to those of PSll under normal labo- 420 pmol of O2 (mg chlorophyll hr)-l. For the apcf-, PSI-less, and ratory light conditions. Also, in our hands, the transformability PSI-1esslapc.E strains, this value is 360, 2460, and 2070 pmol of O2 of strains grown under LAHG conditions was poor (S. Boussiba (mg chlorophyll hr)-l, respectively. PSI-less and apcE- Synechocystis Strains 1861

center proteins exist in cyanobacteria; for example, another functional dissociation of phycobilisomes from PSll (a loss of chlorophyll binding protein can be expressed under conditions light energy transfer from phycobilins to PS II chlorophylls), of iron depletion (Laudenbach and Straus, 1988; Riethman and an easy loss of phycobilins from the thylakoid fraction, and Sherman, 1988a, 1988b), while a 22-kD protein resembling a depletion in the level of phycobilins in Synechocystis 6803. a light-harvestingchlorophyll II protein (Kim et al., 1992; Wedel This appears similar to the situation in Synechococcussp PCC et al., 1992) also appears to be present in Synechocystis 6803 7002, where no intact phycobilisomes can be isolated in an (Nilsson et al., 1990). apcf- strain (Bryant et al., 1990). These results are com- The loss of PSI did not appear to affect PSll assembly and patible with the view of the LcM being critical not only for function: in terms of oxygen evolution, all PSll centers in the attachment of the phycobilisome to the thylakoid and for provid- PSI-less strain were as active as in the wild type, because the ing a pathway of energy transfer from phycobilin in the ratio of the rate of oxygen evolution in the wild type and this phycobilisometo chlorophyll in the membrane but also for sta- strain (on a chlorophyll basis) was similar to the ratio of the ble assembly of the phycobilisome subunits. amount of PSll on a chlorophyll basis as measured through Although deletion of apcf led to a decrease in the phycobi- herbicide binding. Also, in a light-sensitive PSI-less Syn- lin content of the cells, a measurable amount of phycobilins echocystis 6803 mutant (with inactivated psaA) PSll assembles remained. However, this does not impede the usefulness of into functional complexes (Smart et al., 1991). the PSI-lesslapcf- strain for analysis of PSll mutants by It is interesting to note that the level of phycobilisomes in fluorescence, for example, because the phycobilins are eas- the cell did not change upon deletion of PSI (Figure 4). Also, ily and quantitatively washed off upon thylakoid isolation and upon deletion of apcf the PSII-to-PSIratio was not affected are not functionally coupled to PSII. (Figure 6A). This suggests that the syntheses of these three complexes (PSII, PSI, and phycobilisomes) are independently regulated. Decrease of Light Sensitivity upon apcE Deletion

The PSI-IesslapcE- strain can be propagated under standard Light Sensitivity of PSI-less Strains growth conditions (at light intensities used to propagate wild- type Synechocystis 6803) with a doubling time only slightly An important question is what may be responsible for the large longer than that of the wild type. The fact that the PSI-less difference in light sensitivity in our PSI-less strain propagated strain becomes much more light tolerant upon deletion of apcf in dim-light versus in the strain grown under LAHG conditions. implies that PSll is the main reason for the light sensitivity in In the transition from LAHG to dim-light conditions, these two PSI-less strains. It is possible that overreduction of the strains showed a similar requirement for a lengthy adaptation plastoquinone (PQ) pool (as could occur in PSI-less cells if period. After adaptation to LAHG conditions, the relatively light- the respiratory PQH2-oxidizingactivity is insufficient to keep tolerant strain obtained from the segregation under dim light up with PSll activity) directly or indirectly leads to metabolic became light sensitive and needed -4 weeks to recover light imbalances that are detrimental to cell growth. However, the tolerance. This adaptation time is unusually long for an organ- observation that growth of the PSI-less strain in the presence ism with a usual doubling time of approximately a day or less. of 20 pM of atrazine (which blocks PSll electron transfer into It is possible that during propagation under LAHG conditions, the PQ pool) still was impaired (data not shown) suggests that the cells strongly express particular enzymes and accumu- the generation of potentially toxic substances (such as singlet late specific metabolites to deal with dark growth. Transition oxygen, chlorophyll radicals, andlor superoxide) within PSll from LAHG to continuous dim-light conditions is likely to alter may also be a contributing factor, particularly if electron flow metabolic pathways and may lead to particular degradation out of the PSll complex is blocked. or converSion products utilizingenzymes remaining from LAHG In any case, now that a PSI-less cyanobacterial strain has growth and those synthesized in dim continuous light. It is pos- been developed that can be grown under normal light condi- sible that these incongruous enzyme complements yield tions, the stage has been set for introduction of site- and components poisonous to the cells. Only after an extended region-directed mutations into the various PSll genes in this period needed for degradation and dilution of the LAHG en- background. Detailed analysis of resulting mutants can be per- zyme machinery may growth of the PSI-lessstrain in dim light formed in vivo or by using thylakoids without the need to become possible. A similarly long adaptation period upon trans- prepare PSII-enriched particles. fer to LAHG conditions has been observed (Boussiba and Vermaas, 1992). METHODS

Anchor Polypeptide LCM Culture and Growth

Deletion of the apcE gene, encoding the anchor polypeptide Synechocystis sp strain PCC 6803 was cultivated in BGll medium LCM, in the apcE- and PSI-IesslapcE- strains led to a (Rippka et al., 1979) at 3OOC. Mutant cells of this organism were grown 1862 The Plant Cell

in BGll supplemented with 5 mM of glucose. The light intensity at ACKNOWLEDGMENTS which the wild type and the PSI-IesslapcE- strain were grown was 50 pE sec-l, unless indicated otherwise. Strains lacking only photosystem I (PSI) were. propagated at low light intensity (5 pE m2 we are grateful to Dr. Scoti Bingham for synthesis of oligonucleotides. sec-l). Growth of the wild-type and mutant strains was followed un- we thank Dr. Rick Debus for sending us the pRL425 plasmid carrying der photoautotrophic and photomixotrophic (with 5 mM glucose) the erythromycin resistance marker; this plasmid was developed by conditions by monitoring the optical density (cell scattering) at 730 Drs. Jeff Elhai and Peter Wolk. We thank Dr. Anastasios Melis for using nm using a UV-160 spectrophotometer. his PSI antibody. This research is supported by National Science Foun- dation Grant No. DMB 90-58279 to W.F.J.V. This is Publication No. 167 of the Arizona State University Center for the Study of Early Events in Photosynthesis. The Center is funded by U.S. Department of Energy Electmn Transport and Herblcide Binding Measurements Grant No. DE-FG02-88ER13969as part of the Plant Science Centers Program of the U.S. Department of AgriculturelDepartment of Oxygen evolution measurements and herbicide binding experiments Energy/National Science Foundation. were performed as described by Shen et al. (1993). In oxygen evolution measurements, the electron acceptor was 0.5 mM kFe(CN),, while 0.1 mM of 2,5-dimethyl-pbenzoquinonewas added as redox medi- Received July 14, 1993; accepted October 19, 1993. ator between thylakoids and the nonpenetratingferricyanide. Light was provided by a 150-W xenon arc lamp. The light was filtered through water and subsequently passed through a broad-band interference filter (Lmm = 600 nm, 50-nm bandwidth) or red cut-off filters, trans- mitting light with a wavelength above 665 nm. For herbicide binding REFERENCES analysis using I4C-diuron,50 pg of chlorophyll per mL was used for the wild type and the apcE- strain and 10 lgof chlorophyll per mL for PSI-less and PSI-1esslapc.E- strains. Anderson, S.L., and Mclntosh, L. (1991). Light-activated heterotrophic growth of the cyanobacterium Synechocystis sp. strain PCC 6803: A blue-light-requiring process. J. Bacteriol. 73, 2761-2767. Boussiba, S., and Vermaas, W.F.J. (1992). Creation of a mutant with Fluorescence Emission Spectra an enriched photosystem lllpigment ratio in the cyanobacterium Syn- echocystis sp. PCC 6803. In Research in Photosynthesis, Vol. 111, Fluorescence emission spectra were determined using a Fluorolog N. Murata, ed (Dordrecht: Kluwer), pp. 429-432. 2 instrument (SPEX lndustries Inc., Edison, NJ). Cells were diluted Bryant, D.A. (1988). Genetic analysis of phycobilisome biosynthesis, to a concentration of 5 pg (for wild type and apcE-) and 2 lg(for PSI- assembly, structure, and function in the cyanobacterium Syn- less and PSI-IesslapcE-) of chlorophyll per mL in 25 mM of Hepes- echococcus sp. PCC 7002. In Light-Energy Transduction in NaOH, pH 7, in 60% (v/v) glycerol or to a concentration of 50 pg (for Photosynthesis: Higher Plant and Bacterial Models, S.E. Stevens, wild type and apcE-) and 10 lg(for PSI-less and PSI-IesslapcE-) in Jr. and D.A. Bryant, eds (Rockville, MD: American Society of Plant absence of glycerol for spectra measured at liquid nitrogen tempera- Physiologists), pp. 62-90. ture. The excitation and emission slitwidths were 12 and 2.4 nm, Bryant, D.A. (1991). Cyanobacterial phycobilisomes: Progress toward respectively. complete structural and functional analysis via molecular genetics. In The Photosynthetic Apparatus: Molecular Biology and Operation, Vol. 78, L. Bcgorad and I.K. Vasil, eds (San Diego: Academic Press), pp. 257-300. DNA lsolation and DNA Gel Blotting Bryant, D.A. (1992). Molecular biology of photosystem I. In The Pho- tosystems: Structure, Function and Molecular Biology, J. Barber, ed DNA was preparedfrom Synechocystis 6803 essentially as described (Amsterdam: Elsevier), pp. 501-549. by Williams (1988). After restriction digestion of genomic DNA, gel blot- Bryant, D.A., Zhou, J., Gasparich, G.E., de Lorimier, R., Guglielmi, ting to GeneScreen Plus (Du Pont-New England Nuclear) membranes G., and Stirewalt, V.L. (1990). Phycobilisomes of the cyanobacterium was performed, and blots were hybridized with a 3*P-labeled nick- Synechococcus sp. PCC 7002: Structure, function, assembly and translated Synechocystis apcE probe (a 1.9-kb Clal-Kpnl fragment) or expression. In Molecular Biology of Membrane-Bound Complexes a Synechocystis psaAB probe (2.3-kb Ncol-Hindlll fragment) and in Phototrophic Bacteria, G. Drews, ed (New York: Plenum), pp. washed according to the manufacturer‘s recommendations. 129-141. Burnap, R., Koike, H., Sotimpoulou, G., Sherman, L.A., and Inoue, Y. (1989). Oxygen evolving membranes and particles from the trans- Thylakoids Preparation and Protein Gel Blotting formable cyanobacterium Synechocystis sp. PCC 6803. Photosynth. Res. 22, 123-130. The procedurefor the preparation of the thylakoids from the wild type Capuano, V., Braux, A., de Marsac, N.F., and Houmard, J. (1991). and mutants was described by Shen et al. (1993). Methods used for The “anchor polypeptide” of cyanobacterial phycobilisomes: Molec- SDS-polyacrylamide gel electrophoresis and protein gel blotting were ular characterization of the Synechococcus sp. PCC 6301 apcE gene. identical to those described by Vermaas et al. (1988). J. Biol. Chem. 266, 7239-7247. PSI-less and apcE- Synechocystis Strains 1863

Elhai, J., and Wolk, C.P. (1988). A versatile class of positive-selection Noren, G.H., Boerner, R.J., and Barry, B.A. (1991). EPR character- vectors based on the nonviabilityof palindromecontaining plasmids ization of an oxygen-evolving photosystem II preparation from the that allows cloning into long polylinkers. Gene 68, 119-138. transformable cyanobacterium Synechocystis 6803. Biochemistry Fork, D.C., and Satoh, K. (1983). State I-State I1 transitions in the ther- 30, 3943-3950. mophilic blue-green alga (cyanobacterium) Synechococcus lividus. Pakrasi, H.B., and Vermaas, W.F.J. (1992). Protein engineering of Photochem. Photobiol. 37, 421-427. photosystem II. In The Photosystems: Structure, Function and Mo- Fujita, Y., and Murakami, A. (1988). Steady state of photosynthesis lecular Biology, J. Barber, ed (Amsterdam: Elsevier), pp. 231-257. in cyanobacterial photosynthesis systems before and after regula- Riethman, H.C., and Sherman, L.A. (1988a). Purification and tion of electron transport composition: Overall rate of photosynthesis characterization of an iron stress-induced chlorophyll-protein from and PS IlPS II composition. Plant Cell Physiol. 29, 305-311. the cyanobacteriumAnacystis nidulans R2. Biochim. Biophys. Acta Gantt, E. (1988). Phycobilisomes: Assessment of the core structure 935, 141-151. and thylakoid interaction. In Light-Energy Transduction in Pho- Riethman, H.C., and Sherman, L.A. (1988b). lmmunological char- tosynthesis: Higher Plant and Bacterial Models, S.E. Stevens, Jr. acterization of iron-regulated membrane proteins in the cyanobac- and D.A. Bryant, eds(Rockville, MD: American Societyof Plant Phys- terium Anacystis nidulans R2. Plant Physiol. 88, 497-505. iologists), pp. 91-101. Rippka, R., Deruelles, J., Waterbury, J.B., Herdman, M., and Stanier, Golbeck, J.H. (1992). Structure and function of photosystem I. Annu. R.Y. (1979). Generic assignments, strain histories and propertiesof Rev. Plant Physiol. Plant MOI. Biol. 43, 293-324. pure cultures of cyanobacteria. J. Gen. Microbiol. 111, 1-61. Houmard, J., Capuano, V., Colombano, M.V., Coursin, T., and Shen, G., Eaton-Rye, J.J., and Vermaas, W.F.J. (1993). Mutation of Tandeau de Marsac, N. (1990). Molecular characterization of the histidine residues in CP47 leads to destabilization of the photosystem terminal energy acceptor of cyanobacterial phycobilisomes. Proc. II complex and to impairment of energy transfer. Biochemistry 32, Natl. Acad. Sci. USA 87, 2152-2156. 5109-5115. Kim, S., Sandusky, P., Bowlby, N.R., Aebersold, R., Green, B.R., Shestakov, S.V., and Reaston, J. (1987). Gene-transferand hostvector Vlahakis, S., Yocum, C.F., and Pichersky, E. (1992). Character- systems of cyanobacteria. Oxford Surv. Plant MOI. Cell Biol. 4, ization of a spinach psbS cDNA encoding the 22 kDa protein of 137-166. photosystem II. FEBS Lett. 314, 67-71. Smart, L.B., and Mclntosh, L. (1993). Genetic inactivation of thepsaB Kirilovsky, D.L., Boussac, A.G.P., van Mieghem, F.J.E., Ducruet, gene in the cyanobacterium Synechocystis sp. PCC 6803 disrupts J.R.C., Sbtif, P.R., Yu, J., Vermaas, W.F.J., andRutherford, A.W. assembly of photosystem I. Plant MOI. Biol. 21, 177-180. (1992). Oxygen-evolving photosystem II preparation from wild type and photosystem II mutants of Synechocystis sp. PCC 6803. Bio- Smart, L.B., Anderson, S.L., and Mclntosh, L. (1991). Targeted chemistry 31, 2099-2107. genetic inactivation of the photosystem I reaction center in the Laudenbach, D.E., and Straus, N.A. (1988). Characterization of a cyanobacterium Synechocystis sp. PCC 6803. EM60 J. 10, cyanobacterialiron stress-induced gene similar topsbc. J. Bacteriol. 3289-3296. 170, 5018-5026. Tamura, N., and Cheniae, G.M. (1988). Photoactivation of the water Mannan, R.M., Whitmarsh, J., Nyman, P., and Pakrasi, H.B. (1991). oxidizing complex: The mechanism and general consequences to Directed mutagenesis of an iron-sulfur protein of the photosystem photosystem 2. In Light-Energy Transduction in Photosynthesis: I complex in the filamentous cyanobacterium Anabaena variabilis Higher Plant and Bacterial Models, S.E. Stevens, Jr. and D.A. Bryant, ATCC 29413. Proc. Natl. Acad. Sci. USA 88, 10168-10172. eds (Rockville, MD: American Society of Plant Physiologists), pp. Montoya, G., Yruela, I., and Picorel, R. (1991). Pigment stoichiome- 227-242. try of a newly isolated Dl-D2-Cyt b559 complex from the higher plant Toelge, M., Zlegler, K., Maldener, I., and Lockau, W. (1991). Directed Beta vulgaris L. FEBS Lett. 283, 255-258. mutagenesis of the gene psaB of photosystem I of the cyanobac- Mullet, J.E. (1988). Chloroplast development and gene expression. terium Anebaena variabiis ATCC 29413. Biochim. Biophys. Acta 1060, Annu. Rev. Plant Moi. Biol. 39, 475-502. 233-236. Mullineaux, C.W., Bittersmann, E., Allen, J.F., and Holzwarth, A.R. Vermaas, W.F.J., and Ikeuchi, M. (1991). Photosystem II. In The Pho- (1990). Picosecond time-resolved fluorescence emission spectra in- tosynthetic Apparatus: Molecular Biology and Operation, Vol. 78, dicate decreased energy transfer from the phycobilisome to L. Bogorad and I.K. Vasil, eds (San Diego: Academic Press), pp. photosystem II in light-state 2 in the cyanobacteriumSynechococcus 25-1 11. 6301. Biochim. Biophys. Acta 1015, 231-242. Vermaas, W.F.J., Ikeuchi, M., and Inoue, Y. (1988). Protein composi- Nilsson, F., Andersson, B., and Jansson, C. (1990). Photosystem tion of the photosystem 2 core complex in genetically engineered I1 characteristics of a constructed Synechocystis 6803 mutant lack- mutants of the cyanobacterium Synechocystis sp. PCC 6803. Pho- ing synthesis of the D1 polypeptide. Plant MOI.Biol. 14, 1051-1054. tosynth. Res. 17, 97-113. Nilsson, F., Gounaris, K., Styring, S., and Andersson, B. (1992). lsolation and characterization of oxygen-evolving photosystem II Wedel, N., Klein, R., Ljungberg, U., Andersson, B., and Herrmann, membranes from the cyanobacterium Synechocystis 6803. Biochim. R.G. (1992). The single-copy gene psbS codes for a phylogeneti- Biophys. Acta 1100, 251-258. cally intriguing 22 kDa polypeptide of photosystem II. FEBS Lett. 314, 61-66. Nixon, P.J., Chisholm, D.A., and Diner, B.A. (1992). lsolation and functional analysis of random and site-directed mutants of pho- Williams, J.G.K. (1988). Construction of specific mutations in photosystem II. In Plant Protein Engineering, F! Shewry and S. Gutteridge, tosystem II photosynthetic reaction center by genetic engineering eds (Cambridge: Cambridge University Press), pp. 93-141. methods in Synechocystis 6803. Methods Enzymol. 167,766-778.