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A Null Mutant of Synechococcus sp. PCC7942 Deficient in the Sulfolipid Sulfoquinovosyl Diacylglycerol

A Null Mutant of Synechococcus sp. PCC7942 Deficient in the Sulfolipid Sulfoquinovosyl... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 271, No. 13, Issue of March 29, pp. 7501–7507, 1996 © 1996 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. A Null Mutant of Synechococcus sp. PCC7942 Deficient in the Sulfolipid Sulfoquinovosyl Diacylglycerol* (Received for publication, November 17, 1995, and in revised form, January 15, 1996) ¶ i Sinan Gu¨ ler‡, Angela Seeliger§, Heiko Ha¨rtel , Gernot Renger§, and Christoph Benning‡ From the ‡Institut fu¨r Genbiologische Forschung Berlin GmbH, Ihnestrasse 63, 14195 Berlin, the §Max-Volmer-Institut fu¨r Physikalische und Biophysikalische Chemie der Technischen Universita¨t, Strasse des 17 Juni 135, 10623 Berlin, and the ¶Institut fu¨r Biologie/Pflanzenphysiologie, Humboldt-Universita¨t zu Berlin, Philippstrasse 13, 10115 Berlin, Federal Republic of Germany The sulfolipid 6-sulfo-a-D-quinovosyldiacylglycerol is potential and evolve no oxygen. Because of the similar organi- associated with the thylakoid membranes of many pho- zation of the photosynthetic apparatus in cyanobacteria and tosynthetic organisms. Previously, genes involved in higher plants, cyanobacteria have been used as model systems sulfolipid biosynthesis have been characterized only in to genetically dissect the protein complexes of the thylakoid the purple bacterium Rhodobacter sphaeroides. Unlike membrane (for reviews see Refs. 6 and 7). Because some cya- plants and cyanobacteria, photosynthesis in this bacte- nobacterial strains can grow heterotrophically, genes encoding rium is anoxygenic due to the lack of a water splitting individual protein components of the two photosystems can be photosystem II. To test the function of sulfolipid in an inactivated by gene replacement, and the analysis of the re- organism with oxygenic photosynthesis, we isolated and sulting null mutants can reveal the function of the affected inactivated a sulfolipid gene of the cyanobacterium Syn- proteins. This genetic approach can also be applied to investi- echococcus sp. PCC7942. Extensive analysis of the sulfo- gate the role of polar lipids for the formation and maintenance lipid-deficient null mutant revealed subtle changes in of protein lipid complexes required for oxygenic photosynthesis. photosynthesis related biochemistry of O . In addition, a However, no cyanobacterial mutants have been available that slight increase in the variable room temperature chlo- completely lack a class of polar lipids of the thylakoid mem- rophyll fluorescence yield was observed. Regardless of brane. But it should be noted that heterocyst mutants deficient these changes, it seems unlikely that sulfolipid is an in the biosynthesis of glycolipids specifically associated with essential constituent of a functional competent water this specialized, nitrogen-fixing cell type are known (8). Fur- oxidase or the core antenna complex of photosystem II. thermore, the fatty acid composition of cyanobacterial polar However, reduced growth of the mutant under phos- lipids has been altered by genetic engineering in order to study phate-limiting conditions supports the hypothesis that sulfolipid acts as a surrogate for anionic phospholipids the influence of fatty acid composition on thermal tolerance of under phosphate-limiting growth conditions. the organism (9). The almost exclusive occurrence of sulfolipid in photosyn- thetic membranes and its unusual sulfoquinovosyl head group Photosynthesis is a function of highly organized pigment (10) has stimulated debate over a specific role for this lipid in protein complexes that are embedded in the polar lipid matrix photosynthesis (11). Sulfolipid has been identified as integral of thylakoid membranes. Some of the lipids found in this mem- component of photosystem II protein complexes (12, 13). Fur- brane are generally absent from nonphotosynthetic mem- thermore, in reconstitution experiments with chloroplast ATP branes. A typical example is the sulfolipid sulfoquinovosyl synthase, sulfolipid was found to be required in stoichiometric diacylglycerol, which occurs in almost all photosynthetic organ- amounts with other lipids for successful restoration of enzy- isms (1) with the exception of a few photosynthetic bacteria (2) matic activity (14). These results led to the conclusion that and Rhizobium meliloti 2011, a nonphotosynthetic but plant- sulfolipid functions as essential boundary lipid. Based on a associated bacterium (3) with sulfolipid in its membranes. Al- more recent analysis of a sulfolipid-null mutant of the purple though in higher plants and cyanobacteria two photoactive bacterium Rhodobacter sphaeroides, it can be assumed that pigment-containing complexes exist, photosystems I and II, sulfolipid plays no specific role in anoxygenic photosynthesis only one is present in purple bacteria. The photosynthetic (15), because photosynthetic electron transport rates were not reaction center of purple bacteria shows high structural and altered and growth under optimal conditions was not reduced. functional homology to that of photosystem II of cyanobacteria However, upon transfer to phosphate-limiting conditions, and plants (for a review see Ref. 4), but only photosystem II growth of the mutant ceased earlier than that of wild type cells. catalyzes the light-induced reduction of plastoquinone with In addition, a strong reduction in phospholipid content and a electrons from water, thereby releasing oxygen (for a review concomitant increase in novel lipids as well as sulfolipid was see Ref. 5). The water splitting system is absent from purple observed in the phosphate-stressed cells of R. sphaeroides (16). bacteria, which use hydrogen donors with relatively low redox Taken together, these observations led to the conclusion that sulfolipid may play a role as substitute for anionic phospholip- ids under phosphate-limiting growth conditions in purple bac- * This work was supported in part by Grant BMBF 0316301 A, teria and possibly other photosynthetic organisms. Project 2, from the Genzentrum Berlin. The costs of publication of this Recently, a sulfolipid-deficient mutant of Chlamydomonas article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance reinhardtii, which was induced by exposure to UV light, has with 18 U.S.C. Section 1734 solely to indicate this fact. been isolated based on its abnormal chlorophyll fluorescence The nucleotide sequence(s) reported in this paper has been submitted (17). The chlorophyll fluorescence phenotype is an indication TM to the GenBank /EMBL Data Bank with accession number(s) U45308. that photosynthesis is affected in this mutant. However, it has To whom reprint requests should be addressed. Fax: 49-30-830007- 36; E-mail: [email protected]. not clearly been demonstrated that the fluorescence phenotype This is an Open Access article under the CC BY license. 7502 Sulfolipid-deficient Null Mutant of Synechococcus sp. PCC7942 sphaeroides (18). Lipids were separated by one-dimensional thin layer and the sulfolipid deficiency are due to the same genetic defect, chromatography on ammonium sulfate-impregnated plates as de- leaving the causal relation between the two phenotypes open. scribed (18), except that benzene was replaced by toluene. Lipids were To further address the question of whether sulfolipid plays visualized by exposing the plates to iodine vapor. Fatty acid methyl an essential role for oxygenic photosynthesis, we isolated and esters were prepared from each lipid and quantified by gas chromatog- inactivated a gene involved in sulfolipid biosynthesis from the raphy (18). From these data the mol % fraction was calculated for each cyanobacterium Synechococcus sp. PCC7942. The only previ- lipid. Preparation of Radiolabeled Lipids—Cultures (50 ml) of sulfolipid ously known genes encoding sulfolipid biosynthetic enzymes mutant and wild type strains were grown to midlog phase in BG-11 were the sqd genes from R. sphaeroides (18, 19), of which one medium. Cells were washed in sulfate-free BG-11 medium (MgSO was served as a molecular probe to isolate the homologous gene replaced by MgCl ) and resuspended in 1 ml of the same medium. from the cyanobacterium. The resulting sulfolipid null mutant 35 Following the addition of 100 mCi of sodium [ S]sulfate (specific activ- was analyzed with regard to its photosynthetic characteristics ity, 100 mCi/mmol), the cells were incubated for 16 h and harvested by centrifugation. Lipid extracts were prepared and separated as de- and growth under different conditions. Possible modifications scribed above. of the photosystem II reaction kinetics due to the lack of sul- Oxygen Evolution Measurements—Oxygen evolution from whole cells foquinovosyl diacylglycerol were investigated by measuring the under continuous white light was determined polarographically with 10 oxygen yield in response to a regime of short flashes. mM NaHCO as acceptor in a buffer solution of 25 mM Hepes-NaOH, pH 7.0 (26). The concentration of cells was equivalent to 5 mgml chloro- MATERIALS AND METHODS phyll. The oscillation pattern of flash-induced oxygen yield produced by Bacterial Strains, Plasmids, Media, and Growth Conditions—Syn- a series of short flashes from a Xenon lamp (FWHM ' 10 ms) was echococcus sp. PCC7942 wild type (kindly provided by P. Wolk) and monitored with a Joliot-type electrode as described (27). mutant lines were grown photoautotrophically at 28 °C in liquid or Absorption, 77 K, and Room Temperature Fluorescence Emission agar-solidified (1.5%) BG-11 medium (20). Light was provided by fluo- Measurements—In vivo room temperature absorption and 77 K fluores- rescent bulbs at an photon flux density of approximately 50 mmol m cence emission spectra were recorded as described previously (26). s . If required, the growth medium was supplemented with kanamycin Room temperature chlorophyll a fluorescence was determined with a 21 21 (15 mgml ) or ampicillin (1 mgml ). The phosphate content of the pulse amplitude modulation fluorometer (PAM101, Walz, Effeltrich, medium was reduced as indicated in the text by replacing KH PO with Germany) according to the method of Clarke et al. (28) essentially as 2 4 KCl. Growth of the cultures was monitored by determination of the described (26). Phycocyanin and chlorophyll content were determined optical density at 730 nm or by counting colony forming units after in whole cells as described by Collier and Grossman (29). plating. Nucleotide Sequence—The nucleotide sequence reported in this pa- TM For routine cloning experiments, the Escherichia coli strains XL-1 per has been submitted to the GenBank /EMBL Data Bank with Blue, XL-1 Blue MRF9, and XLOLR, as well as the plasmids pBluescript accession number U45308. II-SK and pBK-CMV and the phage l-ZAP-Express and ExAssist RESULTS helper phage were used (Stratagene). The inactivation cassette carry- ing a neomycin phosphotransferase gene was derived from pUC4K (21, Isolation and Characterization of a Cyanobacterial Gene with 22). The origin of plasmids pSY2, pSY3, and pSYB (see Fig. 1) is Sequence Similarity to sqdB of R. sphaeroides—A prerequisite described in the results section. Cultures of E. coli were grown in Luria for the construction of a completely sulfolipid-deficient cya- broth. Kanamycin was usually added as required at 50 mgml , and nobacterial strain by gene replacement is the availability of ampicillin was added at 100 mgml . wild type genes coding for sulfolipid biosynthetic enzymes. Recombinant DNA Techniques—The genomic Synechococcus sp. With the goal to isolate the first cyanobacterial gene involved PCC7942 DNA library was prepared in l-ZAP-Express according to the manufacturer’s instructions. For this purpose, genomic DNA was iso- in sulfolipid biosynthesis, cross-hybridization between the dif- lated from Synechococcus sp. PCC7942 wild type (23), partially digested ferent sqd genes from R. sphaeroides and genomic DNA from with Sau 3A, and 2–4-kilobase pair fragments were ligated with pre- Synechococcus sp. PCC7942 was tested. This cyanobacterium pared phage arms. The library was screened by heterologous DNA was chosen, because it is naturally competent to take up DNA. hybridization (24) using Hybond N filters (Amersham Corp.). Hybrid- In addition, the DNA of Synechococcus sp. PCC7942 has a GC ization was performed at 42 °C overnight in a solution containing 5 3 content intermediate to that of DNA from R. sphaeroides and SSPE, 5 3 Denhardt’s solution, 0.5% (w/v) SDS, 20% (v/v) formamid, and 20 mgml sonicated herring sperm DNA. The filters were washed higher plants (30). Of three sqd genes of R. sphaeroides, only twice at 42 °C for 20 min each in 2 3 SSC, 0.5% (w/v) SDS prior to sqdB gave a strong positive hybridization signal with genomic exposure. The stock solutions were prepared following standard proce- DNA of Synechococcus sp. PCC7942. Probing a genomic library dures (25). Routine recombinant DNA techniques and DNA sequencing prepared in l-ZAP-Express, several clones hybridizing to sqdB were done as described (18, 19). of R. sphaeroides were isolated. Following the excision of plas- Insertional Inactivation of sqdB and Transformation of Synechococ- mids from the phage and restriction analysis, it became appar- cus sp. PCC7942—For inactivation of the sqdB open reading frame, the pBluescript II-SK based plasmid pSYB (see Fig. 1) was partially ent that the inserts of all clones overlapped. The two clones digested with SalI/PstI to remove a 952-base pair fragment internal to containing the longest DNA fragments, pSY2 and pSY3, were the sqdB open reading frame. The deleted fragment was replaced by a recombined by ligation of a 1600-base pair KpnI/SalI fragment 1230-base pair SalI/PstI fragment carrying the neomycin phospho- from pSY3, a 1500 SalI/BamHI fragment from pSY2, and transferase gene of pUC4K. The resulting plasmid pSYBK (see Fig. 1) pBluescript II-SK cut with KpnI and BamHI giving rise to was used to transform wild type Synechococcus sp. PCC7942. For this plasmid pSYB (Fig. 1). The overlapping DNA region of pSY2 purpose, cells (20 ml cultures) were grown to midlog phase in BG-11 medium, harvested by centrifugation for 5 min at 5000 3 g, washed and pSY3 was sequenced on both strands, as indicated in Fig. once, and resuspended in 1 ml of the same medium. Approximately 4 mg 1. Sequence analysis revealed an open reading frame predicted of plasmid DNA were added, and the suspension was incubated under to encode a protein of the molecular mass of 44.6 kDa consist- shaking in the light overnight at 28 °C. Following the addition of 10 ml ing of 402 amino acids (Fig. 2). A GTG triplet is proposed to of BG-11 medium, incubation was continued for 24 h prior to plating the 21 serve as the initiation codon because it is preceded by a perfect cells on selective medium containing 15 mgml kanamycin. Transfor- ribosome binding site, no suitable in frame ATG codon was mants became visible after 1 week and were restreaked at least 4 times. They were tested for ampicillin sensitivity in order to detect double present, and the predicted N-terminal amino acids sequence recombinants. Putative candidates were further analyzed by Southern corresponded well with the N-terminal amino acid sequence hybridization to ensure complete segregation of wild type genome of sqdB of R. sphaeroides. Comparing the amino acid se- copies. quence over its total length with that of sqdB of R. spha- Lipid Analysis—Cultures (50 ml) of sulfolipid mutant or wild type eroides, a sequence identity of 63% and a sequence similarity strains were grown to late log-phase in BG-11 medium containing 0.18 of 77.3%, taking into account conservative substitutions, was or 0.018 mM KH PO as indicated in the text. Cells were harvested by 2 4 centrifugation, and lipid extracts were prepared as described for R. determined. Sulfolipid-deficient Null Mutant of Synechococcus sp. PCC7942 7503 FIG.1. Plasmids used for the characterization and inactiva- tion of the sqdB gene from Synechococcus sp. PCC7942. Plasmids were constructed as described in the text. Small solid arrows, directions of sequence reactions; gray arrow, sqdB open reading frame; open arrow, neomycin phosphotransferase gene; cross-hatched box, fragment used for Southern hybridization. Restriction sites: A, BamHI; E, SpeI; H, HindIII; O, XhoI; P, PstI; S, SalI. The asterisks indicate a Sau3A/ BamHI ligation site. Construction of a Sulfolipid-deficient Null Mutant—As part of a strategy to demonstrate that the isolated open reading frame represents a gene coding for a protein involved in sulfo- lipid biosynthesis in Synechococcus sp. PCC7942, we inacti- vated the wild type copy of the gene by replacing an internal fragment with a kanamycin resistance cassette in opposite orientation. The kanamycin-resistant lines were tested by Southern hybridization (Fig. 3). This experiment revealed the complete disappearance of wild type genome copies and the expected replacement of the targeted open reading frame in all putative mutant lines tested. There was only one hybridizing band of approximately 6 kilobases pairs present in wild type samples, a result that is in agreement with the presence of a single copy DNA sequence. Analysis of the composition of lipid extracts prepared from wild type and mutant cells by thin layer chromatography and quantification of individual lipids by gas chromatography of fatty acid methyl esters derived from the lipids (Table I) revealed no detectable amount of sulfolipid in extracts from the mutant lines. Furthermore, using the most sensitive method available, the analysis of lipid extracts from cells labeled with [ S]sulfate, no traces of residual sulfolipid were detected in mutant samples (Fig. 4). Apparently, the inactivation of the open reading frame abolished sulfolipid biosynthesis in the affected cells and gave rise to a sulfolipid- deficient null mutant of Synechococcus sp. PCC7942. The Effects of Phosphate-limiting Conditions on Growth and Lipid Composition of Mutant and Wild Type—The isolation of genetically pure sulfolipid-deficient mutants of Synechococcus sp. PCC7942 suggests that sulfolipid is not essential for pho- toautotrophic growth of this cyanobacterium. Accordingly, no difference in growth rates was observed under optimal growth conditions (Fig. 5A). However, 10-fold reduction of the phos- phate concentration in the medium caused the mutant to cease growth after 6 days, whereas the wild type continued to grow FIG.2. DNA and deduced amino acid sequence of the Synecho- (Fig. 5B). Comparable results were obtained using optical den- coccus sp. PCC7942 sqdB gene. The nucleotide sequence is shown sity (Fig. 5) or live cell counts (data not shown) for measuring from the KpnI site to the XhoI site of pSYB. The protein sequence is growth of the cultures. given below the DNA sequence. The underlining indicates a putative Under phosphate-limiting growth conditions the relative ribosome binding site. amount of the major phospholipid phosphatidyl glycerol was reduced in wild type cells to 7.2 mol % compared with cells digalactosyl diacylglycerol was observed for the wild type, grown under optimal conditions (16.6 mol %, Table I). Concom- whereas the relative amount of monogalactosyl diacylglycerol itantly, an increase in the relative amount of sulfolipid and was slightly decreased. In the mutant, the relative amount of 7504 Sulfolipid-deficient Null Mutant of Synechococcus sp. PCC7942 FIG.3. Southern hybridization of wild type (WT) and SY-SQDB mutant. Genomic DNA was cut with HindIII and probed with a 1470- base pair SpeI/XhoI fragment from sqdB containing the open reading FIG.5. Growth of Synechococcus sp. PCC7942 wild type (closed frame and adjacent sequences. The approximate length of DNA frag- circles) and SY-SQDB mutant (open circles) under optimal (A) ments is indicated (kilobase pairs). and under phosphate limiting conditions (B). Each value repre- sents the mean of three measurements using independent cultures. TABLE I Error bars were smaller than symbols. Effect of phosphate nutrition on the polar lipid composition of wild type and mutant mined as function of photon flux density. Essentially, identical The values are the means 6 standard errors of three independent cell curves were obtained for the wild type and the mutant (Fig. 6). cultures grown in BG-11 medium supplemented with P as indicated. MGD, monogalactosyl diacylglycerol; PG, phosphatidylglycerol; DGD, To examine the possibility of subtle changes in the reaction digalactosyl diacylglycerol; SQD, sulfoquinovosyl diacylglycerol; nd, not kinetics of photosystem II, the characteristic period four oscil- detected (,0.5 mol %). lation pattern of flash-induced oxygen evolution was compared mol % in dark adapted wild type and mutant cells. The maximum Lipid Wild type SY-SQDB oxygen yield is generated by the fourth flash (Fig. 7). This feature is typical for thoroughly dark-adapted cyanobacteria 0.18 mM P 0.018 mM P 0.18 mM P 0.018 mM P i i i i (31). With regard to the active site tyrosine (Y ) of the D2 MGD 60.6 6 1.1 42.5 6 0.9 56.2 6 0.3 43.7 6 1.9 protein, this pattern is indicative of an apparent population of PG 16.6 6 0.3 7.2 6 0.5 28.4 6 0.2 23.1 6 0.1 OX DGD 12.5 6 0.8 28.0 6 0.6 15.4 6 0.4 33.2 6 1.8 redox states below S Y (see Ref. 32 and references therein). 1 D SQD 10.3 6 0.2 22.3 6 1.0 nd nd Within the frame work of an extended Kok model, in which a cyclic sequence of redox states adopted by the water oxidase during catalysis is postulated (33), the data can be satisfacto- rily described by the probability of misses (a 5 0.23) and double hits (b 5 0.01) and apparent S -state dark populations of [S ] 5 i 1 0.47, [S ] 5 0.39, and [S ] 5 0.12. Preillumination with a 0 21 short saturating flash and subsequent dark incubation for 3 min leads to a shift of oxygen yield maximum to the third flash. This observation shows that the apparent high population of S is mainly due to the presence of Y in its reduced form (32). Both oscillatory patterns exhibited virtually the same features (Fig. 7, A and B) except for the pronounced oxygen uptake in the mutant sample after the first two flashes of the sequence (Fig. 7B). In an attempt to test whether this phenomenon was restricted to the first two flashes, the measurements were repeated in the presence of hydrazine. Under these conditions the redox state S is highly populated, and the maximum of oxygen yield is shifted toward the sixth flash (32). Likewise, virtually no oxygen is evolved during the first four flashes. Contrary to the wild type, the oxygen yield pattern of hy- drazine-treated mutant cells revealed a marked oxygen uptake during the first four flashes in the SY-SQDB mutant (data not FIG.4. Separation of S-labeled lipids of wild type (WT) and shown). SY-SQDB mutant by thin layer chromatography. Approximately Comparing room temperature chlorophyll fluorescence in the equal amounts of total lipids were spotted in case of undiluted extracts (undil.). In addition, 10-, 100-, and 1000-fold dilutions of the wild type wild type and the mutant (Table II), a similar dark level fluo- extracts were loaded for estimation of the reduction of sulfolipid in the rescence yield (F ) was observed for both strains. Because state mutant extract. Radiolabeled lipids were visualized by autoradiogra- 2-state 1 transitions can be important for the determination of phy. F, solvent front; O, origin; SQD, sulfoquinovosyl diacylglycerol; U, the maximum fluorescence yield (F ; Ref. 28), cells were first unidentified compound. m illuminated with low intensity white light to induce state 1 phosphatidylglycerol (28.4 mol %) was increased under optimal prior to the addition of the electron transfer inhibitor 3,4- growth conditions and did not decrease as dramatically under (dichlorophenyl)-1,1-dimethylurea to close photosystem II re- phosphate-limiting conditions (23.2 mol %, Table I). The rela- action centers. Under these conditions, the mutant showed a tive amounts of the galactolipids were comparable with those higher F value and hence a higher variable fluorescence yield found in wild type cells under both growth regimes. (F ). Consequently, the ratio of F /F , which is a measure of the v v m Photosynthetic Characteristics of the Sulfolipid-deficient Mu- photochemical efficiency of photosystem II, was slightly in- tant—To elucidate the effect of sulfolipid-deficiency on oxygenic creased in the mutant (Table II). Based on statistical analysis, photosynthesis, first the rate of oxygen evolution was deter- this increase was significant. In search for further alterations Sulfolipid-deficient Null Mutant of Synechococcus sp. PCC7942 7505 attributed to the core antenna proteins CP43 (34) and CP47 (35), respectively. No difference in the relative amplitudes of the emission maxima were observed between both strains. When excited at a wavelength of 590 nm, which corresponds to the maximum for the excitation of phycobilins, the intensity of the emission at approximately 655 nm was considerably re- duced in the mutant (Fig. 8B). This peak presumably repre- sents overlapping emissions for phycocyanin and allophycocya- nin with maxima at 645 and 665 nm, respectively. Because no difference in the phycocyanin/chlorophyll ratio was observed (data not shown), this result can be taken as an indication for a higher efficiency of excitation energy transfer from phycobi- lins to the photosystem II reaction center chlorophyll a in the mutant. DISCUSSION FIG.6. Rate of oxygen evolution as function of photon flux To study the possible role of sulfolipid in oxygenic photosyn- density by Synechococcus sp. PCC7942 wild type (closed circles) thesis in a definitive way, we created a sulfolipid-deficient null and SY-SQDB mutant (open circles). Each value represents the mutant of Synechococcus sp. PCC7942. During the course of mean of three independent measurements. The standard error was less this work, we isolated for the first time and disrupted a gene than 6% of each value. involved in sulfolipid biosynthesis in an organism with oxy- genic photosynthesis. This gene of Synechococcus sp. PCC7942 shares considerable sequence identity with the sqdB gene of R. sphaeroides and is therefore also designated sqdB. However, further experiments will be required to demonstrate functional homology between the two genes in R. sphaeroides and Syn- echococcus sp. PCC7942. Our current inability to detect cross- hybridization between other sqd genes of the two bacterial strains suggests that these are less conserved. Unfortunately, we still do not know the function of the sqdB gene product, and further experiments to elucidate its biochemical role may also allow us to solve the long standing mystery of sulfolipid biosynthesis. Inactivation of the putative sqdB gene of Synechococcus sp. PCC7942 wild type gives rise to an otherwise isogenic null mutant, which was designated SY-SQDB and completely lacks sulfolipid, one of the four polar lipids found in this bacterium. This deficiency has no lethal consequences. It does not even lead to reduced growth under optimal conditions for photoau- totrophic growth (Fig. 5), suggesting that sulfolipid is not es- sential for oxygenic photosynthesis. Apparently, the loss of the anionic sulfolipid is mainly compensated by an increased rela- tive amount of phosphatidylglycerol (Table I), which is the second anionic lipid found in the membranes of Synechococcus FIG.7. Flash-induced changes of oxygen evolution or uptake sp. PCC7942. Maintaining a certain level of anionic lipids in by Synechococcus sp. PCC7942 wild type (A) and SY-SQDB mu- tant (B). The polarographic signals (arbitrary units) were detected by the membranes seems to be crucial for the organism, because a Joliot-type electrode. Positive peaks indicate oxygen evolution, and the reduction in phosphatidylglycerol under phosphate limita- negative peaks indicate uptake. tion in the wild type is compensated by an increased level of sulfolipid. The sulfolipid-deficient mutant SY-SQDB cannot TABLE II respond in the same way to phosphate limitation and has to Room temperature fluorescence parameters (relative units) maintain a higher level of phosphatidylglycerol. Because it All values represent the means of 20 measurements. Samples were adjusted to a chlorophyll concentration of 20 mgml . F , dark level cannot replace lipid-bound phosphor by sulfur under conditions fluorescence yield; F , maximum fluorescence yield; F , variable fluo- m v of phosphate limitation, it becomes phosphate-depleted and rescence yield (F 5 F 2 F ). v m o enters the stationary growth phase at an earlier time point Parameters Wild type SY-SQDB than the wild type (Fig. 5). The same phenomenon has been previously observed for R. sphaeroides (15). In addition, both F 64.5 6 7.0 65.3 6 6.6 F 131.5 6 13.8 153.9 6 13.9 bacteria accumulate dihexosyl lipids under phosphate limita- F 67.0 6 7.0 88.6 6 9.4 v tion. Although Synechococcus sp. PCC7942 does not accumu- F /F 0.51 6 0.04 0.58 6 0.02 v m late glucosylgalactosyl diacylglycerol, as was observed for phos- phate-limited R. sphaeroides (16), the relative amount of in the antenna system of the mutant, low temperature fluores- digalactosyl diacylglycerol is increased (Table I). cence spectra were recorded. The 77 K fluorescence spectra of Normal growth of the SY-SQDB mutant under optimal lab- wild type and mutant strains obtained after chlorophyll a ex- oratory conditions does not exclude the possibility of a more citation at 440 nm are shown in Fig. 8A. The large emission subtle role of sulfolipid in oxygenic photosynthesis relevant peak at 717 nm is predominantly derived from photosystem I, under natural conditions, e.g. high photon flux densities. How- whereas the two peaks at 685 nm and approximately 695 nm ever, the light response curves for oxygen evolution by wild emanate from photosystem II. The latter two can be mainly type and mutant cells were nearly identical (Fig. 6). 7506 Sulfolipid-deficient Null Mutant of Synechococcus sp. PCC7942 FIG.8. 77 K fluorescence emission spectra of Synechococcus sp. PCC7942 wild type (solid lines) or SY-SQDB mutant (broken lines) after excita- tion at 440 nm (A) or 590 nm (B). Spec- tra in A were normalized to the emission maximum at 717 nm, and spectra in B were normalized to 683 nm. The spectra in A were set off to facilitate comparison. In each case, chlorophyll concentrations were adjusted to 2.5 mgml . This finding indicates that the lack of sulfolipid neither increased in the mutant (Fig. 6). The enhanced light-induced affects the overall electron transport rate nor the optical cross- oxygen uptake in the mutant observed during polarographic section of oxygen evolution. More subtle changes in the reaction measurements with the Joliot-type electrode (Fig. 7) may be a kinetics of photosystem II were expected to become apparent by reasonable explanation for this apparent discrepancy. monitoring the characteristic period four oscillation pattern of The subtle alterations in photosynthesis observed for the flash-induced oxygen evolution in dark-adapted wild type and SY-SQDB mutant would not have been sufficient to isolate this mutant cells. A comparison of oscillatory patterns revealed that mutant from a randomly mutagenized population. On the con- both strains exhibit virtually the same features except for the trary, a leaky sulfolipid-deficient mutant of C. reinhardtii has pronounced oxygen uptake in the mutant sample after the first been isolated based on its high fluorescence phenotype follow- two flashes (Fig. 7B). Because hydrazine-treated mutant cells ing random mutagenesis (17). However, a detailed analysis of showed also a marked increase in oxygen uptake during the the photosynthetic characteristics of this mutant is not avail- able for comparison. Furthermore, it has not clearly been dem- first four flashes, it seems most likely that the enhancement of oxygen uptake in the SY-SQDB mutant is not necessarily di- onstrated that the fluorescence phenotype and the lipid pheno- rectly related to the water splitting activity of photosystem II. type are indeed caused by the same genetic defect. Therefore Instead, increased oxygen uptake could be either due to the further experiments will be required to test whether sulfolipid reduction of O by components of the electron transport chain may play a different role in chloroplasts as compared with or increased respiratory activity. Nevertheless, the data pre- cyanobacterial cells. sented in this study clearly show that sulfolipid is not an In summary, the extensive examination of a sulfolipid-defi- essential constituent of a functionally competent water cient null mutant of Synechococcus sp. PCC7942, suggests that oxidase. sulfolipid does not play a specific role for oxygenic photosyn- Low temperature fluorescence measurements suggest that thesis. A similar conclusion was drawn for nonoxygenic photo- the lack of sulfolipid in the null mutant most likely has no synthesis of R. sphaeroides (15). However, subtle changes in effect on the structural organization of the reaction center/core the biochemistry of O and an increased variable room temper- antenna complex of photosystem II. The similarity of the emis- ature chlorophyll fluorescence yield were observed for the cya- sion spectra following chlorophyll a excitation at 440 nm (Fig. nobacterial mutant. As was concluded for R. sphaeroides, the 8A) indicates that neither the binding environment of the chlo- biosynthesis of sulfolipid may have evolved and been main- rophyll a emitting from the core antenna proteins CP43 and tained during evolution, primarily not to provide an essential CP47, nor the excitation energy transfer to the reaction center component for photosynthetic processes but to provide a surro- is affected in the mutant. Moreover, based on the 77 K fluores- gate anionic lipid for conditions of phosphate limitation. Fur- cence emission spectra following the excitation at 590 nm (Fig. ther experiments with higher plants and algae will be required 8B), it appears that excitation energy transfer from phycobilins to answer the question of whether this concept is ubiquitous. to chlorophyll a of photosystem II reaction centers is increased. REFERENCES This finding can be explained in terms of structural modifica- 1. Harwood, J. L. (1980) in The Biochemistry of Plants (Stumpf P. K., ed) Vol. 4, tions within the phycobilisome complex or an altered coupling pp. 301–320, Academic Press, New York between phycobilisomes and thylakoids. An increase in energy 2. Imhoff, J. F., Kushner, D. J., Kushwaha, S. C., and Kates, M. (1982) J. Bacteriol. 150, 1192–1201 transfer from phycobilins to chlorophyll a may explain the 3. Cedergren, R. A., and Hollingsworth, R. I. (1994) J. Lipid Res. 35, 1452–1461 increased variable chlorophyll fluorescence yield in the mutant 4. Golbeck, J. H. (1993) Proc. Natl. Acad. Sci. U. 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(1994) FEBS Lett. 337, 103–108 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

A Null Mutant of Synechococcus sp. PCC7942 Deficient in the Sulfolipid Sulfoquinovosyl Diacylglycerol

Güler, Sinan; Seeliger, Angela; Härtel, Heiko; Renger, Gernot; Benning, Christoph
Journal of Biological Chemistry – Mar 1, 1996
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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 271, No. 13, Issue of March 29, pp. 7501–7507, 1996 © 1996 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. A Null Mutant of Synechococcus sp. PCC7942 Deficient in the Sulfolipid Sulfoquinovosyl Diacylglycerol* (Received for publication, November 17, 1995, and in revised form, January 15, 1996) ¶ i Sinan Gu¨ ler‡, Angela Seeliger§, Heiko Ha¨rtel , Gernot Renger§, and Christoph Benning‡ From the ‡Institut fu¨r Genbiologische Forschung Berlin GmbH, Ihnestrasse 63, 14195 Berlin, the §Max-Volmer-Institut fu¨r Physikalische und Biophysikalische Chemie der Technischen Universita¨t, Strasse des 17 Juni 135, 10623 Berlin, and the ¶Institut fu¨r Biologie/Pflanzenphysiologie, Humboldt-Universita¨t zu Berlin, Philippstrasse 13, 10115 Berlin, Federal Republic of Germany The sulfolipid 6-sulfo-a-D-quinovosyldiacylglycerol is potential and evolve no oxygen. Because of the similar organi- associated with the thylakoid membranes of many pho- zation of the photosynthetic apparatus in cyanobacteria and tosynthetic organisms. Previously, genes involved in higher plants, cyanobacteria have been used as model systems sulfolipid biosynthesis have been characterized only in to genetically dissect the protein complexes of the thylakoid the purple bacterium Rhodobacter sphaeroides. Unlike membrane (for reviews see Refs. 6 and 7). Because some cya- plants and cyanobacteria, photosynthesis in this bacte- nobacterial strains can grow heterotrophically, genes encoding rium is anoxygenic due to the lack of a water splitting individual protein components of the two photosystems can be photosystem II. To test the function of sulfolipid in an inactivated by gene replacement, and the analysis of the re- organism with oxygenic photosynthesis, we isolated and sulting null mutants can reveal the function of the affected inactivated a sulfolipid gene of the cyanobacterium Syn- proteins. This genetic approach can also be applied to investi- echococcus sp. PCC7942. Extensive analysis of the sulfo- gate the role of polar lipids for the formation and maintenance lipid-deficient null mutant revealed subtle changes in of protein lipid complexes required for oxygenic photosynthesis. photosynthesis related biochemistry of O . In addition, a However, no cyanobacterial mutants have been available that slight increase in the variable room temperature chlo- completely lack a class of polar lipids of the thylakoid mem- rophyll fluorescence yield was observed. Regardless of brane. But it should be noted that heterocyst mutants deficient these changes, it seems unlikely that sulfolipid is an in the biosynthesis of glycolipids specifically associated with essential constituent of a functional competent water this specialized, nitrogen-fixing cell type are known (8). Fur- oxidase or the core antenna complex of photosystem II. thermore, the fatty acid composition of cyanobacterial polar However, reduced growth of the mutant under phos- lipids has been altered by genetic engineering in order to study phate-limiting conditions supports the hypothesis that sulfolipid acts as a surrogate for anionic phospholipids the influence of fatty acid composition on thermal tolerance of under phosphate-limiting growth conditions. the organism (9). The almost exclusive occurrence of sulfolipid in photosyn- thetic membranes and its unusual sulfoquinovosyl head group Photosynthesis is a function of highly organized pigment (10) has stimulated debate over a specific role for this lipid in protein complexes that are embedded in the polar lipid matrix photosynthesis (11). Sulfolipid has been identified as integral of thylakoid membranes. Some of the lipids found in this mem- component of photosystem II protein complexes (12, 13). Fur- brane are generally absent from nonphotosynthetic mem- thermore, in reconstitution experiments with chloroplast ATP branes. A typical example is the sulfolipid sulfoquinovosyl synthase, sulfolipid was found to be required in stoichiometric diacylglycerol, which occurs in almost all photosynthetic organ- amounts with other lipids for successful restoration of enzy- isms (1) with the exception of a few photosynthetic bacteria (2) matic activity (14). These results led to the conclusion that and Rhizobium meliloti 2011, a nonphotosynthetic but plant- sulfolipid functions as essential boundary lipid. Based on a associated bacterium (3) with sulfolipid in its membranes. Al- more recent analysis of a sulfolipid-null mutant of the purple though in higher plants and cyanobacteria two photoactive bacterium Rhodobacter sphaeroides, it can be assumed that pigment-containing complexes exist, photosystems I and II, sulfolipid plays no specific role in anoxygenic photosynthesis only one is present in purple bacteria. The photosynthetic (15), because photosynthetic electron transport rates were not reaction center of purple bacteria shows high structural and altered and growth under optimal conditions was not reduced. functional homology to that of photosystem II of cyanobacteria However, upon transfer to phosphate-limiting conditions, and plants (for a review see Ref. 4), but only photosystem II growth of the mutant ceased earlier than that of wild type cells. catalyzes the light-induced reduction of plastoquinone with In addition, a strong reduction in phospholipid content and a electrons from water, thereby releasing oxygen (for a review concomitant increase in novel lipids as well as sulfolipid was see Ref. 5). The water splitting system is absent from purple observed in the phosphate-stressed cells of R. sphaeroides (16). bacteria, which use hydrogen donors with relatively low redox Taken together, these observations led to the conclusion that sulfolipid may play a role as substitute for anionic phospholip- ids under phosphate-limiting growth conditions in purple bac- * This work was supported in part by Grant BMBF 0316301 A, teria and possibly other photosynthetic organisms. Project 2, from the Genzentrum Berlin. The costs of publication of this Recently, a sulfolipid-deficient mutant of Chlamydomonas article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance reinhardtii, which was induced by exposure to UV light, has with 18 U.S.C. Section 1734 solely to indicate this fact. been isolated based on its abnormal chlorophyll fluorescence The nucleotide sequence(s) reported in this paper has been submitted (17). The chlorophyll fluorescence phenotype is an indication TM to the GenBank /EMBL Data Bank with accession number(s) U45308. that photosynthesis is affected in this mutant. However, it has To whom reprint requests should be addressed. Fax: 49-30-830007- 36; E-mail: [email protected]. not clearly been demonstrated that the fluorescence phenotype This is an Open Access article under the CC BY license. 7502 Sulfolipid-deficient Null Mutant of Synechococcus sp. PCC7942 sphaeroides (18). Lipids were separated by one-dimensional thin layer and the sulfolipid deficiency are due to the same genetic defect, chromatography on ammonium sulfate-impregnated plates as de- leaving the causal relation between the two phenotypes open. scribed (18), except that benzene was replaced by toluene. Lipids were To further address the question of whether sulfolipid plays visualized by exposing the plates to iodine vapor. Fatty acid methyl an essential role for oxygenic photosynthesis, we isolated and esters were prepared from each lipid and quantified by gas chromatog- inactivated a gene involved in sulfolipid biosynthesis from the raphy (18). From these data the mol % fraction was calculated for each cyanobacterium Synechococcus sp. PCC7942. The only previ- lipid. Preparation of Radiolabeled Lipids—Cultures (50 ml) of sulfolipid ously known genes encoding sulfolipid biosynthetic enzymes mutant and wild type strains were grown to midlog phase in BG-11 were the sqd genes from R. sphaeroides (18, 19), of which one medium. Cells were washed in sulfate-free BG-11 medium (MgSO was served as a molecular probe to isolate the homologous gene replaced by MgCl ) and resuspended in 1 ml of the same medium. from the cyanobacterium. The resulting sulfolipid null mutant 35 Following the addition of 100 mCi of sodium [ S]sulfate (specific activ- was analyzed with regard to its photosynthetic characteristics ity, 100 mCi/mmol), the cells were incubated for 16 h and harvested by centrifugation. Lipid extracts were prepared and separated as de- and growth under different conditions. Possible modifications scribed above. of the photosystem II reaction kinetics due to the lack of sul- Oxygen Evolution Measurements—Oxygen evolution from whole cells foquinovosyl diacylglycerol were investigated by measuring the under continuous white light was determined polarographically with 10 oxygen yield in response to a regime of short flashes. mM NaHCO as acceptor in a buffer solution of 25 mM Hepes-NaOH, pH 7.0 (26). The concentration of cells was equivalent to 5 mgml chloro- MATERIALS AND METHODS phyll. The oscillation pattern of flash-induced oxygen yield produced by Bacterial Strains, Plasmids, Media, and Growth Conditions—Syn- a series of short flashes from a Xenon lamp (FWHM ' 10 ms) was echococcus sp. PCC7942 wild type (kindly provided by P. Wolk) and monitored with a Joliot-type electrode as described (27). mutant lines were grown photoautotrophically at 28 °C in liquid or Absorption, 77 K, and Room Temperature Fluorescence Emission agar-solidified (1.5%) BG-11 medium (20). Light was provided by fluo- Measurements—In vivo room temperature absorption and 77 K fluores- rescent bulbs at an photon flux density of approximately 50 mmol m cence emission spectra were recorded as described previously (26). s . If required, the growth medium was supplemented with kanamycin Room temperature chlorophyll a fluorescence was determined with a 21 21 (15 mgml ) or ampicillin (1 mgml ). The phosphate content of the pulse amplitude modulation fluorometer (PAM101, Walz, Effeltrich, medium was reduced as indicated in the text by replacing KH PO with Germany) according to the method of Clarke et al. (28) essentially as 2 4 KCl. Growth of the cultures was monitored by determination of the described (26). Phycocyanin and chlorophyll content were determined optical density at 730 nm or by counting colony forming units after in whole cells as described by Collier and Grossman (29). plating. Nucleotide Sequence—The nucleotide sequence reported in this pa- TM For routine cloning experiments, the Escherichia coli strains XL-1 per has been submitted to the GenBank /EMBL Data Bank with Blue, XL-1 Blue MRF9, and XLOLR, as well as the plasmids pBluescript accession number U45308. II-SK and pBK-CMV and the phage l-ZAP-Express and ExAssist RESULTS helper phage were used (Stratagene). The inactivation cassette carry- ing a neomycin phosphotransferase gene was derived from pUC4K (21, Isolation and Characterization of a Cyanobacterial Gene with 22). The origin of plasmids pSY2, pSY3, and pSYB (see Fig. 1) is Sequence Similarity to sqdB of R. sphaeroides—A prerequisite described in the results section. Cultures of E. coli were grown in Luria for the construction of a completely sulfolipid-deficient cya- broth. Kanamycin was usually added as required at 50 mgml , and nobacterial strain by gene replacement is the availability of ampicillin was added at 100 mgml . wild type genes coding for sulfolipid biosynthetic enzymes. Recombinant DNA Techniques—The genomic Synechococcus sp. With the goal to isolate the first cyanobacterial gene involved PCC7942 DNA library was prepared in l-ZAP-Express according to the manufacturer’s instructions. For this purpose, genomic DNA was iso- in sulfolipid biosynthesis, cross-hybridization between the dif- lated from Synechococcus sp. PCC7942 wild type (23), partially digested ferent sqd genes from R. sphaeroides and genomic DNA from with Sau 3A, and 2–4-kilobase pair fragments were ligated with pre- Synechococcus sp. PCC7942 was tested. This cyanobacterium pared phage arms. The library was screened by heterologous DNA was chosen, because it is naturally competent to take up DNA. hybridization (24) using Hybond N filters (Amersham Corp.). Hybrid- In addition, the DNA of Synechococcus sp. PCC7942 has a GC ization was performed at 42 °C overnight in a solution containing 5 3 content intermediate to that of DNA from R. sphaeroides and SSPE, 5 3 Denhardt’s solution, 0.5% (w/v) SDS, 20% (v/v) formamid, and 20 mgml sonicated herring sperm DNA. The filters were washed higher plants (30). Of three sqd genes of R. sphaeroides, only twice at 42 °C for 20 min each in 2 3 SSC, 0.5% (w/v) SDS prior to sqdB gave a strong positive hybridization signal with genomic exposure. The stock solutions were prepared following standard proce- DNA of Synechococcus sp. PCC7942. Probing a genomic library dures (25). Routine recombinant DNA techniques and DNA sequencing prepared in l-ZAP-Express, several clones hybridizing to sqdB were done as described (18, 19). of R. sphaeroides were isolated. Following the excision of plas- Insertional Inactivation of sqdB and Transformation of Synechococ- mids from the phage and restriction analysis, it became appar- cus sp. PCC7942—For inactivation of the sqdB open reading frame, the pBluescript II-SK based plasmid pSYB (see Fig. 1) was partially ent that the inserts of all clones overlapped. The two clones digested with SalI/PstI to remove a 952-base pair fragment internal to containing the longest DNA fragments, pSY2 and pSY3, were the sqdB open reading frame. The deleted fragment was replaced by a recombined by ligation of a 1600-base pair KpnI/SalI fragment 1230-base pair SalI/PstI fragment carrying the neomycin phospho- from pSY3, a 1500 SalI/BamHI fragment from pSY2, and transferase gene of pUC4K. The resulting plasmid pSYBK (see Fig. 1) pBluescript II-SK cut with KpnI and BamHI giving rise to was used to transform wild type Synechococcus sp. PCC7942. For this plasmid pSYB (Fig. 1). The overlapping DNA region of pSY2 purpose, cells (20 ml cultures) were grown to midlog phase in BG-11 medium, harvested by centrifugation for 5 min at 5000 3 g, washed and pSY3 was sequenced on both strands, as indicated in Fig. once, and resuspended in 1 ml of the same medium. Approximately 4 mg 1. Sequence analysis revealed an open reading frame predicted of plasmid DNA were added, and the suspension was incubated under to encode a protein of the molecular mass of 44.6 kDa consist- shaking in the light overnight at 28 °C. Following the addition of 10 ml ing of 402 amino acids (Fig. 2). A GTG triplet is proposed to of BG-11 medium, incubation was continued for 24 h prior to plating the 21 serve as the initiation codon because it is preceded by a perfect cells on selective medium containing 15 mgml kanamycin. Transfor- ribosome binding site, no suitable in frame ATG codon was mants became visible after 1 week and were restreaked at least 4 times. They were tested for ampicillin sensitivity in order to detect double present, and the predicted N-terminal amino acids sequence recombinants. Putative candidates were further analyzed by Southern corresponded well with the N-terminal amino acid sequence hybridization to ensure complete segregation of wild type genome of sqdB of R. sphaeroides. Comparing the amino acid se- copies. quence over its total length with that of sqdB of R. spha- Lipid Analysis—Cultures (50 ml) of sulfolipid mutant or wild type eroides, a sequence identity of 63% and a sequence similarity strains were grown to late log-phase in BG-11 medium containing 0.18 of 77.3%, taking into account conservative substitutions, was or 0.018 mM KH PO as indicated in the text. Cells were harvested by 2 4 centrifugation, and lipid extracts were prepared as described for R. determined. Sulfolipid-deficient Null Mutant of Synechococcus sp. PCC7942 7503 FIG.1. Plasmids used for the characterization and inactiva- tion of the sqdB gene from Synechococcus sp. PCC7942. Plasmids were constructed as described in the text. Small solid arrows, directions of sequence reactions; gray arrow, sqdB open reading frame; open arrow, neomycin phosphotransferase gene; cross-hatched box, fragment used for Southern hybridization. Restriction sites: A, BamHI; E, SpeI; H, HindIII; O, XhoI; P, PstI; S, SalI. The asterisks indicate a Sau3A/ BamHI ligation site. Construction of a Sulfolipid-deficient Null Mutant—As part of a strategy to demonstrate that the isolated open reading frame represents a gene coding for a protein involved in sulfo- lipid biosynthesis in Synechococcus sp. PCC7942, we inacti- vated the wild type copy of the gene by replacing an internal fragment with a kanamycin resistance cassette in opposite orientation. The kanamycin-resistant lines were tested by Southern hybridization (Fig. 3). This experiment revealed the complete disappearance of wild type genome copies and the expected replacement of the targeted open reading frame in all putative mutant lines tested. There was only one hybridizing band of approximately 6 kilobases pairs present in wild type samples, a result that is in agreement with the presence of a single copy DNA sequence. Analysis of the composition of lipid extracts prepared from wild type and mutant cells by thin layer chromatography and quantification of individual lipids by gas chromatography of fatty acid methyl esters derived from the lipids (Table I) revealed no detectable amount of sulfolipid in extracts from the mutant lines. Furthermore, using the most sensitive method available, the analysis of lipid extracts from cells labeled with [ S]sulfate, no traces of residual sulfolipid were detected in mutant samples (Fig. 4). Apparently, the inactivation of the open reading frame abolished sulfolipid biosynthesis in the affected cells and gave rise to a sulfolipid- deficient null mutant of Synechococcus sp. PCC7942. The Effects of Phosphate-limiting Conditions on Growth and Lipid Composition of Mutant and Wild Type—The isolation of genetically pure sulfolipid-deficient mutants of Synechococcus sp. PCC7942 suggests that sulfolipid is not essential for pho- toautotrophic growth of this cyanobacterium. Accordingly, no difference in growth rates was observed under optimal growth conditions (Fig. 5A). However, 10-fold reduction of the phos- phate concentration in the medium caused the mutant to cease growth after 6 days, whereas the wild type continued to grow FIG.2. DNA and deduced amino acid sequence of the Synecho- (Fig. 5B). Comparable results were obtained using optical den- coccus sp. PCC7942 sqdB gene. The nucleotide sequence is shown sity (Fig. 5) or live cell counts (data not shown) for measuring from the KpnI site to the XhoI site of pSYB. The protein sequence is growth of the cultures. given below the DNA sequence. The underlining indicates a putative Under phosphate-limiting growth conditions the relative ribosome binding site. amount of the major phospholipid phosphatidyl glycerol was reduced in wild type cells to 7.2 mol % compared with cells digalactosyl diacylglycerol was observed for the wild type, grown under optimal conditions (16.6 mol %, Table I). Concom- whereas the relative amount of monogalactosyl diacylglycerol itantly, an increase in the relative amount of sulfolipid and was slightly decreased. In the mutant, the relative amount of 7504 Sulfolipid-deficient Null Mutant of Synechococcus sp. PCC7942 FIG.3. Southern hybridization of wild type (WT) and SY-SQDB mutant. Genomic DNA was cut with HindIII and probed with a 1470- base pair SpeI/XhoI fragment from sqdB containing the open reading FIG.5. Growth of Synechococcus sp. PCC7942 wild type (closed frame and adjacent sequences. The approximate length of DNA frag- circles) and SY-SQDB mutant (open circles) under optimal (A) ments is indicated (kilobase pairs). and under phosphate limiting conditions (B). Each value repre- sents the mean of three measurements using independent cultures. TABLE I Error bars were smaller than symbols. Effect of phosphate nutrition on the polar lipid composition of wild type and mutant mined as function of photon flux density. Essentially, identical The values are the means 6 standard errors of three independent cell curves were obtained for the wild type and the mutant (Fig. 6). cultures grown in BG-11 medium supplemented with P as indicated. MGD, monogalactosyl diacylglycerol; PG, phosphatidylglycerol; DGD, To examine the possibility of subtle changes in the reaction digalactosyl diacylglycerol; SQD, sulfoquinovosyl diacylglycerol; nd, not kinetics of photosystem II, the characteristic period four oscil- detected (,0.5 mol %). lation pattern of flash-induced oxygen evolution was compared mol % in dark adapted wild type and mutant cells. The maximum Lipid Wild type SY-SQDB oxygen yield is generated by the fourth flash (Fig. 7). This feature is typical for thoroughly dark-adapted cyanobacteria 0.18 mM P 0.018 mM P 0.18 mM P 0.018 mM P i i i i (31). With regard to the active site tyrosine (Y ) of the D2 MGD 60.6 6 1.1 42.5 6 0.9 56.2 6 0.3 43.7 6 1.9 protein, this pattern is indicative of an apparent population of PG 16.6 6 0.3 7.2 6 0.5 28.4 6 0.2 23.1 6 0.1 OX DGD 12.5 6 0.8 28.0 6 0.6 15.4 6 0.4 33.2 6 1.8 redox states below S Y (see Ref. 32 and references therein). 1 D SQD 10.3 6 0.2 22.3 6 1.0 nd nd Within the frame work of an extended Kok model, in which a cyclic sequence of redox states adopted by the water oxidase during catalysis is postulated (33), the data can be satisfacto- rily described by the probability of misses (a 5 0.23) and double hits (b 5 0.01) and apparent S -state dark populations of [S ] 5 i 1 0.47, [S ] 5 0.39, and [S ] 5 0.12. Preillumination with a 0 21 short saturating flash and subsequent dark incubation for 3 min leads to a shift of oxygen yield maximum to the third flash. This observation shows that the apparent high population of S is mainly due to the presence of Y in its reduced form (32). Both oscillatory patterns exhibited virtually the same features (Fig. 7, A and B) except for the pronounced oxygen uptake in the mutant sample after the first two flashes of the sequence (Fig. 7B). In an attempt to test whether this phenomenon was restricted to the first two flashes, the measurements were repeated in the presence of hydrazine. Under these conditions the redox state S is highly populated, and the maximum of oxygen yield is shifted toward the sixth flash (32). Likewise, virtually no oxygen is evolved during the first four flashes. Contrary to the wild type, the oxygen yield pattern of hy- drazine-treated mutant cells revealed a marked oxygen uptake during the first four flashes in the SY-SQDB mutant (data not FIG.4. Separation of S-labeled lipids of wild type (WT) and shown). SY-SQDB mutant by thin layer chromatography. Approximately Comparing room temperature chlorophyll fluorescence in the equal amounts of total lipids were spotted in case of undiluted extracts (undil.). In addition, 10-, 100-, and 1000-fold dilutions of the wild type wild type and the mutant (Table II), a similar dark level fluo- extracts were loaded for estimation of the reduction of sulfolipid in the rescence yield (F ) was observed for both strains. Because state mutant extract. Radiolabeled lipids were visualized by autoradiogra- 2-state 1 transitions can be important for the determination of phy. F, solvent front; O, origin; SQD, sulfoquinovosyl diacylglycerol; U, the maximum fluorescence yield (F ; Ref. 28), cells were first unidentified compound. m illuminated with low intensity white light to induce state 1 phosphatidylglycerol (28.4 mol %) was increased under optimal prior to the addition of the electron transfer inhibitor 3,4- growth conditions and did not decrease as dramatically under (dichlorophenyl)-1,1-dimethylurea to close photosystem II re- phosphate-limiting conditions (23.2 mol %, Table I). The rela- action centers. Under these conditions, the mutant showed a tive amounts of the galactolipids were comparable with those higher F value and hence a higher variable fluorescence yield found in wild type cells under both growth regimes. (F ). Consequently, the ratio of F /F , which is a measure of the v v m Photosynthetic Characteristics of the Sulfolipid-deficient Mu- photochemical efficiency of photosystem II, was slightly in- tant—To elucidate the effect of sulfolipid-deficiency on oxygenic creased in the mutant (Table II). Based on statistical analysis, photosynthesis, first the rate of oxygen evolution was deter- this increase was significant. In search for further alterations Sulfolipid-deficient Null Mutant of Synechococcus sp. PCC7942 7505 attributed to the core antenna proteins CP43 (34) and CP47 (35), respectively. No difference in the relative amplitudes of the emission maxima were observed between both strains. When excited at a wavelength of 590 nm, which corresponds to the maximum for the excitation of phycobilins, the intensity of the emission at approximately 655 nm was considerably re- duced in the mutant (Fig. 8B). This peak presumably repre- sents overlapping emissions for phycocyanin and allophycocya- nin with maxima at 645 and 665 nm, respectively. Because no difference in the phycocyanin/chlorophyll ratio was observed (data not shown), this result can be taken as an indication for a higher efficiency of excitation energy transfer from phycobi- lins to the photosystem II reaction center chlorophyll a in the mutant. DISCUSSION FIG.6. Rate of oxygen evolution as function of photon flux To study the possible role of sulfolipid in oxygenic photosyn- density by Synechococcus sp. PCC7942 wild type (closed circles) thesis in a definitive way, we created a sulfolipid-deficient null and SY-SQDB mutant (open circles). Each value represents the mutant of Synechococcus sp. PCC7942. During the course of mean of three independent measurements. The standard error was less this work, we isolated for the first time and disrupted a gene than 6% of each value. involved in sulfolipid biosynthesis in an organism with oxy- genic photosynthesis. This gene of Synechococcus sp. PCC7942 shares considerable sequence identity with the sqdB gene of R. sphaeroides and is therefore also designated sqdB. However, further experiments will be required to demonstrate functional homology between the two genes in R. sphaeroides and Syn- echococcus sp. PCC7942. Our current inability to detect cross- hybridization between other sqd genes of the two bacterial strains suggests that these are less conserved. Unfortunately, we still do not know the function of the sqdB gene product, and further experiments to elucidate its biochemical role may also allow us to solve the long standing mystery of sulfolipid biosynthesis. Inactivation of the putative sqdB gene of Synechococcus sp. PCC7942 wild type gives rise to an otherwise isogenic null mutant, which was designated SY-SQDB and completely lacks sulfolipid, one of the four polar lipids found in this bacterium. This deficiency has no lethal consequences. It does not even lead to reduced growth under optimal conditions for photoau- totrophic growth (Fig. 5), suggesting that sulfolipid is not es- sential for oxygenic photosynthesis. Apparently, the loss of the anionic sulfolipid is mainly compensated by an increased rela- tive amount of phosphatidylglycerol (Table I), which is the second anionic lipid found in the membranes of Synechococcus FIG.7. Flash-induced changes of oxygen evolution or uptake sp. PCC7942. Maintaining a certain level of anionic lipids in by Synechococcus sp. PCC7942 wild type (A) and SY-SQDB mu- tant (B). The polarographic signals (arbitrary units) were detected by the membranes seems to be crucial for the organism, because a Joliot-type electrode. Positive peaks indicate oxygen evolution, and the reduction in phosphatidylglycerol under phosphate limita- negative peaks indicate uptake. tion in the wild type is compensated by an increased level of sulfolipid. The sulfolipid-deficient mutant SY-SQDB cannot TABLE II respond in the same way to phosphate limitation and has to Room temperature fluorescence parameters (relative units) maintain a higher level of phosphatidylglycerol. Because it All values represent the means of 20 measurements. Samples were adjusted to a chlorophyll concentration of 20 mgml . F , dark level cannot replace lipid-bound phosphor by sulfur under conditions fluorescence yield; F , maximum fluorescence yield; F , variable fluo- m v of phosphate limitation, it becomes phosphate-depleted and rescence yield (F 5 F 2 F ). v m o enters the stationary growth phase at an earlier time point Parameters Wild type SY-SQDB than the wild type (Fig. 5). The same phenomenon has been previously observed for R. sphaeroides (15). In addition, both F 64.5 6 7.0 65.3 6 6.6 F 131.5 6 13.8 153.9 6 13.9 bacteria accumulate dihexosyl lipids under phosphate limita- F 67.0 6 7.0 88.6 6 9.4 v tion. Although Synechococcus sp. PCC7942 does not accumu- F /F 0.51 6 0.04 0.58 6 0.02 v m late glucosylgalactosyl diacylglycerol, as was observed for phos- phate-limited R. sphaeroides (16), the relative amount of in the antenna system of the mutant, low temperature fluores- digalactosyl diacylglycerol is increased (Table I). cence spectra were recorded. The 77 K fluorescence spectra of Normal growth of the SY-SQDB mutant under optimal lab- wild type and mutant strains obtained after chlorophyll a ex- oratory conditions does not exclude the possibility of a more citation at 440 nm are shown in Fig. 8A. The large emission subtle role of sulfolipid in oxygenic photosynthesis relevant peak at 717 nm is predominantly derived from photosystem I, under natural conditions, e.g. high photon flux densities. How- whereas the two peaks at 685 nm and approximately 695 nm ever, the light response curves for oxygen evolution by wild emanate from photosystem II. The latter two can be mainly type and mutant cells were nearly identical (Fig. 6). 7506 Sulfolipid-deficient Null Mutant of Synechococcus sp. PCC7942 FIG.8. 77 K fluorescence emission spectra of Synechococcus sp. PCC7942 wild type (solid lines) or SY-SQDB mutant (broken lines) after excita- tion at 440 nm (A) or 590 nm (B). Spec- tra in A were normalized to the emission maximum at 717 nm, and spectra in B were normalized to 683 nm. The spectra in A were set off to facilitate comparison. In each case, chlorophyll concentrations were adjusted to 2.5 mgml . This finding indicates that the lack of sulfolipid neither increased in the mutant (Fig. 6). The enhanced light-induced affects the overall electron transport rate nor the optical cross- oxygen uptake in the mutant observed during polarographic section of oxygen evolution. More subtle changes in the reaction measurements with the Joliot-type electrode (Fig. 7) may be a kinetics of photosystem II were expected to become apparent by reasonable explanation for this apparent discrepancy. monitoring the characteristic period four oscillation pattern of The subtle alterations in photosynthesis observed for the flash-induced oxygen evolution in dark-adapted wild type and SY-SQDB mutant would not have been sufficient to isolate this mutant cells. A comparison of oscillatory patterns revealed that mutant from a randomly mutagenized population. On the con- both strains exhibit virtually the same features except for the trary, a leaky sulfolipid-deficient mutant of C. reinhardtii has pronounced oxygen uptake in the mutant sample after the first been isolated based on its high fluorescence phenotype follow- two flashes (Fig. 7B). Because hydrazine-treated mutant cells ing random mutagenesis (17). However, a detailed analysis of showed also a marked increase in oxygen uptake during the the photosynthetic characteristics of this mutant is not avail- able for comparison. Furthermore, it has not clearly been dem- first four flashes, it seems most likely that the enhancement of oxygen uptake in the SY-SQDB mutant is not necessarily di- onstrated that the fluorescence phenotype and the lipid pheno- rectly related to the water splitting activity of photosystem II. type are indeed caused by the same genetic defect. Therefore Instead, increased oxygen uptake could be either due to the further experiments will be required to test whether sulfolipid reduction of O by components of the electron transport chain may play a different role in chloroplasts as compared with or increased respiratory activity. Nevertheless, the data pre- cyanobacterial cells. sented in this study clearly show that sulfolipid is not an In summary, the extensive examination of a sulfolipid-defi- essential constituent of a functionally competent water cient null mutant of Synechococcus sp. PCC7942, suggests that oxidase. sulfolipid does not play a specific role for oxygenic photosyn- Low temperature fluorescence measurements suggest that thesis. A similar conclusion was drawn for nonoxygenic photo- the lack of sulfolipid in the null mutant most likely has no synthesis of R. sphaeroides (15). However, subtle changes in effect on the structural organization of the reaction center/core the biochemistry of O and an increased variable room temper- antenna complex of photosystem II. The similarity of the emis- ature chlorophyll fluorescence yield were observed for the cya- sion spectra following chlorophyll a excitation at 440 nm (Fig. nobacterial mutant. As was concluded for R. sphaeroides, the 8A) indicates that neither the binding environment of the chlo- biosynthesis of sulfolipid may have evolved and been main- rophyll a emitting from the core antenna proteins CP43 and tained during evolution, primarily not to provide an essential CP47, nor the excitation energy transfer to the reaction center component for photosynthetic processes but to provide a surro- is affected in the mutant. Moreover, based on the 77 K fluores- gate anionic lipid for conditions of phosphate limitation. 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Published: Mar 1, 1996

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