The sulfolipid sulfoquinovosyldiacylglycerol is not required for ...

Proc. Natl. Acad. Sci. USA Vol. 90, pp. 1561-1565, February 1993 Biochemistry

The sulfolipid sulfoquinovosyldiacylglycerol is not required for photosynthetic electron transport in Rhodobacter sphaeroides but enhances growth under phosphate limitation (sqdl gene/directed mutagenesis/photosynthesis/lipid)

CHRISTOPH BENNING*, J. THOMAS BEATTYt, ROGER C. PRINCE*, AND CHRIS R. SOMERVILLE*§ *Michigan State University-Department of Energy Plant Research Laboratory, Michigan State University, East Lansing, MI 48824-1312; tDepartments of Microbiology and Medical Genetics, University of British Columbia, Room 300, 6174 University Boulevard, BC V6T 1Z3, Canada; and tExxon Research and Engineering Company, Annandlale, NJ 08801

Communicated by William L. Ogren, November 17, 1992

All photosynthetic organisms, with the excepABSTRACT tion of several species of photosynthetic bacteria, are thought to contain the sulfolipid 6-sulfo-a-D-quinovosyldiacylglycerol. The association of this lipid with photosynthetic membranes has led to the assumption that it plays some role in photosynthesis. Stable null mutants of the photosynthetic bacterium Rhodobacter sphaeroides completely lacking sulfolipid were obtained by disruption of the sqdB gene. The ratios of the various components of the photosynthetic electron transport chain, as well as the electron transfer rates during cyclic electron transport, were not altered in the mutants, when grown under optimal conditions. Growth rates of wild type and mutants were identical under a variety of growth conditions, with the exception of phosphate limitation, which resulted in reduced growth of the mutants. Phosphate limitation of the wild type caused a significant reduction in the amount of all phospholipids and an increased amount of sulfolipid. By contrast, the sulfolipid-deficient mutant had reduced levels of phosphatidylcholine and phosphatidylethanolamine but maintained a normal level of phosphatidylglycerol. In addition, two unidentified lipids lacking phosphorus accumulated in the membranes of both wild-type and mutant strains under phosphate limitation. We conclude that sulfolipid plays no significant unique role in photoheterotrophic growth or photosynthetic electron transport in R. sphaeroides but may function as a surrogate for phospholipids, particularly phosphatidylglycerol, under phosphate-limiting conditions.

Since the discovery (1) and structural elucidation (2) of sulfoquinovosyldiacylglycerol (sulfolipid) by Benson and colleagues more than 30 years ago, it has been repeatedly suggested that sulfolipid may play some vital role in photosynthesis (3). This hypothesis is based primarily on the fact that sulfolipid is found exclusively associated with the photosynthetic membranes of all photosynthetic organisms, except for a few species of photosynthetic bacteria (4, 5). The proportion of sulfolipid in total, ether-extractable lipids varies from 2.6% in Rhodobacter sphaeroides up to 18.6% in the brown alga Fucus vesiculosus (6) and can be as high as 40%o of the glycolipids in marine red algae (7). In higher plants, sulfolipid typically represents -5% of the polar lipids. Experimental evidence in support of a vital role for sulfolipid in photosynthesis has been inferred from correlations between developmentally or environmentally induced changes of sulfolipid levels and photosynthesis (8-10). Also, inclusion of sulfolipid has been reported to have stimulatory effects in reconstitution experiments with integral proteins of photosynthetic membranes (11-13). The requirement for The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

sulfolipid in stoichiometric amounts with other lipids for successful reconstitution of enzymatic activity led to the conclusion that it functions as an essential boundary lipid (3). Based on measurements of the pH gradient across protonpumping membranes, Haines (14) proposed a model in which the charged, anionic head groups of phospholipids, or the sulfated lipids of Ochromonas, could act as a protonconducting pathway. This model implies that sulfolipid located in the inner leaflet of the thylakoid membrane might play an essential and direct role in photosynthetic electron transport. In support of this hypothesis, it has been shown that antibodies raised against sulfolipid can inhibit photosynthetic electron transport (15). In summary, although several experiments suggest a role for the plant sulfolipid in photosynthesis, the evidence is indirect and not sufficient to support a specific model for its function. However, with the recent isolation of genes involved in sulfolipid biosynthesis in the photosynthetic purple bacterium R. sphaeroides (16, 17), a new approach to address the question of sulfolipid function became feasible. Here we describe the construction and properties of lines of R. sphaeroides in which sulfolipid is completely absent due to directed inactivation of sqdB, a gene that is specifically required for sulfolipid biosynthesis (17).

MATERIALS AND METHODS Bacterial Strains, Plasmids, Media, and Growth Conditions. Escherichia coli strains used for propagation of the plasmids were HB101 (F- proA2 recA13 mcrB) (18) and S17-1 [pro(A or B) endAl hsdRl7 (RP4-2::Mu::Tn7)J (19). R. sphaeroides mutants were derived from wild-type strain 2.4.1 (20). Plasmids pCHB16201 (17), pUC4K (21, 22), and pSUP202 (19) were used in the construction of pSB1K1 and pSB1K2 (Fig. 1A). R. sphaeroides cultures were grown on Sistrom's medium (23, 24) as described (16), or in the minimal malate/ inorganic salts medium RCV (25, 26), modified by the inclusion of D-biotin and niacin (each at 15 pug/liter). For experiments requiring defined initial phosphate concentration in the medium, 100 mM potassium Hepes (pH 6.8) was added and the phosphate concentration was adjusted with potassium phosphate. Growth was measured turbidometrically with a Klett-Summerson photometer (filter 66). E. coli cultures were grown in Luria broth. Kanamycin was usually added as required at 50 ,ug/ml (E. coli) or 25 gg/ml (R. sphaeroides), and tetracycline at 10 pug/ml (E. coli) or 0.1 p.g/ml (R. sphaeroides). For the selection of exconjugants following mating and during the screening for double crossAbbreviation: PAR, photosynthetically active radiation. §To whom reprint requests should be addressed.

1561

Proc. Natl. Acad. Sci. USA 90 (1993)

Biochemistry: Benning et al.

1562

A

were prepared as described (16). Polyhydroxybutyrate in extracts from cells grown under phosphate-limiting conditions was removed by precipitation following the addition of 4 volumes of hexane. Lipids were separated by twodimensional TLC and fatty acid methyl esters were prepared from each lipid and quantified by gas chromatography (16). From these data the mol % fraction of the analyzed polar lipids was calculated for each lipid. Isolation of Chromatophores and Analysis by Spectroscopy. Chromatophores (inner-membrane vesicles) were prepared (27) from cells grown photosynthetically. Reduced-minusoxidized differential absorption spectroscopy and flash spectroscopy of the chromatophores were carried out as described (28, 29).

sqdB

pSB1

PAOS

pSB1K1

PAOS

0

A

A

-E~

~

~

BBP

E

O* B1B P

E

~~

PS'

H

0W

Kanr PAOS

pSB1K2

BBP

500 bp

BBP EEP

E

E

"K' '1 Kanr

H

B WT

O

A

E

aqdB aqdD WqdC

SB1K1

H PBBP EEP

E I

mi

E a

sqdB::Kanr aqdD eqdC

SB1K2

E

P H

BBP EEP

E

aqdBu:Kanr aqdD sqdC 1

kb

FIG. 1. Restriction maps of the plasmids used to inactivate sqdB (A) and of the chromosome before and after insertion of the kanamycin cassette (B). (A) Vector sequences are drawn with thicker line. The open reading frame for the sqdB gene is indicated as a hatched arrow. The solid bar underneath pSB1 represents the 650-bp fragment used as probe for DNA hybridizations. The kanamycin cassette is drawn as an open box with the open reading frame for the kanamycin-resistance gene drawn as a solid arrow. (B) The promoter (open arrow) preceding three known open reading frames for sqd genes (hatched boxes) in the wild type (WT) as well as the kanamycin cassette (open box with solid arrow representing the open reading frame) in the mutants is indicated. The insertion site in the wild-type sqdB gene for the kanamycin cassette is marked with a solid triangle. Restriction sites: A, BamHI; B, Bgl II; E, EcoRI; H, HindIII; 0, Xho I; P, Pst I; S, Sal I. Sites which were lost during the cloning procedure are marked with a star.

kanamycin (100 ,ug/ml) and tetracycline (0.5 ,ug/ml) added to Sistrom's medium. Genetic Procedures and Recombinant DNA Techniques. Plasmids were transferred from E. coli S17-1 into R. sphaeroides by diparental mating as described (16), except that due to the properties of the donor strain S17-1 (19), no helper strain was required. Routine recombinant DNA techniques were performed as described (16, 17). Analysis of -"S-Labeled Lipids. Cultures (8 ml) of sulfolipid mutant or wild-type strains inoculated from single colonies were grown photoheterotrophically for 24 hr in Sistrom's medium lacking sulfate. Carrier-free [35S]sulfate [10 uCi (370 kBq)] was added and the cultures were further incubated for 16 hr. Lipids were extracted and 20% of the total amounts were separated by TLC on ammonium sulfate-impregnated silica plates (16). Quantitative Lipid Analysis. Cultures (200 ml) of sulfolipid mutant or wild-type strains were grown photoheterotrophically to late logarithmic phase in the medium indicated in the text. Cells were harvested by centrifugation and lipid extracts

overs, were

RESULTS Insertional Inactivation of the sqdB Gene. To construct null mutants of R. sphaeroides specifically lacking sulfolipid, the sqdB gene, essential for the biosynthesis ofthis lipid (17), was inactivated by insertion of the kanamycin-resistance gene from Tn903 (22). A 2-kb Pst I-EcoRI fragment obtained from a partial digest of pCHB16201 was cloned into pSUP202 to give rise to pSB1 (Fig. 1A). This clone was partially digested with Xho I, and a 1.2-kb Sal I fragment from pUC4K, carrying the kanamycin cassette, was inserted in both orientations into the Xho I site internal to the sqdB gene, resulting in clones pSB1K1 and pSB1K2 (Fig. 1A). These two plasmids, conferring resistance to kanamycin and tetracycline but lacking an origin of replication recognized by R. sphaeroides, were independently mated from E. coli S17-1 into R. sphaeroides wild-type 2.4.1. Double crossovers resulting in the loss of the pSUP202 vector and replacement of the wild-type copy of the sqdB gene with a disrupted gene were identified as Kanr Tets clones. To test for the complete segregation of wild-type and mutant chromosomes and to confirm the orientation of the kanamycin cassette with respect to sqdB, genomic DNA was isolated, digested with EcoRI and HindIII, and hybridized to a 650-bp BamHI-Bgl II fragment from pBS1 (Fig. 1A). Two mutant lines, SB1K1 and SB1K2 (Fig. 1B), showing the diagnostic restriction and hybridization pattern for each of the two orientations as well as complete absence of a 2.9-kb fragment characteristic for the wild type (data not shown) were retained for further experiments. Mutant Lines SB1K1 and SB1K2 Do Not Contain Detectable Amounts of Sulfolipid. To examine the effect of the gene disruptions on sulfolipid content by the most sensitive method available, wild-type and mutant cells were labeled in vivo with [35S]sulfate, and lipids were extracted and separated by TLC (Fig. 2). When equal amounts of total lipids from the mutant and the wild-type were loaded, no labeled sulfolipid was detectable in either of the mutant extracts, whereas a band running at the position of sulfolipid could still be detected in 100-fold diluted wild-type extracts. These results indicate that insertional inactivation of sqdB resulted in null mutants, which were unable to synthesize sulfolipid and were completely devoid of this lipid. Relative Amounts of b-I and c-Type Cytochrome Are Not Altered in the Sulfolipid-Deficient Mutants. As part of an evaluation of the effect of the absence of sulfolipid on the structure and function of the photosynthetic membranes, optical difference spectra were used to compare the relative amounts of total b- and c-type cytochromes, which are components of the electron transport chain in chromatophores of wild-type and mutant strains (30). Ascorbatereduced minus ferricyanide-oxidized difference spectra reveal the high-redox-midpoint-potential cytochromes, mostly of the c type (absorption maxima at -550 nm), including cytochromes cl and C2, but also some b-type cytochromes

Biochemistry: Benning et al. WT

Proc. Natl. Acad. Sci. USA 90 (1993)

-

-

o -

o 0

O

0

c 0

0

SB1 K1

wT

lKl 1K2

A

1563

SB1 K2

-XA 0.03 A

c

-- F

0.03 A

530

Wbqa

-

-

0

0

-

SL

530 570 Wavelength, nm

530

570

FIG. 3. Absorption spectra of chromatophores isolated from wild type (WT) and the SB1K1 and SB1K2 mutant strains grown in RCV medium. The bacteriochlorophyll concentration was 113 uM in a buffer containing 20 mM Mops, 100 mM KCl, and 1 mM MgCl2 at pH 7.0. (A) Ascorbate-reduced minus ferricyanide-oxidized difference spectra. (B) Dithionite-reduced minus ferricyanide-oxidized difference spectra.

u

o

FIG. 2. Separation of35S-labeled polar lipids from R. sphaeroides wild type (WT) and SB1K1 and SB1K2 mutants by TLC. A series of 10-fold dilutions of wild-type extracts were spotted as indicated. Approximately equal amounts of total lipids were contained in undiluted (1.0) extracts from the wild type and the two mutants SB1K1 (iKi) and SB1K2 (1K2). 35S-labeled material contained in the extracts was visualized by autoradiography. F, solvent front; SL, sulfolipid; U, unidentified compound; 0, origin.

(560 nm), including the terminal oxidases of the respiratory pathway. Dithionite reduction reveals the lower-potential cytochromes, which are mostly of the b type. The two sets of difference spectra obtained for each of the sulfolipid-deficient mutant and wild-type strains revealed no differences (Fig. 3), indicating that the ratios of b- and c-type cytochromes are not altered in the mutants. Using an extinction coefficient of 100 mM-1 cm-1 at 852 nm for bacteriochlorophyll, and reducedminus-oxidized difference spectra extinction coefficients of 29.8 mM-1-cm-l at 605 540 nm for reaction centers and of 19 mM-1 cm-1 at 550 - 540 nm for photo-oxidizable c-type cytochromes (31), we estimated the number of reaction centers per 1000 bacteriochlorophylls to be 11 for the wild type and 12 for the two sulfolipid-deficient mutants, and the number of photo-oxidizable c-type chromophores per 1000 bacteriochlorophylls to be 8 for wild type and mutants. Electron Transfer Rates During Cyclic Electron Transport Are Not Altered in the Sulfolipid-Deticient Mutants. The abilities of the sulfolipid-deficient mutants to perform cyclic electron transport were investigated by flash spectroscopy. The spectroscopic traces in Fig. 4A show the response of the carotenoid bandshift (523-509 nm) to eight flashes of actinic light spaced 32 ms apart. Independent experiments (data not shown) indicated that these flashes were >94% saturating. The bandshift is well known to exhibit three phases after a single flash; phase I accompanies the initial charge separation in the reaction center, phase II the re-reduction of the reaction center by the c-type cytochromes, and phase III the electrogenic reactions in the cytochrome bc1 complex (30). The wild-type and the mutant strains exhibited all three -

570

phases of the bandshift after the first flash, and all strains showed a similar asymptotic response to subsequent flashes due to approaching equilibrium membrane potential, which prevented further electrogenic electron transfer. To examine the responses of the reaction center and the band c-type cytochromes to flash activation, the response traces shown in Fig. 4 B-D were measured after the addition of valinomycin, to collapse the membrane potential, and antimycin, to inhibit the cytochrome bc, complex by preventing the oxidation of cytochrome bh. The oxidation of the reaction-center bacteriochlorophyll dimer, measured at 605 - 540 nm (Fig. 4B), was partially obscured in the first few flashes by the prompt re-reduction by cytochromes cl and C2. These were measured at 550 - 540 nm (Fig. 4C) and their oxidation was in turn partially obscured after the first flash by their re-reduction by the Rieske iron-sulfur protein. Cytochrome bh, measured at 560 - 540 nm (Fig. 4D), was reduced after the first flash, concomitant with this reduction (see ref. 30 for review of the described phenomena). Clearly, the flash activation responses of wild-type and sulfolipiddeficient mutant strains were indistinguishable. Measurements of Growth Rate and Culture Yield. To test the effect of sulfolipid on photoheterotrophic growth, growth of wild-type and mutant lines was compared under different conditions. Growth rates and yields under optimal conditions at 150 Amol of photosynthetically active radiation (PAR) per m2 per s, measured at the surface of the incubation vessel and 340C (Fig. 5A), were found to be identical for wild-type and both mutants. To test whether the lack of sulfolipid is critical for photoheterotrophic growth under light and temperature extremes, cells of wild-type and mutant lines were grown in complete medium under low-light conditions at 15 pumol-M2s-1 and 340C (Fig. SB), under high-temperature conditions with PAR at 150 AM.mom-2-s-1 and 400C (data not shown), and under low-temperature conditions with PAR at 150 IAmoIlm-2ms- and 100C (Fig. SC). However, growth rates and yields for the wild-type and the mutant cells were indistinguishable under these conditions. To test the hypothesis that sulfolipid may serve as a surrogate for one or more phospholipids under conditions unfavorable for phospholipid biosynthesis, the growth ofwild type and mutants was compared under phosphate-limiting conditions. Although the initial growth rates of the wild-type

1564

Biochemistry: Benning et al.

Proc. Natl. Acad Sci. USA 90 (1993)

WT

SB1K1

SB1 K2

A 0.04A 523-509nm

T

0.004A T

B

605-540nm

C

550-540nm

0.002A

-T

D

I-

0.002A

560-540nm

-T

2 200 ms

and the mutant strains were identical at all concentrations of phosphate tested (20-100 AM), there was a reproducible decrease in growth rates and cell yields of the mutants at later stages of the cultures, well before the wild type was affected (Fig. 5D). In contrast to these results, in experiments in which cultures were subjected to sulfate limitation the wildtype and the mutant strains exhibited the same growth rate and yield (data not shown). Lipid Composition of Wild Type and Sulfolipid-Deficient Mutant. Comparison of the polar lipid compositions of the sulfolipid-deficient mutant SB1K1 and the wild-type grown photoheterotrophically under phosphate saturation and lim-

A ,i0 a~~f

4

A

FIG. 4. Responses of the carotenoid bandshift (A), reaction center (B), c-type cytochromes (C), and b-type cytochromes (D) of chromatophores to eight actinic flashes spaced 32 ms apart. Chromatophores of wild-type (WT) and SB1K1 and SB1K2 mutant strains grown in RCV medium were suspended at a concentration of 25 #M bacteriochlorophyll in a buffer containing 20 mM Mops, 100 mM KCI, and 1 mM MgCl2 at pH 7.0 with the addition of a few crystals of ascorbate. Following the recording of the carotenoid bandshift (A), antimycin was added at 2 j.M and valinomycin at 5 juM (B-D).

itation revealed that both strains dramatically changed their lipid composition in response to phosphate limitation (Table 1). Under phosphate-limiting conditions, the mutant and the wild type showed greatly reduced levels (10- to 20-fold) of phosphatidylcholine and phosphatidylethanolamine and high levels of two novel lipids (Table 1, Li and L2) not previously described in this bacterium. Preliminary results based on diagnostic staining of TLC plates indicated that one of the novel lipids was a glycolipid, while the second contained a quaternary amine. The amount of phosphatidylglycerol was reduced -2-fold in the wild type and did not change in the mutant under phosphate limitation. Under the same conditions, the wild type showed an -8-fold increase in sulfolipid, which was undetectable in the mutant. The ornithine lipid increased 2-fold in the wild type but 4-fold in the sulfolipiddeficient mutant grown under phosphate limitation.

DISCUSSION

1 100 .

00

A

0 c

In

20

15 40 65 90

60 100 140

To address the question of a possible role of sulfolipid in photosynthesis, sulfolipid-deficient mutants of the purple bacterium R. sphaeroides were constructed and compared with the wild type. The wild-type allele of one of the genes required for sulfolipid biosynthesis (sqdB) was inactivated by insertion of a kanamycin-resistance cassette in both orienta-

E

j2 300 C

400 -D

a-

00

100

00 oQ 00

St aa

43

100 r 9

63

0

AP

9

A

50 100150 200 250

50 150 250 350

Time, hr FIG. 5. Photoheterotrophic growth of wild-type (o) and SB1K1 (o) and SB1K2 (A) mutant strains with light intensity (PAR) of 150 ,umolm-2*s-', temperature of 34°C (A and D); 15 ,umolm-2-s-1, 34°C (B); or 150 ,umolm-2-s-1, 10°C (C). Cells were grown in complete RCV medium (A-) or cells were inoculated in modified RCV medium supplemented with 50 ,uM phosphate (D).

Table 1. Effect of phosphate nutrition on the polar lipid composition of mutant and wild type mol % Wild type SBlKl Lipid 1.0 mM Pi 0.1 mM P1 1.0 mM Pi 0.1 mM Pi Li 1.2 ± 0.5 31.1 ± 5.7 1.2 ± 0.1 33.4 ± 5.2 PE 39.6 ± 2.5 6.8 ± 5.6 44.6 ± 16.1 2.5 ± 0.4 OL 5.5 ± 1.3 11.2 ± 3.5 4.9 ± 2.7 23.3 ± 3.7 L2 1.1 ± 0.6 19.2 ± 5.2 0.6 ± 0.5 11.0 ± 1.3 SL 2.2 ± 0.0 16.6 ± 3.7 nd nd PC 27.7 ± 1.8 2.9 ± 1.8 25.0 ± 8.9 2.3 ± 0.8 PG 22.8 ± 3.8 12.2 ± 4.8 23.8 ± 5.7 27.4 ± 2.7 Values are the means + standard errors of three independent cell cultures grown in modified Sistrom's medium supplemented with phosphate (P) as indicated. L1, unidentified lipid 1; PE, phosphatidylethanolamine; OL, ornithine lipid; L2, unidentified lipid 2; SL, sulfolipid; PC, phosphatidylcholine; PG, phosphatidylglycerol; nd, not detected (