(Received for publication, July 5, 1995)
From the
The sulfolipid 6-sulfo--D-quinovosyl
diacylglycerol is found in the photosynthetic membranes of all plants
and most photosynthetic bacteria. Progress toward the elucidation of
the pathway for sulfolipid biosynthesis has been slow in the past.
However, the recent isolation of three genes of the photosynthetic
bacterium Rhodobacter sphaeroides known to be involved in
sulfolipid biosynthesis provides promising new opportunities. Two of
the genes flank an open reading frame predicted to encode a protein
with amino acid sequence similarity to sugar nucleotide-dependent
glycosyltransferases. The UDP-sulfoquinovose:diacylglycerol
sulfoquinovosyltransferase thought to catalyze the last step of
sulfolipid biosynthesis belongs to this group of glycosyltransferases.
To test whether this open reading frame encodes the
sulfoquinovosyltransferase of R. sphaeroides, it was
inactivated by gene replacement avoiding polar mutagenesis. The
resulting sulfolipid-deficient mutant defines a new gene, designated sqdD. Mutant cells grown in the presence of
[
S]sulfate accumulate a water-soluble
S-labeled compound. The purified compound was tentatively
identified by co-chromatography with standards and enzymatic conversion
as UDP-sulfoquinovose, the final precursor of sulfolipid biosynthesis.
This result strongly suggests that the inactivation of sqdD causes a metabolic block in the last step of sulfolipid
biosynthesis.
The sulfolipid sulfoquinovosyl diacylglycerol is found in all
plants and most photosynthetic bacteria(1, 2) . In
higher plants it is confined to the chloroplast(3) . Since the
discovery (4) and structural elucidation (5) of
sulfoquinovosyl diacylglycerol by Benson and co-workers, different
pathways for the biosynthesis of the 6-sulfoquinovose-containing lipid
have been suggested (see summary in (1) ). Experimental
verification of the proposed steps of the pathway is generally lacking,
but evidence is accumulating that the immediate precursors from which
sulfolipid is assembled are UDP-sulfoquinovose and diacylglycerol. The
tentative identification of nucleotide sulfoquinovose in extracts of
[S]sulfate-labeled cells of Chlorella(6) led Benson (5) to suggest that the transfer of
the sulfoquinovose moiety from a nucleotide sulfoquinovose onto
diacylglycerol may represent the last step of sulfolipid biosynthesis.
More recently, it was demonstrated that chemically synthesized
UDP-sulfoquinovose could stimulate sulfolipid biosynthesis by spinach
chloroplast membranes which were biochemically preloaded with
radioactive diacylglycerol(7) . Since the effect was specific
for UDP-sulfoquinovose as compared to other sulfosugar nucleotides, it
was concluded that in spinach chloroplasts a
UDP-sulfoquinovose:diacylglycerol sulfoquinovosyltransferase catalyzes
the last step of sulfolipid biosynthesis. Using synthetic
UDP-sulfoquinovose as substrate, the biochemical properties of the
enzyme and its association with the inner envelope membrane of the
chloroplast could be determined(8) .
Currently, the only genes known to encode proteins involved in the biosynthesis of sulfoquinovosyl diacylglycerol are sqdA, sqdB, and sqdC isolated from the photosynthetic bacterium Rhodobacter sphaeroides(9, 10) . Two of the genes, sqdB and sqdC, were found to flank an open reading frame predicted to encode a protein with an amino acid sequence similar to that of glycogenin(9) , a sugar nucleotide-dependent glycosyltransferase, but no mutant affecting this open reading frame was available. Given this sequence similarity and the location of the open reading frame within one of the sulfolipid operons of R. sphaeroides, it is tempting to predict that the open reading frame may encode a protein catalyzing the transfer of sulfoquinovose during final assembly of the sulfolipid in analogy to the reaction in spinach chloroplasts. As a first step to test this hypothesis, we had to specifically inactivate this open reading frame, avoiding polar mutagenesis. This experiment would answer the general question whether the open reading frame represents a novel sqd gene of R. sphaeroides essential for sulfolipid biosynthesis. In addition, we set out to identify UDP-sulfoquinovose in the mutant cells which we expected to accumulate due to a metabolic block in the final step of sulfolipid biosynthesis, if the protein encoded by the open reading frame would be involved in catalyzing this final reaction.
R. sphaeroides cell cultures were grown photoheterotrophically in Sistrom's medium (17, 18) as described previously(9) . To obtain low sulfate medium, sulfate salts in Sistrom's medium were substituted with chloride salts. E. coli strains were grown in Luria broth. Antibiotics were added at concentrations of 100 µg/ml ampicillin, 50 µg/ml kanamycin, or 10 µg/ml tetracycline for E. coli cultures or 25 µg/ml kanamycin for R. sphaeroides cultures.
Figure 1:
Restriction map of the inactivation
cassette for nonpolar mutagenesis. The neomycin phosphotransferase gene (npt) is drawn as solid bar, the cytochrome c promoter from R. capsulatus (Pcyc) is presented as a gray bar with the
orientation indicated by the arrow. Polylinker sequences are
indicated by open boxes. Restriction sites are A, BamHI; E, EcoRI; H, HindIII; O, XhoI; P, PstI; S, SalI.
Construction of
the plasmid pMR4D used to inactivate the open reading frame flanked by
the sqdB and sqdC genes was accomplished in three
steps. A 3.6-kb partial PstI/HindIII fragment from
pCHB160018 containing the sulfolipid operon from R. sphaeroides was cloned into pBluescript-KSII giving rise to
plasmid pMR2. This plasmid was cut with EcoRI and ligated with
a 1.6-kb EcoRI fragment of pMR1 containing the new
inactivation cassette. In the resulting plasmid pMR2D1, a 53-bp
fragment of the targeted open reading frame was replaced with the
inactivation cassette. The proper orientation of the cassette was
confirmed by restriction analysis with XhoI. Finally, a
partial 3.6-kb PstI fragment of pMR2D1 containing the
interrupted open reading frame followed by the sqdC gene was
ligated with pSUP202 cut with PstI. The resulting plasmid
pMR4D1 was used to inactivate the respective gene in R. sphaeroides wild type cells.
Lipids were separated by one-dimensional thin layer chromatography on ammonium sulfate-impregnated silica plates as described previously (9) with one modification to the solvent, the substitution of benzene with toluene.
Radiolabeled water-soluble compounds were
separated on Silica Gel 60 F plates (0.2 mm, Merck)
developed with one of the following solvents as indicated in the text:
solvent A, ethanol/water/acetic acid (20:10:1, by volume); solvent B,
isopropanol/dioxane/H
O/acetic acid (20:10:10:1, by volume);
and solvent C, isopropanol/H
O/acetic acid (35:10:1, by
volume). The R
values for UDP-sulfoquinovose in
the different solvents were: A, 0.7; B, 0.2; and C, 0.6. The
S-labeled compounds were visualized by autoradiography.
Their relative abundance was determined by densitometry scanning of the
respective lanes on the autoradiography employing a laser densitometer
(LKB, Ultroscan XL). For preparation of labeled UDP-sulfoquinovose, the
compound accumulating in the MRD mutant was extracted with water from
the silica powder prepared by scraping the respective spot.
Analysis of sulfosugars and
nucleotides was accomplished by reversed phase high performance liquid
chromatography using a RP-18 column (µBondapak C18 from Waters)
with a particle size of 10 µm and the dimensions of 3.9 300
mm. As mobile phase a mixture of 30 mM
KH
PO
, 2 mM tetrabutylammonium
hydroxide, adjusted to pH 6.0 with KOH (solvent A) and acetonitrile
(solvent B) was used. For gradient elution at a flow rate of 1 ml/min a
linear gradient from 0 to 30% of solvent B in A was run for 30 min, and
the solvent mixture was kept for an additional 15 min at this ratio.
The column was returned to start conditions with a linear gradient over
10 min ending with 100% solvent A followed by equilibration for 40 min.
Since an on-line mass detector for sugar analysis was not available,
different modes of detection were used: recording the UV absorption at
254 nm followed in line by radiodetection employing a solid-phase
flow-through scintillation detector (Berthold LB 507) or manual
collection of fractions for colorimetric determination of sugar content
and parallel scintillation counting. The second combination was used to
detect sulfoquinovose and sulfoquinovose 1-phosphate. Approximately
0.4-ml fractions were collected and dried in vacuum for 3 h. The
residues were dissolved in 300 µl of water. An aliquot of 100
µl was subjected to scintillation counting. The remaining solution
was rapidly mixed with 400 µl of anthrone reagent (3 mg/ml in
concentrated H
SO
). Following incubation for 20
min at room temperature, the extinction was measured at 600 nm.
Glycosyltransferase reaction mixtures of 30 µl total volume
contained approximately 5,000 dpm of the S-labeled
compound accumulating in the MRD mutant or 100,000 dpm
UDP-[U-
C]galactose (2.5 Ci/mol, Amersham Corp.)
and broken chloroplast membranes equivalent to 35 µg of chlorophyll
in reaction buffer (50 mM Tricine/KOH, 30 mM MgCl
, pH 7.5). Following incubation for 1 h at 25
°C, the reaction was stopped by the addition of 250 µl of
methanol/chloroform (1:1, v/v) and 100 µl of 1 M KCl, 0.2 M phosphoric acid. Chloroform-soluble material was analyzed by
thin layer chromatography as described above.
Quantitation of total chlorophyll in 80% acetone was done according to Lichtenthaler(23) .
Figure 2: Insertional inactivation of the open reading frame for sqdD. A, restriction maps for the central portion of the sulfolipid operon in R. sphaeroides wild type 2.4.1 and the MRD mutant line. The open arrows indicate the sqd genes. The cross-hatched box marks the position of the probe used for Southern hybridization. The inactivation cassette and the restriction sites are as described in the legend to Fig. 1. Additional restriction site is B, BglII. B, Southern hybridization of MRD and wild type 2.4.1. Genomic DNA was cut with XhoI and probed with a 1.1-kb BglII/BamHI fragment containing the entire open reading frame of sqdD.
No
sulfolipid band could be detected by iodine staining on thin layer
chromatograms of lipids extracted from three independent MRD lines. To
use the most sensitive detection method available, we grew cells of one
of the MRD lines and wild type in the presence of
[S]sulfate. Comparison of a serial dilution of
wild type lipid extract and undiluted MRD lipid extract by thin layer
chromatography and autoradiography revealed that in MRD the sulfolipid
content is reduced at least by a factor of 1000 (Fig. 3).
Therefore, inactivation of the open reading frame flanked by sqdB and sqdC in the MRD lines results in a sulfolipid
deficient mutant of R. sphaeroides.
Figure 3:
Separation of S-labeled
lipids from wild type and MRD mutant line by thin layer chromatography.
Approximately equal amounts of total lipids were loaded in case of
undiluted extracts (undil.). In addition, 10-, 100-, and
1000-fold dilutions of the wild type extracts were loaded for
estimation of the reduction of sulfolipid in the mutant extracts.
Radiolabeled lipids were visualized by autoradiography. F,
solvent front; O, origin; SL, sulfolipid; U,
unidentified compound.
Figure 4:
Complementation analysis of sqd mutants using different subfragments of the sulfolipid operon. The
chemically induced mutants carrying defects in the sqdB gene (CHB16) or the sqdC gene (CHB18) as well as
the plasmids were constructed during a previous study(10) . The open arrows indicate the sqd genes. Vector sequences
are drawn with a thick line; the solid arrows indicate the position and orientation of the cytochrome c promotor (Pcyc) driving the expression
of some subfragments of the operon. The gray box marks the
position of the open reading frame of sqdD. The result of the
complementation analysis is indicated as presence (+) or absence
(-) of sulfolipid in lipid extracts separated by TLC. The gray box highlights the result for the MRD mutant line. *,
point mutation;
, insertional mutation. Restriction sites are as
described in Fig. 1and Fig. 2.
Figure 5:
Thin layer chromatography of S-labeled water-soluble compounds isolated from wild type
and mutant line MRD. Solvent was ethanol/H
O/acetic acid
(20:10:1, by volume). The compounds were visualized by autoradiography.
Their relative abundance was determined by densitometry scanning of the
respective lanes on the autoradiography. The arrowhead marks
the position of the compound accumulating in MRD. F, front; O, origin; SL,
sulfolipid.
Figure 6:
Identification of the compound
accumulating in MRD using high performance liquid chromatography. A, UV trace and B, radio detector trace following the
injection of a mixture of the S-labeled compound and
different synthetic sulfosugar nucleotides (50 nmol each). The radio
signal is recorded with slight delay, since the column effluent passes
first through the optical cell before reaching the scintillation
detector via a short connecting capillary. ADPS,
ADP-sulfoquinovose; CDPS, CDP-sulfoquinovose; GDPS,
GDP-sulfoquinovose; UDPS,
UDP-sulfoquinovose.
The nucleotide structure of the labeled compound was confirmed by cleavage with specific enzymes and subsequent identification of the cleavage products by co-chromatography with standards. The labeled compound was mixed with an excess of synthetic unlabeled UDP-sulfoquinovose and treated either with nucleotide pyrophosphatase or a combination of nucleotide pyrophosphatase and alkaline phosphatase. The nucleotide pyrophosphatase cleaves sugar nucleotides releasing a sugar phosphate and a nucleoside monophosphate. In the presence of alkaline phosphatase, these products are converted to free sugar and the corresponding nucleoside. Following the enzymatic hydrolysis, the products were analyzed by high performance liquid chromatography. The elution of the base containing part of synthetic UDP-sulfoquinovose was monitored on-line by UV absorption, and the radioactive portion of the unknown compound by radio detection. Treatment with nucleotide pyrophosphatase resulted in complete disappearance of the UV signal for UDP-sulfoquinovose and the overlapping radio signal characteristic for the unknown compound. Concomitantly, a UV signal for uridine monophosphate and a radio signal for a radioactive compound escaping UV detection and eluting after uridine monophosphate appeared (data not shown). Combined treatment with nucleotide pyrophosphatase and alkaline phosphatase resulted in a UV signal for uridine and a radio signal for a new compound escaping UV detection and eluting ahead of uridine (data not shown). Under the conditions employed, a complete conversion of substrates was observed for the two different reactions.
To demonstrate that the radioactive hydrolysis products co-chromatographed with the expected compounds, synthetic sulfoquinovosyl 1-phosphate and sulfoquinovose (40 µg each) were added after termination of the reactions to the hydrolysis mixtures and subjected to high performance liquid chromatography. For this purpose the effluent was manually collected, and the fractions were monitored for the presence of sulfoquinovose (colorimetry with anthrone) and radioactive compounds. Nucleotide pyrophosphatase released a labeled product co-chromatographing with sulfoquinovosyl 1-phosphate (Fig. 7, right traces). The additional treatment with alkaline phosphatase produced a labeled product co-chromatographing with sulfoquinovose (Fig. 7, left traces). Taken together, these results present strong biochemical evidence for the identity of the unknown compound with UDP-sulfoquinovose.
Figure 7:
Analysis of the radioactive cleavage
products by high performance liquid chromatography following treatment
of the S-labeled unknown compound with hydrolytic enzymes.
A mixture of the labeled unknown compound and synthetic
UDP-sulfoquinovose (100 nmol) was treated either with nucleotide
pyrophosphatase (triangles) or with a combination of
nucleotide pyrophosphatase and alkaline phosphatase (circles).
Following the termination of the reactions synthetic sulfoquinovose or
sulfoquinovose 1-phosphate standards were added (40 µg each). The
two reaction mixtures were analyzed separately by high performance
liquid chromatography. Sulfosugars (open symbols) and
radioactivity (closed symbols) were monitored in manually
collected fractions. The elution profiles from both experiments were
combined in one figure since neither radioactivity nor sugars were
detected at different retention times than indicated for each
experiment. SQVP, sulfoquinovosyl phosphate; SQV,
sulfoquinovose.
Figure 8:
Separation of lipid extracts from spinach
chloroplast membranes incubated with the S-labeled unknown
compound or UDP-[U-
C]galactose. Lipids were
visualized by A, autoradiography and by B, charring. F, solvent front; MGD, monogalactosyl diacylglycerol; DGD, digalactosyl diacylglycerol; O, origin; SL, sulfolipid.
Our objective was to test whether the open reading frame
flanked by sqdB and sqdC in the sulfolipid operon of R. sphaeroides represents a new gene crucial for sulfolipid
biosynthesis. Our strategy was to disrupt the open reading frame
without affecting the expression of the neighboring sulfolipid genes.
As a precautionary measure we designed an inactivation cassette
containing the outward reading cytochrome c promoter from R. capsulatus at the 3`-end of a neomycin
phosphotransferase gene. Insertion of this cassette in proper
orientation resulted in specific inactivation of the targeted open
reading frame which was confirmed by complementation analysis. However,
further experiments will be required to decide whether this cassette
can be used in a general way for nonpolar mutagenesis in R.
sphaeroides. The specific disruption of the targeted open reading
frame led to sulfolipid deficiency thereby defining a new sulfolipid
gene, which we tentatively designated sqdD. It is the fourth
gene of R. sphaeroides which was shown to be crucial for
sulfolipid biosynthesis using mutational analysis combined with genetic
complementation(9, 10) . Three of the genes, sqdB, sqdD, and sqdC are located in one
transcriptional unit (Fig. 4).
Of all four genes, only for sqdD a clearer picture emerges about the possible function of
the gene product. First, the predicted amino acid sequence encoded by
this gene shows sequence similarity to a UDP-glucose-dependent
glycosyltransferase(10) , an enzyme with functional analogy to
the UDP-sulfoquinovose-dependent sulfoquinovosyltransferase known to
catalyze the last step of sulfolipid biosynthesis in spinach
chloroplasts(8) . Second, the MRD mutant described here which
is unable to express sqdD accumulates a S-labeled
compound tentatively identified as UDP-sulfoquinovose. Since we were
unable to purify a sufficient amount of unlabeled compound for
structural analysis by spectroscopy, we alternatively applied two
established experimental approaches to determine the possible chemical
nature of the
S-labeled compound. One line of evidence was
based on co-chromatography with synthetic standards, the second on the
conversion of the UDP-sulfoquinovose by enzymes with well defined
substrate specificities. The compound clearly co-chromatographed in the
three different thin layer systems tested, as well as in the high
performance liquid chromatography system with synthetic
UDP-sulfoquinovose. It was completely cleaved by nucleotide
pyrophosphatase, confirming a nucleotide structure. The resulting
S-labeled cleavage product co-chromatographed with
sulfoquinovose 1-phosphate. Further dephosphorylation using alkaline
phosphatase resulted in co-elution of the labeled cleavage product with
sulfoquinovose. These independent observations were all in agreement
with a UDP-sulfoquinovose structure for the compound accumulating in
MRD. In addition, the labeled portion of the molecule was incorporated
into a lipophilic compound co-chromatographing with sulfolipid
following the incubation with isolated spinach chloroplast membranes.
These membranes contain a sulfoquinovosyltransferase highly specific
for UDP-sulfoquinovose as compared to other nucleotide
sulfoquinovoses(7) . Taken together, these results provide
convincing evidence for a UDP-sulfoquinovose structure of the compound
accumulating in the sqdD insertion mutant MRD. More than 30
years ago, a sulfur-containing sugar nucleotide, presumably
UDP-sulfoquinovose, was observed by paper chromatography of Chlorella extracts(6) , a result which has not been
reproduced since.
To address the question whether sqdD encodes the sulfoquinovosyltransferase of R. sphaeroides we tried to express this gene in E. coli in a functional form using different strategies. Currently, all our attempts have failed. Although the accumulation of presumably UDP-sulfoquinovose in a sqdD-inactivated line supports the idea that the sqdD protein at least may be involved in some aspect of this transfer reaction, it remains to be seen whether the holoenzyme requires additional proteins or factors missing in E. coli.
The MRD mutant line provides a new tool for sulfolipid research. Since it apparently accumulates UDP-sulfoquinovose it can be utilized as a source for radiolabeled UDP-sulfoquinovose, particularly if the yield can be increased. Currently, the multistep chemical synthesis of UDP-sulfoquinovose (7) represents no feasible alternative to the biochemical preparation of labeled UDP-sulfoquinovose with high specific activity as required for biochemical studies.
The accumulation of UDP-sulfoquinovose in a sulfolipid-deficient mutant of R. sphaeroides and the incorporation of the labeled part of this molecule into sulfolipid by isolated spinach chloroplast membranes provide further experimental evidence for the hypothesis that the head group of sulfolipid is derived from UDP-sulfoquinovose in photosynthetic bacteria and higher plants. Whether the transfer reaction itself proceeds in the same way in photosynthetic bacteria and plants must await further experiments. The question for the pathway of sulfolipid biosynthesis can be reduced to the question for the biosynthesis of UDP-sulfoquinovose. Further analysis of the proteins encoded by the four known sulfolipid genes of R. sphaeroides, particularly in vitro reconstitution experiments, may provide the answer to this question in the future.
Dedicated to Professor Richard R. Schmidt on the occasion of his 60th birthday.