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J Bacteriol, January 1998, p. 100-106, Vol. 180, No. 1
Department of Biochemistry and Molecular
Biology,
Received 1 July 1997/Accepted 15 October 1997
The psd gene of Bacillus subtilis Marburg,
encoding phosphatidylserine decarboxylase, has been cloned and
sequenced. It encodes a polypeptide of 263 amino acid residues (deduced
molecular weight of 29,689) and is located just downstream of
pss, the structural gene for phosphatidylserine synthase
that catalyzes the preceding reaction in phosphatidylethanolamine
synthesis (M. Okada, H. Matsuzaki, I. Shibuya, and K. Matsumoto, J. Bacteriol. 176:7456-7461, 1994). Introduction of a plasmid
containing the psd gene into temperature-sensitive Escherichia coli psd-2 mutant cells allowed growth at
otherwise restrictive temperature. Phosphatidylserine was not detected
in the psd-2 mutant cells harboring the plasmid; it
accumulated in the mutant up to 29% of the total phospholipids without
the plasmid. An enzyme activity that catalyzes decarboxylation of
14C-labeled phosphatidylserine to form
phosphatidylethanolamine was detected in E. coli psd-2
cells harboring a Bacillus psd plasmid. E. coli
cells harboring the psd plasmid, the expression of which was under the control of the T7 Biosynthesis of membrane lipids in
gram-positive Bacillus species has not been studied
extensively, and it has been assumed to follow the standard pathways
established in Escherichia coli (7, 29, 39, 40).
As for the synthesis of phosphatidylethanolamine in
Bacillus species, two enzymes involved,
CDP-diacylglycerol-dependent phosphatidylserine synthase
and phosphatidylserine decarboxylase, are associated with
membranes (11, 12, 26, 40), whereas upon disruption of the
cell, the phosphatidylserine synthase of E. coli is mainly
associated with ribosomes, not with the membrane (31, 42)
because of its hydrophilic and basic nature (6). This
property of the E. coli synthase appears to be related to the regulation mechanism for phosphatidylethanolamine synthesis (30, 46). In Bacillus subtilis, with the
membrane-localized enzymes, the phosphatidylethanolamine synthesis
should be regulated in a very different way (7, 32);
however, no data on the biosynthetic pathway and regulation have been
available, despite the wealth of its genetic information.
In order to get pertinent information on the physiological roles of
phosphatidylethanolamine and the possible regulatory mechanism of its
synthesis in Bacilli, as well as for use as a heterologous gene probe for elucidation of the regulatory mechanism in E. coli, we have cloned and characterized the structural gene
pss for the phosphatidylserine synthase of
B. subtilis Marburg (38, 43). During the
course of cloning the pss gene, a reading frame showing a
homology with known phosphatidylserine decarboxylase sequences was
found downstream of the pss gene. This report describes the characterization of the B. subtilis gene for
phosphatidylserine decarboxylase, psd, and its translation
product. By constructing the strain bearing an interrupted chromosomal
allele of psd or pss, we further examined the
effect of phosphatidylethanolamine deprivation from the B. subtilis membrane.
Bacterial strains and plasmids.
B. subtilis
Marburg and E. coli K-12 strains and plasmids used in this
study are listed in Table 1. For
structures of plasmids, see Fig. 1. The reference B. subtilis strains for Southern hybridization mapping are described
in the following section.
Media and bacterial growth.
Luria-Bertani broth contained
1% tryptone (Difco, Detroit, Mich.), 0.5% yeast extract (Difco), and
1% NaCl. NBY medium (35) contained 0.8% nutrient broth
(Difco), 0.5% polypeptone (Dainihon Seiyaku, Tokyo), 0.2%
yeast extract, and 0.1% NaCl and was adjusted to pH 7.2 with NaOH. TY
broth, which contained 1% tryptone, 0.5% yeast extract, and 0.5%
NaCl; synthetic media CI and CII for development of competence
(2); and Penassay broth (Difco) were used for B. subtilis. When required, the following supplements were added to
the media (per liter): 50 mg of thymine, 5 mg of
thiamine-hydrochloride, 50 mg of ampicillin (Sigma), 20 mg of neomycin
(Wako Pure Chem.), and 50 mg of spectinomycin (Sigma). Growth of
bacteria was monitored by measuring turbidity with a Klett-Summerson
photoelectric colorimeter (no. 54 filter). For solid media, 1.5% agar
(Difco) was included.
DNA preparation and manipulations.
For construction,
isolation, and identification of recombinant DNA, the methods described
by Sambrook et al. (45) were used. Plasmid DNAs were
prepared from XL1-Blue cells harboring the respective plasmids by the
alkaline lysis method. Fragments of DNA were recovered from agarose
gels with a GENECLEAN II kit from BIO 101 (La Jolla, Calif.).
Restriction endonuclease digestion, filling of cohesive ends, and
ligation were performed with enzymes from Takara Shuzo (Kyoto), Nippon
Gene (Tokyo), and New England Biolabs (Beverly, Mass.) under the
conditions recommended by the suppliers. Competent cells of strains
OS2101 and XL1-Blue were prepared by the standard CaCl2
method.
DNA sequencing.
Subclones for DNA sequencing were obtained
by cloning the defined restriction fragments and overlapping deletion
fragments made by using exonuclease III and Sl nuclease into pWSK29
vector (51). Single-stranded DNA templates were prepared
from phage M13KO7-infected XL1-Blue cells harboring each of the
subclone plasmids (45). Chain termination reactions were
performed as described in the manual supplied by the United States
Biochemical Corporation (Cleveland, Ohio) with Sequenase version 2.0 DNA polymerase and a fluorescein-labeled 21-mer M13 universal primer
(Yuki Gosei Kogyo, Tokyo, Japan). Nucleotide mixtures containing
7-deaza-dGTP and 7-deaza-dATP (Pharmacia) were used to prevent
formation of intrastrand secondary structures. Reaction products were
analyzed with an automated laser fluorescent DNA sequencer, DSQ-1
(Shimadzu, Kyoto). To verify the sequence data, both strands were
sequenced. The DNA sequence and the deduced amino acid sequence were
examined with sequence analysis programs of GENETYX software (Software Development Co., Tokyo, Japan).
Analysis of plasmid-encoded protein.
Detection and molecular
weight determination of phosphatidylserine decarboxylase were performed
by the T7 RNA polymerase-promoter system (10). Strain
JM109(DE3) harboring pBM281 was grown in LB medium supplemented with
100 µg of ampicillin per ml, and at 70 Klett units (about 2 × 108 cells per ml),
isopropyl- Phosphatidylserine decarboxylase assay.
Cells grown to late
log phase for 6 h at 43°C in LB medium were collected and
suspended in a potassium phosphate buffer (100 mM [pH 7.4]) and
disrupted by sonication with a Branson sonifier followed by
fractionation into supernatant and crude membrane fractions by
centrifugation for 1 h at 100,000 × g after
removal of cell debris. The tightly packed pellet was suspended in 100 mM potassium phosphate buffer (pH 7.4) and used as the crude membrane fraction. The enzyme activities in the membrane and supernatant fractions were assayed essentially by the method of Hawrot and Kennedy
(15). The assay mixture (0.1 ml) contained 0.1 M potassium phosphate buffer (pH 7.0), 0.1% Triton X-100 (wt/vol), 0.2 mM L-phosphatidyl[U-14C]serine (800 dpm/nmol),
and the enzyme fraction (40 ng). After incubation at 30°C for 30 min,
the reaction was terminated by the addition of methanol containing 0.1 N HCl. Chloroform-soluble materials were extracted and dried, and the
lipids were then redissolved in chloroform-methanol (2:1 [vol/vol]),
applied to a silica gel plate (no. 60; Merck, Darmstadt, Germany), and
developed in chloroform-methanol-acetic acid (65:25:10
[vol/vol/vol]). The positions of the radioactive phospholipids were
determined and quantified with the bioimaging analyzer.
Lipid analysis.
Membrane lipids were labeled with 0.5 µCi
of [1-14C]acetic acid per ml (57.2 mCi/mmol [Amersham])
for at least six generations of cultivation of the mutant cells in
Penassay broth (5 ml) or cultivated in LB broth with no radioisotope
and then were harvested in the late exponential phase. Lipids were
extracted by the method of Bligh and Dyer (1), and halves of
the lipid fractions were separated by two-dimensional thin-layer
chromatography on silica gel (no. 60; Merck, Darmstadt), first
(x dimension) with chloroform-methanol-water (65:25:4
[vol/vol/vol]) and then (y dimension) with
chloroform-methanol-acetic acid (65:25:10 [vol/vol/vol]). Spots for
14C-labelled lipid were visualized and quantified with the
BAS 1000 bioimaging analyzer. Phospholipids were visualized by
uniformly spraying Dittmer-Lester reagent (8), and spots
were quantified with a high-speed thin-layer chromatography (TLC)
scanner (model CS-920; Shimadzu, Kyoto). Glycolipids were visualized
with a spray of orcinol-sulfuric acid mixture, and lipid species having
free amino groups were detected with a ninhydrin spray. Molar
percentages of each component were calculated.
Southern hybridization mapping of psd.
The
procedures for labelling of pBM102 (pss-psd) and pBM281
(psd) probes with digoxigenin (Boehringer Mannheim),
blotting, hybridization, and identification of hybridized restriction
fragments of reference genomes were described previously
(21). For the hybridization with NotI-digested
fragments, reference genome DNAs from the CU741, BEST4041, BEST4087,
and BEST4133 strains were used, and for the hybridization with
SfiI-digested fragments, reference genome DNAs from the
OA101, BEST3015, BEST3028, and BEST3055 strains (21) were
used.
Nucleotide sequence accession number.
The nucleotide
sequence data reported in this paper have appeared in the GSDB, DDBJ,
EMBL, and NCBI nucleotide databases under accession no. D38022.
Cloning and sequencing of the structural gene for
phosphatidylserine decarboxylase of B. subtilis.
During
the course of the cloning of a B. subtilis structural
gene for phosphatidylserine synthase, pss (38), a
reading frame which showed homology with phosphatidylserine
decarboxylases of E. coli, Saccharomyces
cerevisiae, and CHO-K1 cells was found 461 nucleotides downstream
of the coding frame of the pss gene (Fig.
1). We therefore sequenced the downstream
region to the end of the reading frame. The open reading frame,
starting at nucleotide 587 and extending up to the stop codon at 1,375 (Fig. 2), encoded a polypeptide of 263 amino acid residues with a deduced molecular weight of 29,689. Six base
pairs upstream of the initiation codon was a putative 5'-AGGG-3'
ribosome binding site. A potential promoter consensus sequence
(13) starting at nucleotide 1,157 was found and designated
as P3, and those with lower homology scores were also found downstream
of P3. Immediately upstream of P3, a possible
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Cloning, Sequencing, and Disruption of the
Bacillus subtilis psd Gene Coding for
Phosphatidylserine Decarboxylase
ABSTRACT
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Abstract
Introduction
Materials & Methods
Results & Discussion
References
10 promoter, produced proteins of 32 and 29 kDa upon induction. A pulse-labeling experiment suggested that
the 32-kDa protein is the primary translation product and is processed
into the 29-kDa protein. The psd gene, together with pss, was located by Southern hybridization to the 238- to
306-kb SfiI-NotI fragment of the chromosome. A
B. subtilis strain harboring an interrupted psd
allele, psd1::neo, was constructed.
The null psd mutant contained no phosphatidylethanolamine
and accumulated phosphatidylserine. It grew well without
supplementation of divalent cations which are essential for the
E. coli pssA null mutant lacking phosphatidylethanolamine.
In both the B. subtilis null pss and psd mutants, glucosyldiacylglycerol content increased two-
to fourfold. The results suggest that the lack of
phosphatidylethanolamine in the B. subtilis membrane may be
compensated for by the increases in the contents of
glucosyldiacylglycerols by an unknown mechanism.
INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results & Discussion
References
MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results & Discussion
References
TABLE 1.
Bacterial strains and plasmids used in this study
-D-thiogalactopyranoside (Takara Shuzo) was
added to a final concentration of 5 mM, and after 20 min of incubation,
this was followed by an addition of rifampin (Sigma, St. Louis, Mo.
[final concentration of 100 µg per ml]). After a further 12 min of
incubation, 50 µCi of L-[35S]methionine
(1,152 Ci/mmol [American Radiolabeled Chemicals, Inc., St. Louis,
Mo.]) was added, and the mixture was incubated for 10, 50, and 100 min. The cells were then collected and solubilized in a sample buffer
(3% sodium dodecyl sulfate, 5% mercaptoethanol) at 100°C for 3 min.
Labeled proteins were subjected to sodium dodecyl sulfate-12.5%
polyacrylamide gel electrophoresis, and they were visualized and
quantified with the BAS 1000 Mac bioimaging analyzer (Fuji Photo Film,
Tokyo, Japan).
RESULTS AND DISCUSSION
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Abstract
Introduction
Materials & Methods
Results & Discussion
References
-independent
terminator signal of pss was observed, and the reading frame
was also followed by a possible -independent terminator signal.
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FIG. 1.
Physical map of the B. subtilis pss and
psd gene loci and complementation of pssA1 and
psd-2 mutations. The physical map of the 4.6-kbp fragment of
B. subtilis chromosome cloned on pWSK29, designated
pBM201, is presented. The solid line in the map indicates the regions
sequenced. The horizontal arrows show the direction and extent of the
reading frames of the pss and psd genes. P1, P2,
and P3 indicate putative promoter sites. Horizontal bars indicate the
regions subcloned on the respective plasmids; their complementation
abilities are also listed (see Table 1 and Materials and Methods for
more details). Except for pBM22, the T7 10 promoter of pWSK29 was
placed on the left ends of the subcloned fragments. Complementation of
temperature-sensitive mutations of OS2101 (pssA1) and EH150
(psd) was examined at 42°C on NBY plates. A,
AvaII; E, EcoRI; H, HindIII; P,
PstI; M, MunI.
View larger version (64K):
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FIG. 2.
Nucleotide sequence of the B. subtilis
psd locus. Numbering begins at nucleotide 1,001 of the sequence
presented previously (38), and the newly sequenced region in
this work starts from nucleotide 422. Possible promoter P3 is
underlined. A putative ribosome binding site is double underlined, and
the start and stop codons are boxed. The dotted arrows indicate
inverted repeats of a possible -independent terminator. The
PstI site used for the disruption of psd is shown
by bold dashed lines. From nucleotide 106 to nucleotide 591 (overlapping with the end of pss and the start of
psd), an open reading frame predicting a polypeptide of 162 amino acid residues is possible. The deduced sequence has partial
similarity (31%) to the product of the E. coli dedA gene,
the function of which is presently unknown (GenBank accession no.
S53037).
Analysis of the B. subtilis psd gene product.
The deduced primary sequence of the product of B. subtilis
psd gene was compared with those of the phosphatidylserine
decarboxylases of E. coli (27), S. cerevisiae (5, 48), and CHO cells (24). The
B. subtilis product, a 263-amino-acid residue protein,
was the smallest among the known enzymes; the numbers of residues of
E. coli, S. cerevisiae, and CHO cells are
322, 500, and 370, respectively. The B. subtilis
sequence shared 27% identical amino acids with those of E. coli and CHO cells and 29% identical residues with that of
S. cerevisiae. When the related amino acid substitutes were
included, the similarity values were 43, 40, and 41% with the products
from E. coli, CHO cells, and S. cerevisiae,
respectively. There were several conserved segments among these four
products. The similarity at the carboxyl-terminal end of the
B. subtilis product included the conserved segment
GX3GXFX2GST(V/I)(V/I)X2F, corresponding to the posttranslational processing site in E. coli decarboxylase, which is cleaved into the subunit of
28,579 Da and the small pyruvoyl-containing 
subunit of 7,332 Da
(27). Putative processing of the product of B. subtilis psd (29,689 Da) would result in a pyruvoyl-containing
subunit of 3,700 Da and a 25,973-Da subunit. Hydropathy analysis
(25) of the sequence indicated that the conserved segment
for the processing is hydrophobic, with the remainder primarily
hydrophilic (data not shown), as in the cases of the E. coli (27), CHO cell, and S. cerevisiae (5) counterparts.
-D-thiogalactopyranoside indicated that
E. coli JM109(DE3) cells harboring pBM281 produced a large amount of 32- and 29-kDa proteins (Fig.
3). Prolonged labeling led to a decrease
in the proportion of 32-kDa protein with a concomitant increase in
29-kDa protein; when the labeling was for 10 min, the proportion of 29- versus 32-kDa protein was 2.2, and with 100 min of labeling, the ratio
was 3.5. After 4 h of incubation, a ratio of 10 was observed
(Coomassie brilliant blue staining [data not shown]). The molecular
weights of these induced proteins coincided with the predicted
molecular weights for the primary product and its processed large
subunit, respectively. Therefore, the results suggested that the
translated product of B. subtilis psd was processed in
E. coli cells into a pyruvoyl-containing small subunit
and the remaining large subunit, as in the case of E. coli (27). The majority of the overproduced primary
product was observed in the membrane fraction, and a large amount of
the processed 29-kDA protein was observed in the 100,000 × g supernatant fraction of the sonically disrupted cells
(data not shown), as in the case of the E. coli
counterpart (49). This loose association of the overproduced
decarboxylase with the E. coli membrane might be
correlated with its primarily hydrophilic nature (Fig.
4).
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Expression of the B. subtilis psd gene in E. coli. When strain EH150 (psd-2) was kept at 42°C for 6 h, phosphatidylserine accumulated to 29% of the total phospholipids, with the concomitant decrease in the phosphatidylethanolamine level to 51%, as previously described (15). After introduction of pBM281 containing the Bacillus psd gene, the cells of EH150 regained normal growth and recovered the phosphatidylethanolamine content to the wild-type level of 73%, with the decrease of phosphatidylserine to below the detectable limit. The phosphatidylserine decarboxylase activity of EH150 cells harboring pBM281 was then examined. The cells of strain EH150 harboring pBM281 were cultivated at 43°C for 6 h to inactivate the thermosensitive host phosphatidylserine decarboxylase (16, 17). The membrane fraction of the cells harboring pBM281 showed higher phosphatidylserine decarboxylase activity (8.5 nmol/min/mg) than that from cells without the plasmid (0.2 nmol/min/mg). This value was about two times higher than that of the wild-type E. coli cells, and the activity in the soluble fraction was 7% of that of the membrane fraction. This result indicated that the cloned DNA fragment really contained the structural gene for phosphatidylserine decarboxylase of B. subtilis and that the enzyme activity of the B. subtilis decarboxylase was detectable under the conditions described for the E. coli counterpart (9).
It should be noted that the amino acid sequences of the four decarboxylases are highly homologous and that the genes of S. cerevisiae (5) and B. subtilis were able to complement the temperature-sensitive defect of the E. coli (psd-2) mutant. Therefore, the primary product of B. subtilis psd probably undergoes a similar processing to become an active phosphatidylserine decarboxylase like the E. coli product, the processing to the active form is expected to be an autocatalytic process (27, 28), as for the prohistidine decarboxylase (50).Chromosomal locus of pss-psd genes. To determine the locus of psd gene on the SfiI and NotI restriction map of the B. subtilis chromosome (21), Southern hybridization analysis was conducted. The procedures for blotting and hybridization with digoxigenin-labeled probe and identification of hybridized restriction fragment were performed as described previously (21). Plasmid pBM102, which contained both the pss and psd genes, hybridized with the 22N fragment of the NotI digest. In the case of the SfiI digest, the BS fragment hybridized with the probe. Experiments with gels of different running conditions and with the probe (pBM281) containing the psd gene alone were consistent with this assignment. Accordingly, both pss and psd genes were unambiguously localized within the overlapping segment (68 kbp) of the 22N and BS fragments: the 238- to 306-kb region on the 4,188-kb physical map (19).
A B. subtilis mutant that showed temperature-sensitive net synthesis of phosphatidylethanolamine was reported (29). Because the in vitro phosphatidylethanolamine synthesis with the membrane of this mutant was no more temperature sensitive than the wild-type preparation, the mutation was considered to affect the synthesis indirectly. The mutation was linked by transformation to aroD, which is located at around 2,600 kb on the physical map (19), and, therefore, it is not in the pss or psd gene region. In E. coli, pss and psd genes are located at different loci on the chromosome; pss and psd were mapped at 49 min (37, 41) and at 83 min (15), respectively. The result of the Southern mapping with the probes of pss and psd genes showing no sign of multiple signals of hybridization indicated an unequivocal close linkage of the two genes, in accordance with the data from nucleotide sequencing. The close linkage may suggest an organized control of phosphatidylserine synthase and decarboxylase, constituting an operon in which the both genes are transcribed into a single mRNA. Although the presence of a putative-independent terminator signal and a
promoter sequence between the two genes (Fig. 2) might oppose an
organization in an operon, there are many examples of complex operons
having sets of promoters and terminators for dynamic and fine control.
In E. coli, all phospholipid enzymes appear to be
synthesized constitutively, and lipid synthesis is considered to be
regulated by far more complex mechanisms than those presently known
(46). Studies of transcription of B. subtilis cells to define the nature of the linkage of the
pss and psd genes will answer this question.
Disruption of the psd gene and its effect on lipid composition. To construct a strain harboring an interrupted chromosomal allele of psd, we first prepared a B. subtilis strain, 160, transformed with a covering plasmid, pLUCK100, which has a spac promoter-controllable pss-psd gene combination. This strain, 160/pLUCK100, and the parental strain, 160, were then transformed with the DNA fragment of the psd gene region from plasmid pYH01, in which the gene was interrupted with the neo (20) gene at its unique PstI site (Fig. 2). From both recipient strains, neomycin-resistant transformants appeared at frequencies comparable to those with chromosomal markers. Unexpectedly, all of the neomycin-resistant transformants of strain 160 without the covering plasmid required no divalent cation, which is essential for E. coli pssA null mutants (6, 44). Analysis of the chromosomal DNA of the neomycin-resistant transformants by the PCR method using primers for the psd gene produced an amplified DNA product corresponding to the size of the neo-interrupted psd gene, and no product corresponding to the intact psd gene was detected, indicating that the chromosomal psd gene was correctly disrupted by homologous recombination with the insertion of neo as constructed on plasmid pYH01. The resistant strain, designated as SDB01 (psd1::neo), showed the same growth rates as the wild-type strain in LB, TY, and NBY media without supplementation with divalent cations and synthetic medium CI. In all cases, no difference in final cell densities was observed.
The composition of lipids from the psd disruptant (SDB01) was compared with that of the wild type after labelling with [1-14C]acetic acid followed by two-dimensional TLC on a silica gel plate (Fig. 4). No significant radiolabel was detected in the region corresponding to phosphatidylethanolamine, and a concomitant accumulation of phosphatidylserine (corresponding to 40% of the amount of phosphatidylethanolamine of the wild type) was observed (Table 2). The absence of phosphatidylethanolamine in B. subtilis cells, therefore, does not have any adverse effect on their growth, which is quite different from the case of E. coli phosphatidylethanolamine-deficient mutants, which require divalent cations (6, 44). Phosphatidylethanolamine, therefore, seems dispensable for the growth of B. subtilis cells. This is compatible with the previous report that phosphatidylethanolamine was practically absent from the membrane of B. subtilis cells that had been cultured in a chemostat under Mg2+-limited conditions at pH 8.0 (33). Phosphatidylethanolamine is a major component of the membrane of E. coli, and the different phenotypes of the disruptants of E. coli and B. subtilis could be due to the difference in the membrane. The B. subtilis null pss mutant SDB02 (
pss10::spc) constructed in
the same way lacked both phosphatidylethanolamine and
phosphatidylserine in the membrane, yet grew normally without further
addition of divalent cations. This indicated that the accumulated
phosphatidylserine in place of phosphatidylethanolamine
in the psd disruptant had little effect for the growth of
the disruptant. In the psd-disrupted strain, apparent
increases in monoglucosyldiacylglycerol (2-fold),
diglucosyldiacylglycerol (3.1-fold), and triglucosyldiacylglycerol
(1.6-fold) were observed. Similar increases in
diglucosyldiacylglycerol (3.1-fold) and triglucosyldiacylglycerol (4.1-fold), but not in monoglucosyldiacylglycerol, were observed in the lipid fraction of the pss null mutant strain (Table
2). This is consistent with the previous observation that the membrane levels of phosphatidylethanolamine and diglucosyldiacylglycerol changed inversely in response to changes in the culture conditions (34). The need for phosphatidylethanolamine in the
B. subtilis membrane may be satisfied by increases in
the contents of glucosyldiacylglycerols. A careful inspection of the
proportion of the lipids in Table 2 revealed that the sums of
glucosyldiacylglycerols plus phosphatidylethanolamine (including phosphatidylserine) of the three strains were quite equal;
wild-type, psd null, and pss null mutant lipids
were 78.8, 80.1, and 79.7% of the total lipids, respectively. In
B. subtilis, glucosyldiacylglycerols are believed to be
synthesized by transfer of glucose from UDP-glucose to diacylglycerol,
which is produced by dephosphorylation from phosphatidic acid
according to the reactions detected in other organisms
(7). Therefore, it is possible that the regulated cellular
level (3) of CDP-diacylglycerol results in the accumulation
of phosphatidic acid in the absence of phosphatidylserine synthesis,
which in turn accelerates the synthesis of diacylglycerol, the putative
substrate for glucosyldiacylglycerols. However, the reasons for the
apparently specific increase of glucosyldiacylglycerols but not of
phosphatidylglycerol derivatives in the two null mutants, as well
as for the decrease of monoglucosyldiacylglycerol in the pss
null mutant, are unknown.
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ACKNOWLEDGMENTS |
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We thank W. Dowhan for providing us with E. coli EH150 and S. Kushner and P. Stragier for vector plasmids. Thanks are also due to S. Fuchizawa and K. Komori for construction of B. subtilis SDB02.
This work was supported in part by grants-in-aid for scientific research from the Ministry of Education, Science, and Culture of Japan.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, Faculty of Science, Saitama University, 255 Shimo-ohkubo, Urawa, Saitama 338, Japan. Phone: 81 (048)858-3406. Fax: 81(048)858-3698. E-mail. koumatsu{at}sacs.sv.saitama-u.ac.jp.
Present address: National Institute of Genetics, Mishima, Shizuoka
411, Japan.
Present address: Immunobiological Laboratories Co., Ltd.,
Fujioka, Gunma 375, Japan.
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Abstract
Introduction
Materials & Methods
Results & Discussion
References
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