Bacillus subtilis spore coat protein LipC is a phospholipase B

Article abstract of DOI:10.1271/bbb.90391

In Bacillus subtilis, the germination-related lipase LipC is located in the spore coat, and mutant spores are defective in L-alanine-stimulated germination. To determine the physiological role of LipC, the recombinant LipC expressed in Escherichia coli wa

Full text of DOI:10.1271/bbb.90391

Biosci. Biotechnol. Biochem., 74 (1), 24–30, 2010
Bacillus subtilis Spore Coat Protein LipC Is a Phospholipase B
1
1
1
2
1
Atsushi MASAYAMA, Shiro KATO, Takuya TERASHIMA, Anne MØLGAARD, Hisashi HEMMI,
1
3;y
Tohru YOSHIMURA, and Ryuichi MORIYAMA
1
Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University,
Chikusa, Nagoya, Aichi 464-8601, Japan
2
Department of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen Ø, Denmark
Department of Food and Nutritional Sciences, College of Bioscience and Biotechnology, Chubu University,
3
1
200 Matsumoto-cho, Kasugai, Aichi 487-8501, Japan
Received June 3, 2009; Accepted October 8, 2009; Online Publication, January 7, 2010
[doi:10.1271/bbb.90391]
In Bacillus subtilis, the germination-related lipase
The Bacillus subtilis genome-sequencing project
6
)
LipC is located in the spore coat, and mutant spores are
defective in L-alanine-stimulated germination. To de-
termine the physiological role of LipC, the recombinant
LipC expressed in Escherichia coli was purified and
characterized. The enzyme hydrolyzes p-nitrophenyl
ester substrates with various acyl-chain lengths. Thin-
layer chromatography and gas chromatography-mass
spectrometry analysis indicated that LipC cleaves the
fatty acids at the sn-1 and sn-2 positions of phospho-
lipids as phospholipase B, and that the enzyme shows no
selectivity for the polar head groups of lipid molecules.
When the amounts of free fatty acids in dormant wild-
type and lipC mutant (YCSKd) spores were measured,
the amount of free fatty acids in the YCSKd spores was
about 35% less than in the wild-type spores. These
results suggest the possibility that Bacillus subtilis LipC
plays an important role in the degradation of the outer
spore membrane during sporulation.
revealed about 4,100 protein-encoding genes, and
B. subtilis has been used as a model organism to
understand metabolism, cell division, and macromolec-
ular synthesis, mainly by genetic approaches. Among
the many types of cellular development and differ-
entiation, sporulation is one of the best understood. The
temporal and spatial control of gene expression, inter-
cellular communication, and various aspects of cell
morphogenesis during sporulation have been studied in
7
)
detail, but the mechanism of germination is still
unclear. Many gene products involved in germination
have been identified and characterized: receptor-like
proteins (defined as Ger family proteins), spore coat
8
–10)
proteins, cortex-lytic enzymes, and so on.
Strains
with mutations in these genes are blocked at various
stages of spore germination. In a previous study, we
found that LipC, a protein component of the spore coat,
is a member of a GDSL family of lipolytic enzymes and
that lipC mutant spores are defective in L-alanine-
1
1)
Key words: bacterial spores; Bacillus subtilis; spore
germination; sporulation; phospholipase B
stimulated spore germination. LipC is expressed in the
mother cell compartment at the late stage of sporulation
and is localized around forespores in a CotE-, SafA-, and
SpoIVD-dependent manner, suggesting that LipC is
Members of the gram-positive Bacillus and Clostri-
dium genera produce metabolically dormant endospores
in response to unfavorable conditions. Bacterial endo-
spores are resistant to severe physical and chemical
conditions, including heat, ultraviolet, lytic enzymes,
and solvents. These properties are attributed mainly to
the unique structure of the spore coat and cortex, as
1
1)
probably targeted around the outer spore membrane.
Kawai et al. found that spore membranes of B. subtilis
Marburg had significantly higher cardiolipin contents
1
2)
than the membranes of exponentially growing cells,
but lipid biosynthesis and metabolism in spore formation
and/or germination are poorly understood.
1
–4)
well as to the physical state of the spore core.
In
In this study, we purified recombinant LipC expressed
in E. coli and biochemically characterized the enzy-
matic properties of LipC. In addition, analysis of the free
fatty acid contents in dormant spores suggested that
LipC hydrolyzes phospholipids during sporulation as a
phopholipase B.
addition, bacterial spores contain two lipid membrane
systems: the inner spore membrane, which surrounds
the spore core, and the outer spore membrane, which
1
–4)
encases the cortex.
Despite these resistance proper-
ties, spore germination is triggered by specific germi-
nants and leads to an irreversible loss of spore
dormancy, followed by outgrowth and the formation
of a vegetative cell. Spore germination is controlled
by the sequential activation of a set of pre-exist-
ing germination-related enzymes but not by protein
synthesis,3 and is clearly governed by a new class
of sensory and transducer systems that are distinct
from the numerous response mechanisms identified
to date.5
Materials and Methods
Materials. Restriction enzymes were purchased from Takara Shuzo
Kyoto, Japan). The p-nitrophenyl (pNP) esters were from Nacalai
(
Tesque (Kyoto, Japan), Sigma (St. Louis, MO), and Wako Pure
,4)
Chemicals (Osaka, Japan). His Bindꢀ Resin was from Novagen
(Madison, WI). Phospholipids were from NOF (Tokyo) and Sigma (St.
Louis, MO).
)
y
To whom correspondence should be addressed. Tel/Fax: +81-568-51-6084; E-mail: moriyama@isc.chubu.ac.jp

Phospholipase of B. subtilis Spores
25
Table 1. Bacterial Strains and Plasmids Used in This Study
sonication. After the cell debris was removed by centrifugation, the
supernatant was applied to a column of His Bindꢀ Resin, which had
been equilibrated with buffer A. After the column was washed with
Source or
reference
Strains
Genotype and/or description
2
0 mM Tris–HCl buffer (pH 7.9) containing 0.5 M NaCl and 20 mM
imidazole, the enzyme was eluted with 20 mM Tris–HCl buffer
pH 7.9) containing 0.5 M NaCl and 0.5 M imidazole. The active
B. subtilis
(
Bacillus
Genetic
Stock
Center
11
1
68
trpC2
fractions were dialyzed against 20 mM Tris–HCl buffer (pH 7.9)
containing 10% v/v glycerol.
Enzyme and protein assays. Enzyme activity was measured with
pNP esters with fatty-acid chain lengths of C4–C18. Stock solutions
YCSKd
E. coli
JM109
trpC2 ycsk (lipC)::pMutin3
(
20 mM) of pNP esters were prepared by dissolving the substrates in
relA supE44 endA1 hsdR17 gyrA96 11
0
acetonitrile. The standard assay mixture (1.0 ml) contained 0.5 mM
pNP ester, 50 mM Tris–HCl buffer (pH 8.5), 150 mM NaCl, 0.2%
Triton X-100, 2.5% acetonitrile (final concentrations), and the enzyme.
mcrA mcrB + thiꢁ(lac-proAB)/F
traD36 proAB + lacIq lacZꢁM15)
(
ꢁ
ꢁ
ꢁ
BL21 (DE3)
F
gal dcm (DE3)
ompT hsdSB (rB mB )
Novagen
The reaction mixture without the enzyme was preincubated for 3 min at
ꢀ
3
7 C. The reaction was initiated by the addition of the enzyme and
Plasmids
terminated after 30 min by the addition of 2.0 ml of acetone. After the
addition of 1.0 ml of 2 M Tris–HCl buffer (pH 8.5), the absorbance was
measured at 400 nm. One unit of enzyme activity was defined as the
formation of 1 mmol p-nitrophenol per min. An extinction coefficient,
pET22b(+)
pET-LipC-His
pET-LipC(S11A)- lipC (S11A) bla
bla
Novagen
This study
This study
lipC bla
His
ꢁ1
ꢁ1
1
4,500 M cm of p-nitrophenol at 400 nm, was used in unit
calculations. The protein concentration was determined by the method
of Bradford15) with bovine serum albumin as the standard.
Bacterial strains and plasmids. The bacterial strains and plasmids
used in this study are listed in Table 1. Escherichia coli BL21(DE3)
was used as host for expression of the B. subtilis lipC gene. E. coli
Thermal stability and pH activity profiles. In the thermal stability
studies, the enzyme was incubated in 50 mM sodium phosphate buffer
ꢀ
transformants were grown at 37 C in Luria-Bertani (LB) broth
containing 100 mg/ml of ampicillin (final concentration).
ꢀ
(
pH 7.5) at 4, 20, 30, 40, 50, 55, 60, and 70 C. At 30-min intervals, the
ꢀ
remaining activity was assayed at 37 C using pNP-myristate as the
substrate. The effect of pH on enzyme activity was examined by
standard assay using pNP-myristate as the substrate, except that the
Spore preparation. B. subtilis spores were prepared as described
previously.13) The spores were harvested and purified by washing with
sterilized water until the cell debris and vegetative cells could not be
observed by microscopy. The purity was confirmed by phase-contrast
microscopy. More than 98% of the spores prepared were refractive,
and almost no dark or gray spores were visible by phase-contrast
microscopy.
ꢀ
reaction mixture (without the substrate) was preincubated at 25 C for
1
0 min. The optimal pH for enzyme activity was determined by
assaying the enzyme at 37 ꢀC in a pH range from 4.0 to 10.0 (50 mM
sodium citrate, pH 4.0 to 6.5; 50 mM sodium phosphate, pH 6.0 to 8.0;
5
0 mM Tris–HCl, pH 7.5 to 9.0; 50 mM glycine–NaOH, pH 9.0 to 10.0).
Effects of various reagents on enzyme activity. The enzyme was
preincubated at 25 ꢀC for 10 min in a buffer containing 50 mM Tris–
Cloning and expression of the B. subtilis lipC gene in E. coli. The
B. subtilis lipC gene was amplified by PCR using B. subtilis
chromosomal DNA as the template with the following primers:
HCl (pH 8.5), 150 mM NaCl, 0.2% Triton X-100, and one or two of the
following additives: phenylmethylsulphonyl fluoride (PMSF), EDTA,
DTT, CaCl2, MgCl2, MnCl2, ZnCl2, FeCl2, CuCl2, HgCl2, L-alanine,
D-alanine, dipicolinate (DPA), and calcium dipicolinate (Ca-DPA).
The additives were incubated with the enzyme at a final concentration
0
0
exYCSK1 (5 -TTTTGAATTCATATGGTGCTTCGATATACAGC-3 ,
0
NdeI site underlined.) and exYCSK3 (5 -ATTTGAATTCTCGAG-
0
TGAAACTGATAATTCTTTATAGCC-3 , XhoI site underlined.). The
resulting PCR product was digested with NdeI and XhoI and cloned
between the same sites of pET22b(+) (Novagen), yielding plasmid
pET-LipC-His. E. coli BL21(DE3) cells transformed with pET-LipC-
His were cultivated in LB medium containing ampicillin. Expression
of lipC was induced by the addition of 1.0 mM isopropyl ꢀ–D-
thiogalactopyranoside (IPTG) when the tubidity of culture at 620 nm
of 1 mM or 5 mM. The effects of various reagents on enzyme activity
were assayed at 37 ꢀC using pNP-myristate as the substrate. The
activity of the enzyme without any additives was taken to be 100%.
Phospholipase assays and gas chromatography-mass spectrometry
GC-MS) analysis. Phospholipids were suspended in 50 mM Tris–HCl
pH 8.5) buffer containing 0.15 M NaCl and 0.1% Triton X-100 and
(
ꢀ
reached 0.5. After further cultivation for 3 h at 37 C, the cells were
ꢀ
(
harvested and stored at ꢁ20 C until they were used in enzyme
dispersed in a bath sonicator until the solution cleared. Phospholipids
were mixed with the enzyme in 1.0 ml of buffer solution, and the
purification.
ꢀ
mixture was incubated overnight at 37 C with shaking. After
Site-directed mutagenesis. Site-directed mutagenesis of the cloned
lipC gene was performed by the strategy of overlap extension using
PCR, as described by Higuchi et al.14) The lipC gene was amplified by
PCR using pET-LipC-His as template with the following primers:
extraction with 3.0 ml of a chloroform-methanol solution (4:1), the
chloroform layer was dried under nitrogen gas. The products were
resuspended in 25 ml of a chloroform-methanol solution (2:1) and
spotted on a Silica Gel 60 plate (Whatman, Springfield), along with
100 mg of linoleic acid and 100 mg of phosphatidylcholine (PC). Using
0
exYCSK1, exYCSK3, YCSK(S11A)-FW (5 -GGGCGATGCATTGA-
0
0
16)
CGACA-3 ), and YCSK(S11A)-RV (5 -TGTCGTCAATGCATCG-
0
the solvent system of Matsuzawa and Hostetler, we developed the
CCC-3 ). The first round of PCR was carried out with primers exYCSK1
plate to a height of 8.5 cm from the origin in a chloroform-methanol-
water solution (65:35:5), allowed the plate to dry, and developed it to
15 cm from the origin in a heptane-diethylether-formic acid solution
(90:60:4). Sulfuric acid (50%) was used to visualize phospholipids and
their digested products.
and YCSK(S11A)-RV or primers exYCSK3 and YCSK(S11A)-FW.
The reaction products from the two PCRs were diluted and used as
templates in a second round of PCR. The second PCR was performed
using exYCSK1 and exYCSK3 as the primer pair. The resulting PCR
product was digested with NdeI and XhoI and cloned between the same
sites of pET22b(+), yielding plasmid pET-LipC(S11A)-His. The
presence of the mutation and the fidelity of mutagenesis was confirmed
by sequencing.
In GC-MS analysis, 1-palmitoyl-2-myristoyl-sn-glycero-3-phospha-
tidylcholine was used as substrate. The substrate was mixed with the
enzyme as described above. In methylation, the reaction products were
incubated with 10 vol. of 1.25 M hydrogen chloride methanol solution
ꢀ
(
Fluka, Buchs) at 90 C for 2 h. After the products were extracted with
Purification of LipC. The harvested transformant cells were
suspended in 10 ml of 20 mM Tris–HCl buffer (pH 7.9) containing
hexane, the methylesters were subjected to GC-MS analysis. This was
performed with a Hewlett-Packard 6890 gas chromatograph interfaced
with an MStation JMS-700 mass spectrometry system (JEOL, Tokyo).
0.5 M NaCl and 5 mM imidazole (buffer A) and disrupted by ultra-

26
A. MASAYAMA et al.
M
1
2
3
0.5
0.4
A
kDa
9
6
7.4
6.2
0
0
.3
.2
4
5.0
31.0
0
.1
0
2
1.5
4.4
1
4
6
10 14 16 18
p-Nitrophenyl acyl-chain length
6
7.0 14.2
B
(kDa)
1
48 45.0
Fig. 2. Substrate Specificity of LipC towards p-Nitrophenyl Esters of
Various Acyl-Chain Lengths.
Activities were determined by spectrophotometric assay. The data
are averages for three independent experiments.
Vo
Vt
that estimated by SDS–PAGE. When the purified LipC
was subjected to gel filtration chromatography on
Superdex 200 HR 10/30, it eluted at an elution volume
between ovalbumin (45.0 kDa) and cytochrome c (14.2
kDa) (Fig. 1B). This suggests that LipC is a monomer.
Substrate specificity of LipC
Our previous work indicated that LipC is a lipolytic
1
1)
0
5
10
15
20
25
enzyme.
To determine the substrate specificity of
LipC, we tested the ability of the enzyme to hydrolyze
triglycerides and p-nitrophenyl esters as substrates. The
recombinant enzyme exhibited lipolytic activity towards
p-nitrophenyl esters, while no activity was observed
towards tributyrin (C4), tricaprilin (C8), or triolein
Elution volume (ml)
Fig. 1. Purification of Recombinant LipC.
A, SDS–PAGE of the recombinant LipC expressed in E. coli.
Lane 1, proteins from whole cells of E. coli BL21(DE3) harboring
pET22b(+); lane 2, proteins from whole cells of E. coli BL21(DE3)
harboring pET-LipC-His; lane 3, purified LipC. B, Gel filtration
analysis of the enzyme. The purified recombinant LipC was
subjected to gel filtration chromatography on Superdex 200 HR
(C14) (data not shown). When the substrate specificity
of LipC was examined using p-nitrophenyl esters of
fatty acids of various acyl-chain lengths, the enzyme
showed the highest activity towards pNP-myristate as
the substrate (0.46 units/mg) (Fig. 2). The lipolytic
activity of LipC decreased with substrates of shorter
and longer chain lengths.
1
0/30.
A J&W DBꢀ-1 capillary column (30 m ꢂ 0:25 mm, d.f. ¼ 0:25 mm)
ꢀ
was used for the GC. Samples were injected at 80 C, and the
ꢀ
temperature was increased at a rate of 12 C/min. Electron impact-MS
was performed at 70 eV with a mass range from m=z 50–500 and a
cycle time of 1 s in the positive ion mode.
Effects of temperature, pH, and various reagents on
enzyme activity
After the enzyme was incubated at temperatures
Extraction and measurement of free fatty acids from B. subtilis
dormant spores. The purified B. subtilis spores were disrupted in
ꢀ
ꢀ
between 4 and 70 C for 30 min, the residual activity of
LipC was measured. The stability of the enzyme
decreased sharply, less than 20% activity remaining
ꢀ
sterilized water at 4 C with a bead beater, and the suspensions were
ꢀ
mixed with a chloroform-methanol-water solution (1:2:0.8) at 4 C for
1
h. After this, free fatty acids (FFAs) were extracted with a
ꢀ
when the temperature was over 60 C, as shown in
Fig. 3A. The effect of pH on enzyme activity was
chloroform-methanol-water solution (1:1:1). FFA concentrations were
measured using free fatty acids, half-micro test (Roche Diagnostics,
Basel) according to the manufacturer’s instructions. The data were
obtained from at least three independent experiments.
ꢀ
measured at 37 C between pH 4.0 and 10.0. The pH
profile indicated that the optimum pH was 8.5 (Fig. 3B).
The effects of various reagents on enzyme activity was
measured as described in ‘‘Materials and Methods.’’ As
Results
2
þ
2þ
2
shown in Table 2, Fe and Cu significantly activated
þ
Overexpression of lipC and purification of the lipC
gene product
LipC, whereas the addition of Ca inhibited activity to
40%. To determine whether the active site of LipC is a
serine residue, activity was measured in the presence of
serine-specific inhibitor PMSF. Enzyme activity was not
significantly inhibited by PMSF. Other serine hydrolases
To determine the lipolytic activity of LipC, we cloned
and overexpressed the lipC gene in E. coli. The over-
produced recombinant LipC with a C-terminal hexa-
histidine-tag was purified by Ni-chelating column
chromatography, as described in ‘‘Materials and Meth-
ods’’ (Fig. 1A). The molecular weight of 24,669,
deduced from the amino acid sequence with the
histidine-tag (LEHHHHHH), was in agreement with
1
7–19)
have shown similar resistance to such inhibitors.
Additionally, to determine the relationship between
LipC activation and general germinants, we assayed
enzyme activity in the presence of L-alanine, D-alanine,
DPA, and Ca-DPA chelate. The effects of L-alanine and

Phospholipase of B. subtilis Spores
27
1
1
25
00
PC
PE
PA
A
1
2
3
4
5
6
7
8
9
10 11 12 13 14
2
nd
FFA
products)
S.F.
(
75
5
2
0
5
1
st
S.F.
PA
PE
PC
*
0
0
20
40
60
80
Temperature (°C)
Fig. 4. Thin-Layer Chromatography Analysis of LipC Phospholipase
Activity.
Lane 1, linoleic acid (free fatty acid; FFA); lane 2, reaction buffer
1
25
B
100
(
50 mM Tris–HCl pH 8.5 buffer containing 0.15 M NaCl and 0.1%
Triton X-100); lanes 3 to 6, 7 to 10, and 11 to 14, PC, PE, and PA
respectively as substrates; lanes 3, 7, and 11, undigested phospho-
lipids; lanes 4, 8, and 12, reaction buffer containing undigested
phospholipids; lanes 5, 9, and 13, reaction products generated by
LipC (wild type); lanes 6, 10, and 14, reaction products generated by
the LipC mutant (S11A). The asterisk indicates the position of
Triton X-100.
7
5
5
2
0
5
0
D-alanine on enzyme activity were subtle, whereas DPA
and Ca-DPA decreased it. These results suggest that
LipC is not activated directly by germinants during
spore germination.
2
4
6
8
10
12
pH
Fig. 3. Effects of Temperature (A) and pH (B) on LipC Activity
towards the Substrate pNP-Myristate.
A, Thermal stability was carried out by incubating the enzyme in
a 50 mM sodium phosphate buffer (pH 7.5) for 30 min at various
temperatures before the remaining activity was assayed. B,
LipC has phospholipase B activity
To determine the physiological role of LipC, we
studied the ability of LipC to cleave phospholipids.
After the enzyme was incubated with phospholipids, as
described in ‘‘Materials and Methods,’’ thin-layer chro-
matography (TLC) was used to analyze the digested
products of phosphatidylcholine (PC), phosphatidyle-
thanolamine (PE), and phosphatidic acid (PA). TLC
analysis indicated that the digestion of phospholipids by
LipC released FFAs (lanes 5, 9, and 13 in Fig. 4),
suggesting that the enzymatic activity of LipC is
phospholipase A or B. On the other hand, the S11A
variant did not release FFAs from all phospholipids
Optimum pH profile was determined by incubating the enzyme at
ꢀ
2
(
5
5 C for 10 min at various pHs: 50 mM sodium citrate, pH 4.0 to 6.5
squares); 50 mM sodium phosphate, pH 6.0 to 8.0 (diamonds);
0 mM Tris–HCl, pH 7.5 to 9.0 (circles); 50 mM glycine–NaOH,
pH 9.0 to 10.0 (triangles). The data obtained from three independent
experiments are shown.
Table 2. Effects of Various Reagents on LipC Activity
Relative activity
%)a
Compound
Final concentration
(
(
lanes 6, 10, and 14 in Fig. 4), suggesting that part of the
None
100
40
putative catalytic triad in LipC, Ser11, is a catalytic
residue. To further investigate the enzymatic specificity
of LipC, the digested products of phospholipids were
methylated and subjected to GC-MS analysis. As shown
in Fig. 5A, two main peaks appeared, with retention
times of 11.74 min and 13.47 min. The estimated
molecular masses of peak 1 and peak 2 were 242 and
270 respectively. These values were identical to the
exact molecular weights of methyl myristate and methyl
palmitate (Fig. 5B and C). Thus LipC cleaved both ester
linkages of the sn-1 and sn-2 positions of phospholipids,
suggesting that LipC has phospholipase B activity.
CaCl2
MgCl2
MnCl2
ZnCl2
FeCl2
CuCl2
HgCl2
1 mM
1 mM
1 mM
1 mM
1 mM
1 mM
1 mM
98
67
84
124
127
61
PMSF
EDTA
DTT
1 mM
5 mM
5 mM
72
81
78
L-Alanine
D-Alanine
DPA
1 mM
1 mM
1 mM
1 mM
93
94
82
71
Physiological role of LipC in B. subtilis spores
In a previous study, we noted that the integrity of
the outer spore membrane in sporulating cells was
doubtful. To determine whether LipC hydrolyzes the
phospholipid bilayer during sporulation, we measured
the FFA content in dormant spores. Extracts from
dormant spores were assayed using free fatty acids by
Ca-DPA
aThe assay was performed as described in ‘‘Materials and Methods.’’ The
activity of the enzyme in the absence of additives was taken to be 100%. The
average values obtained from three independent experiments are shown.
2
0)

28
A. MASAYAMA et al.
(
2)
A
B
C
1
00
(1)
5
0
0
0
5
10
15
Retention time (min)
7
4 87
1
00
5
0
0
1
29
43
1
99
2
42
1
185
0
100
200
300
400
500
m/z
7
4 87
100
5
0
0
143
29
2
27 270
1
185
0
100
200
300
400
500
m/z
Fig. 5. GC-MS Analysis of Reaction Products Generated by LipC.
A, Chromatogram of reaction products generated by LipC. In this assay, 1-palmitoyl-2-myristoyl-sn-glycero-3-phosphatidylcholine was used
as the substrate. B, ESI-MS spectrum of peak 1 from Fig. 5A, corresponding to that of methyl C14:0 (myristate). C, ESI-MS spectrum of peak 2
from Fig. 5A, corresponding to that of methyl C16:0 (palmitate).
the half-micro test (Roche Diagnostics), which can
determine the amount of FFAs in serum and plasma.
Serum and plasma contain mainly straight-chain acids,
while the major components of fatty acids in Bacillus
belong to the ꢂ/ꢀ-hydrolase family.23,24) Unlike LipC,
LipA, and LipB are tolerant of alkaline pH. Their
optimum pH values are 10 and 12 respectively. LipA
has phospholipase (EC 3.1.1.3) activity, and LipB
functions as esterase (EC 3.1.1.1). Furthermore, LipA
and LipB prefer middle chain (C8) fatty acid esters as
substrate. The differences among LipA, LipB, and LipC,
in their enzymatic properties and structural features
suggest that expression of the lipA and/or lipB genes
during sporulation would not compensate for the
deletion of lipC.
2
1,22)
species are anteiso- and iso-branched-chain acids.
We confirmed that the assay was able to measure
branched-chain acids. In this study anteiso-C15 was used
as the standard. When the extracts from dormant spores
were assayed, the amount of FFAs in the wild-type and
YCSKd (lipC mutant) spores were 120 ꢃ 4 ng/mg
spores (wet weight) and 77 ꢃ 2 ng/mg spores (wet
weight) respectively. This suggests that LipC hydrolyzes
phospholipids and releases FFAs in wild-type spores.
Based on the speculation that LipC might affect the
outer and/or inner spore membranes, we tested to
determine whether the enzyme would hydrolyze phos-
pholipids. Phospholipases are hydrolytic enzymes that
cleave ester bonds in phospholipids. These enzymes are
categorized into four groups: A to D. Phospholipase A
cleaves fatty acid residues from the sn-1 or sn-2 position
of the glycerol backbone. Phospholipase B cleaves both
fatty acid residues from the sn-1 or sn-2 position.
Phospholipase C and D are phosphodiesterases that
release phosphomonoesters and alcohol respectively.
In this study, we indicated that LipC hydrolyzed a
variety of phospholipids as phospholipase B. In vivo, the
amount of FFAs in the lipC mutant spores was less than
that in the wild-type spores, suggesting the possibility
that the phospholipase activity of LipC modifies the
outer or inner spore membrane and releases FFAs during
sporulation. Taking into account the fact that LipC
expressed in the mother cell compartment does not
contain a signal sequence and is localized around the
Discussion
LipC is a component of the spore coat in B. subtilis,
which protects the spore from numerous assaults. In a
previous study, we found that LipC is a lipolytic enzyme
and that lipC mutant spores are defective in L-alanine-
1
1)
stimulated spore germination.
In this study, we
purified recombinant LipC expressed in E. coli and
provided the first biochemical characterization of this
lipase. The enzyme hydrolyzed pNP ester substrates and
a variety of phospholipids. The thermal stability and
pH-activity profiles of the enzyme indicated that LipC
does not possess the extreme resistance properties of
spores. Furthermore, calcium ions and germinants did
not activate lipase activity, suggesting that the enzyme
dose not play a crucial role in spore germination, but
comprises part of the spore germination machinery,
which includes the spore coat and membranes. It is
known that vegetative cells of B. subtilis secrete two
major lipolytic enzymes, LipA and LipB, both of which
1
1)
outer spore membrane, our results suggest that LipC
modifies the outer spore membrane. In that case, the
outer spore membrane in the lipC mutant spores are

Phospholipase of B. subtilis Spores
29
catalytic residue and can serve as the nucleophile.
Brumlik and Buckley proposed that Asp83 from Block
Variable loop2:
Pro139-Ile150
Block V:
Asp186 & His189
DGVH
31)
III is a putative active site, but structural analysis of
other SGNH hydrolases suggested that Asp187 from
Variable loop1:
D86-H106
2
8,32,33)
Block V is the acidic residue of the catalytic triad.
Gly50 from Block II and Asn82 from Block III appear to
donate H-bonds to the oxyanion hole. In this structural
model, we noticed that two surface loops, Asp86-His106
and Pro139-Ile150, might cover the active site in a lid-
like fashion. Lipolytic enzymes are characterized by
their drastically increased activity when acting at the
lipid-water interface of micellar or emulsified substrates,
a phenomenon called interfacial activation. In the
absence of lipid-water interfaces, the active site of
lipases is covered in the closed formation by the lid
domain. On the other hand, in the presence of hydro-
phobic substrates, the lid is opened, making the catalytic
residues accessible to the substrate and exposing a
Block III:
Asp83
CGND
Block I:
Ser11
GDSL
3
4)
hydrophobic surface. Structural analysis showed that
B. subtilis LipA and LipB lack a lid domain, and that
2
3,24)
their active sites are solvent exposed.
Thus, it is
Fig. 6. Homology Modeling of LipC.
The active site is conserved as in all the known structures of
SGNH hydrolases. The catalytic triad is underlined. The two loop
regions might be involved in the activation of LipC in acting at the
lipid-water interface of substrates.
possible that the two surface loops of LipC contribute to
regulation of switching from the inactive form to the
active form during sporulation. There are other possi-
bilities, that the two surface loops of LipC function
as domains that have other roles, such as substrate
targeting, substrate selectivity, and thermostability.
Santarossa et al. reported that mutations in lid region
affected the substrate preference and thermostability of
intact as a permeability barrier, unlike that in wild-type
spores, which is a defective barrier. The germination
defect in the lipC mutant spores might be caused by a
decrease in the passage of germinants across the outer
spore membrane to reach the inner spore membrane
3
5)
Pseudomonas fragi lipase. Elucidation of the detailed
role of those surface loops in the physiological function
of LipC is our future task.
2
5,26)
where the Ger receptors are localized.
reported, lipC mutant spores are not defective in AGFK
As previously
E. coli thioesterase I (TAP) of the SGNH-hydrolase
family has a single compact domain without a lid and
has a broad substrate specificity, including thioesterase,
arylesterase, protease, and lysophospholipase activi-
(
lated spore germination.
L-asparagine, D-glucose, D-fructose, and KCl)-stimu-
1
1)
As is well known, the
germinant receptors for the AGFK system are totally
different from those for the L-alanine, and the perme-
ation pathways of the germinants differ from each
3
6–38)
ties.
Since the catalytic triad of lipases is similar
to that of serine proteases, the different specificity
between TAP and other lipases may be due to the
presence of a lid domain like the surface loops of LipC.
Although the lipolytic enzymes of the GDSL family
play important roles in the bacterial metabolic pathway
4
)
other, although the details are not known. Therefore,
investigation of the permeability of the lipC spores to
germinants should provide insight into the spore
germination pathways.
3
6,39,40)
Based on the similar sequence position and context
of the catalytic triad, B. subtilis LipC belongs to the
and virulence factors,
family show low lipolytic activity, unlike TAP.
several enzymes in this
3
7,39–42)
1
1)
SGNH-hydrolase family. A subgroup of the GDSL
family is classified as the SGNH-hydrolase family due to
the presence of four strictly conserved residues, Ser-Gly-
Asp-His, in four conserved blocks, I, II, III, and V.27)
Rhamnogalacturonan acetylesterase from Aspergillus
aculeatus (AaRGAE) also belongs to the SGNH-hydro-
lase family. The structural features of AaRGAE and
Structural comparison between LipC and TAP may
provide new insights into the structure-activity relation-
ship in this family. Since many microbial lipases are
available for industrial applications as biocatalysts,
improving the activity and specificity of the LipC by
protein engineering and in vitro evolution will also be a
focus of our further studies.
several other SGNH-hydrolase members have been
Based on two templates (PDB accession
studied.2
7,28)
nos. 1k7c A, and 2o12 A), we created a homology
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