Microbiology (2015), 161, 330–340
DOI 10.1099/mic.0.000011
The PASTA domain of penicillin-binding protein
SpoVD is dispensable for endospore cortex
peptidoglycan assembly in Bacillus subtilis
Ewa Bukowska-Faniband and Lars Hederstedt
Correspondence
Ewa Bukowska-Faniband
[email protected].
se
Received 28 October 2014
Accepted 4 December 2014
Microbiology Group, Department of Biology, Lund University, Biology Building A, Sölvegatan 35,
SE-223 62 Lund, Sweden
Peptidoglycan is the major structural component of the bacterial cell wall. Penicillin-binding
proteins (PBPs), located at the exterior of the cytoplasmic membrane, play a major role in
peptidoglycan synthesis and remodelling. A PASTA domain (penicillin-binding protein and serine/
threonine kinase associated domain) of about 65 residues is found at the C-terminal end of some
PBPs and eukaryotic-like protein serine/threonine kinases in a variety of bacteria. The function of
PASTA domains is not understood, but some of them are thought to bind uncross linked
peptidoglycan. Bacillus subtilis has 16 different PBPs, but only 2 of them, Pbp2b and SpoVD,
contain a PASTA domain. SpoVD is specific for sporulation and essential for endospore cortex
peptidoglycan synthesis. We have studied the role of the PASTA domain in SpoVD by deleting
this domain and analysing the effects on endospore formation and subcellular localization of
SpoVD. Our results demonstrate that the PASTA domain in SpoVD is not essential for cortex
synthesis and not important for targeting SpoVD to the forespore outer membrane during
sporulation.
INTRODUCTION
The bacterial cell wall is a rigid layer located outside the
cytoplasmic membrane. It provides structural support and
protection to the cell. This cellular sacculus consists mainly
of long chains of peptidoglycan that are cross-linked via
flexible peptide bridges. Peptidoglycan assembly depends
on the activity of penicillin-binding proteins (PBPs) that
are located at the exterior of the cell membrane. PBPs
catalyse transglycosylation, transpeptidation and carboxypeptidation reactions. Some PBPs are bifunctional, having
both transglycosylase and transpeptidase activity (Typas
et al., 2012). As essential components of the peptidoglycan
synthesis machinery, PBPs constitute an effective target for
many antibiotics in clinical use (Bugg et al., 2011).
The PASTA domain (penicillin-binding protein and serine/
threonine kinase associated domain) is found at the Cterminus of some PBPs and eukaryotic-like protein serine/
threonine kinases (PSTKs) in a variety of bacteria. The
domain has 60–70 amino acid residues and occurs singly or
as a few consecutive copies. Although PASTA domains
show low amino acid sequence similarity, they share strong
structural conservation. Each domain has a globular fold
Abbreviations: PBP, penicillin-binding protein; PSTK, protein serine/
threonine kinase.
One supplementary table and one supplementary figure are available
with the online Supplementary Material.
330
formed by three b-strands and one a-helix (Yeats et al.,
2002). Several and inconsistent functions have been
ascribed to PASTA domains. Since the time of discovery
of a cefuroxime molecule non-covalently bound between
the transpeptidase domain and one of the two PASTA
domains in Streptococcus pneumoniae Pbp2x (Gordon et al.,
2000), the PASTA domain has been classified as a blactam-binding motif. Structural similarity of the b-lactam
ring to the D-alanyl-D-alanine residues of the stem pentapeptide of peptidoglycan precursors implied that the
PASTA domain is likely to bind uncross linked peptidoglycan. Several studies have demonstrated interaction of
PASTA domains of some PSTKs, e.g. Bacillus subtilis
PrkC, S. pneumoniae StkP and Mycobacterium tuberculosis
PknB, with crude peptidoglycan or synthetic muropeptides
(Maestro et al., 2011; Mir et al., 2011; Shah et al., 2008;
Squeglia et al., 2011). To our knowledge, there is only one
published study that characterizes the binding properties of
the PASTA domain of a PBP. In this recent report the
authors showed that the PASTA domain of M. tuberculosis
PBP PonA2 does not bind muropeptides, b-lactams or
polymeric peptidoglycan (Calvanese et al., 2014). Thus, the
question whether the ability to interact with peptidoglycan
is a general property of PASTA domains remains open.
What would be the importance of peptidoglycan interaction with a PASTA domain? So far, two different roles
have been suggested for such interaction: (i) localization of
the protein to the site of active peptidoglycan synthesis
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Role of the PASTA domain in SpoVD
(Mir et al., 2011; Morlot et al., 2013), and (ii) activation of
a novel endospore germination pathway in response to
peptidoglycan fragments (Shah et al., 2008).
B. subtilis (strain 168) has three PASTA domain-containing
proteins. Two of them, Pbp2b and SpoVD, are PBPs, and
the third, PrkC, is a PSTK. Pbp2b (two PASTA domains)
and SpoVD (one PASTA domain) are class B high molecular mass PBPs. Pbp2b is the only known essential PBP
in B. subtilis and is involved in the formation of septal
peptidoglycan at cell division (Daniel et al., 2000). SpoVD
is a sporulation-specific protein (Daniel et al., 1994) and
its transpeptidase activity is crucial for endospore cortex
peptidoglycan synthesis (Bukowska-Faniband & Hederstedt,
2013). The fact that both proteins are necessary for
peptidoglycan synthesis, but under different physiological
circumstances, and are the only PASTA-containing PBPs in
B. subtilis, prompted us to study the functional importance
of the PASTA domain in SpoVD.
B. subtilis can form endospores in response to nutrient
starvation. At the outset, cells destined to sporulate divide
asymmetrically, resulting in two differently sized cells: the
larger mother cell and the smaller forespore, which will
eventually become a dormant spore. In the subsequent development, the forespore becomes engulfed by the mother
cell, which results in the formation of a double-membraneenclosed forespore inside the mother-cell cytoplasm. In
the final stage, the mother cell lyses, releasing the mature
endospore into the environment (Eichenberger, 2012;
Piggot & Hilbert, 2004).
The endospore cortex is a thick layer of modified peptidoglycan surrounding the spore core and assembled in the
intermembrane compartment of the forespore. The cortex
layer is required for spore core dehydration and heat resistance (Warth, 1978; Warth & Strominger, 1972). Unlike
vegetative cell wall peptidoglycan, endospore cortex is not
essential for growth of B. subtilis; thus it constitutes a
convenient model for studies on peptidoglycan synthesis.
Deficiency in the cortex layer can easily be monitored by
the resulting heat sensitivity of spores (Todd et al., 1986).
SpoVD is synthesized in the mother-cell cytoplasm and
targeted to the forespore, where it is inserted into the outer
membrane with the transpeptidase catalytic domain and
the PASTA domain facing the intermembrane compartment. In this paper, we have studied the effects of deletion
of the PASTA domain of SpoVD on endospore cortex
synthesis and subcellular localization of SpoVD in B.
subtilis. Our results demonstrate that the PASTA domain is
not necessary for cortex assembly.
medium with phosphate (NSMP) (Fortnagel & Freese, 1968), growth
medium and resuspension medium for induction of sporulation
(Nicholson & Setlow, 1990), Spizizen’s minimal medium (SMM)
(Harwood & Archibald, 1990) or on tryptose blood agar base (TBAB)
plates (Difco). Antibiotics were used when appropriate at the
following concentrations: 100 mg ampicillin ml21, 12.5 mg chloramphenicol ml21, for E. coli; and 100 mg spectinomycin ml21, 0.5 mg
erythromycin ml21 combined with 12.5 mg lincomycin ml21, 5 mg
chloramphenicol ml21, for B. subtilis. TBAB medium supplemented
with 1 % (w/v) starch was used to test the amylase activity of B.
subtilis colonies.
DNA techniques. DNA manipulation was performed by standard
methods (Sambrook & Russell, 2001). Plasmid DNA from E. coli and
B. subtilis was isolated using a Quantum miniprep kit (Bio-Rad). In
the case of B. subtilis, the plasmid isolation procedure was slightly
modified by the addition of lysozyme at the final concentration of
4 mg ml21 to the resuspension solution. B. subtilis chromosomal
DNA was isolated according to the procedure described by Marmur
(1963). PCR was carried out using Phusion high-fidelity DNA
polymerase (Finnzymes) and either B. subtilis chromosomal DNA or
plasmid DNA as the template. The sequences of the oligonucleotides
used in the PCRs are listed in Table S1 (available in the online Supplementary Material). DNA ligation was performed using T4 DNA
ligase (New England Biolabs) at 14 uC overnight. Ligates were used to
transform E. coli TOP10 either by electroporation or by chemical
transformation (Hanahan et al., 1991). B. subtilis was grown to
natural competence as described by Hoch (1991) and ~0.5 mg plasmid
DNA was added to 0.5 ml competent cells. Transformation with
chromosomal DNA was carried out at a limiting amount of DNA to
restrict recombination to a single locus per cell. DNA concentrations
were determined using a NanoDrop spectrophotometer (Thermo
Scientific). All DNA fragments cloned in plasmids were verified by
sequencing. The plasmids used in this study are listed in Table 1.
Construction of pLEB4. Primers Ewa5 and Ewa6 were used to
amplify by PCR a 2125 bp fragment of the B. subtilis 1A1
chromosome comprising the promoter region and the coding
sequence of spoVD. After digestion with EcoRI and BamHI, the
PCR product was cloned into pHPSK, yielding plasmid pLEB4. This
plasmid encodes full-length SpoVD.
Construction of pLEB7. The spoVD-mCherry in-frame gene fusion
including the native spoVD promoter region was amplified from
pLEB5 using primers Ewa5 and Ewa13, generating a 2800 bp fragment. After digestion with EcoRI and BamHI, the PCR product was
cloned into pHPSK, yielding plasmid pLEB7. This plasmid encodes
full-length SpoVD fused to mCherry at the C-terminus.
Construction of pLEB34. Primers Ewa52 and Ewa53 were used to
amplify a 1988 bp fragment of the B. subtilis 1A1 chromosome
comprising the spoVD ORF without the start and stop codons. Primer
Ewa52 generated a restriction site for BamHI, and primer Ewa53
generated an EcoRI restriction site as well as the strepII-tag sequence
and the translational stop codon. The PCR fragment was digested
with BamHI and EcoRI and cloned into pSG1729, resulting in plasmid
pLEB34. This plasmid encodes the full-length SpoVD fused to GFP at
the N-terminus and with the StrepII-Tag at the C-terminus.
Construction of pLEB35. Primers Ewa52 and Ewa55 were used to
METHODS
Bacterial strains and growth media. The bacterial strains used in
this work are listed in Table 1. Escherichia coli TOP10 was used for
plasmid DNA propagation. E. coli strains were grown at 37 uC in LB
medium or on LB agar plates (Sambrook & Russell, 2001). B. subtilis
strains were grown at 30 or 37 uC in LB medium, nutrient sporulation
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amplify a 1793 bp fragment of the B. subtilis 1A1 chromosome
comprising the spoVD ORF without the start codon, the sequence for
the PASTA domain and the stop codon. Primer Ewa52 generated a
restriction site for BamHI, and primer Ewa55 generated an EcoRI
restriction site as well as the strepII-tag sequence and the translational
stop codon. The PCR fragment was cloned into pSG1729, resulting in
plasmid pLEB35. This plasmid encodes a truncated version of SpoVD
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331
E. Bukowska-Faniband and L. Hederstedt
Table 1. Strains and plasmids used in this work
Strain/plasmid
Strains
E. coli
TOP10
B. subtilis
1A1
LMD101
LMD104
LMD132B
LMD134
LMD141
LMD142
Plasmids
pSG1729
Genotype/description
F2 mcrA D(mrr–hsdRMS–mcrBC) w80lacZDM15 DlacX74 nupG recA1 araD139 D(ara–leu)7697
galE15 galK16 rpsL (StrR) endA1 l2; StrR
Invitrogen
trpC2
trpC2 DspoVD
BGSC*
Bukowska-Faniband
& Hederstedt (2013)
Bukowska-Faniband
& Hederstedt (2013)
This work
This work
This work
This work
trpC2 DspoVD amyE : : PsE-spoVD-mCherry; SpcR
trpC2
trpC2
trpC2
trpC2
DspoVD
DspoVD
DspoVD
DspoVD
amyE : : Pxyl-gfp-spoVD; SpcR
amyE : : Pxyl-gfp-spoVDD582–646; SpcR
amyE : : Pxyl-spoVDD582–646-pbpB599–716; SpcR
amyE : : PsE-spoVDD582–646-mCherry; SpcR
pHPSK
amyE integration vector designed to generate N-terminal GFP fusions under the xylose-inducible
promoter; ApR SpcR
E. coli/B. subtilis shuttle vector; EmR CmR
pLEB4
pLEB5
pHPSK with a 2.1 kb fragment containing PsE-spoVD; EmR CmR
pKS-mCherry-E-T3 with a 2.0 kb fragment containing PsE-spoVD; ApR
pLEB6
pDG1730 with a 2.8 kb fragment containing PsE-spoVD-mCherry gene fusion; ApR SpcR EmR
pLEB7
pLEB34
pLEB35
pLEB38
pLEB38-Stop
pHPSK with a 2.8 kb fragment containing PsE-spoVD-mCherry gene fusion; EmR CmR
pSG1729 with a 2.0 kb fragment containing spoVD-strepII-tag; ApR SpcR
pSG1729 with a 1.8 kb fragment containing spoVDD582–646-strepII-tag; ApR SpcR
pLEB7 with a deletion of 0.2 kb fragment encoding the PASTA domain of SpoVD; EmR CmR
pLEB38 with a single nucleotide deletion at the junction of spoVD and mCherry sequences
resulting in a stop codon after the spoVD sequence; EmR CmR
pSG1729 with a 2.1 kb fragment containing spoVDD582–646-pbpB599–716-strepII-tag; ApR SpcR
pLEB6 with a deletion of a 0.2 kb fragment encoding the PASTA domain of SpoVD; ApR SpcR EmR
pLEB41
pLEB42
Origin/reference
BGSC*
Johansson &
Hederstedt (1999)
This work
Bukowska-Faniband
& Hederstedt (2013)
Bukowska-Faniband
& Hederstedt (2013)
This work
This work
This work
This work
This work
This work
This work
*Bacillus Genetic Stock Center, Columbus, OH, USA.
without the PASTA domain, fused to GFP at the N-terminus and the
StrepII-Tag at the C-terminus.
plasmid encodes a truncated version of SpoVD without the PASTA
domain fused to the twin PASTA domains of Ppb2b and the StrepIITag at the C-terminal end and to GFP at the N-terminal end.
Construction of pLEB38, pLEB38-Stop and pLEB42. Deletion of
the sequence encoding the PASTA domain of SpoVD in pLEB7 and
pLEB6 was achieved by site-directed mutagenesis (Phusion sitedirected mutagenesis kit; Finnzymes) using primers Ewa58 and
Ewa62, resulting in plasmids pLEB38 and pLEB42, respectively. These
two plasmids encode a truncated version of SpoVD without the PASTA
domain, fused to mCherry at the C-terminus. During sequencing of the
plasmid clones obtained, one, pLEB38-Stop, was found to have a single
nucleotide deletion at the ligation site (corresponding to the junction of
the spoVD and mCherry sequences), resulting in a tryptophan codon
(amino acid residue 582) followed by a translational stop codon.
Construction of pLEB41. A 1035 bp synthetic DNA fragment was
designed, containing a part of the spoVD ORF (encoding residues
366–581), a part of the B. subtilis pbpB ORF (codons 599–716 and a
destroyed EcoRI restriction site), a strepII-tag sequence and a translational stop codon followed by an EcoRI site at the 39 end. Gene
synthesis was carried out by GenScript. The synthetic fragment
was digested with HindIII and EcoRI and cloned in-frame into the
corresponding sites of pLEB34, resulting in plasmid pLEB41. This
332
Construction of B. subtilis strains. B. subtilis strains used in this
work are derivatives of strain 1A1 and are listed in Table 1. B. subtilis
LMD101 was transformed with pLEB34, pLEB35, pLEB41 and pLEB42
resulting in strains LMD132B, LMD133, LMD141 and LMD142,
respectively. Chromosomal DNA from LMD133 was used to transform
strain LMD101, giving rise to strain LMD134. Spectinomycin-resistant
clones were confirmed for integration of the gene cassette by a double
cross-over event at the amy locus by the loss of amylase activity, i.e.
inability to degrade starch on TBAB plates supplemented with 1 %
(w/v) starch. Plasmids pLEB7, pLEB38 and pLEB38-Stop were used
to transform B. subtilis LMD101 and the strains obtained were
named LMD101/pLEB7, LMD101/pLEB38 and LMD101/pLEB38Stop, respectively.
Light microscopy. A 100 ml sample was taken from the culture of
sporulating cells. The cells were collected by centrifugation at 14 000 g
for 30 s and suspended in such a volume of PBS that the density of
cells was appropriate for microscopy. A total of 5 ml of the cell
suspension was mounted on an agarose-coated microscopy glass slide
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Microbiology 161
Role of the PASTA domain in SpoVD
(1 % agarose in PBS) and covered with a coverslip. Phase-contrast
and fluorescent images were acquired using a Zeiss Axio Imager.Z2
microscope with X-Cite 120 Illumination (EXFO Photonic Solutions),
an ORCA-flash 4.0 LT sCMOS camera (Hamamatsu Photonics) and
Volocity software (PerkinElmer). Typical exposure times used to
obtain fluorescence images of cells were 2 s for mCherry and 1 s for
GFP. Images were processed and analysed with Volocity software
(PerkinElmer). The background fluorescence was subtracted from each
image using the autofluorescence of the WT strain (1A1) as a reference.
Background-subtracted images were further used to calculate mean
fluorescence intensity within the cells.
Sporulation and heat-resistance assay. Cells were induced to
sporulate either by nutrient exhaustion in NSMP or by using the
resuspension method (Nicholson & Setlow, 1990). For strains
LMD132B, LMD134 and LMD141, all media were supplemented
with 0.2 % (w/v) xylose from the start of the cultures. The spore heatresistance assay was carried out on 2-day-old cultures in the case of
the nutrient exhaustion method and 24 h after resuspension in the
case of the resuspension method. The heat resistance of spores was
analysed by heating 5 ml culture at 80 uC for 10 min. Serial dilutions
of heated and unheated samples were spread on TBAB plates. The
number of colonies was counted after incubation of the plates at
37 uC overnight and the yield of heat-resistant spores was calculated.
Total cell lysate preparation. Five hours after the induction of
sporulation by resuspension, 50 ml cell culture was collected by
centrifugation at 5000 g and 4 uC for 15 min. The cell pellet was
washed with 20 ml ice-cold 50 mM potassium phosphate buffer,
pH 8.0, and centrifuged. The pellet was resuspended in 1 ml ice-cold
20 mM Na-MOPS buffer, pH 7.4, containing 0.5 mM EDTA and
complete protease inhibitor cocktail (Roche). The cell suspension was
transferred into a pre-chilled screw-cap microcentrifuge tube containing approximately 500 mg of 0.1 mm diameter Zirconia-glass beads.
PMSF was added to the cell suspension at the final concentration of
1 mM. Cells were disrupted using a FastPrep-24 (MP Biomedicals)
bead beating system: setting 6.5 m s21, three cycles of 45 s. Samples
were kept for 5 min on ice between each cycle. Finally, the glass beads
were sedimented by brief centrifugation and the cell lysate was
transferred to a fresh tube.
Immunoblot analysis. Proteins in extracts were separated by Tricine
SDS-PAGE with 4 % stacking gel and 8 % separating gel (Schägger &
von Jagow, 1987). After SDS-PAGE, the proteins were transferred to a
PVDF membrane (Immobilon P; Millipore) using a wet blot. The
transfer buffer was 20 mM Tris, 150 mM glycine containing 20 % (v/v)
methanol. The antibodies used were rabbit anti-dsRed (Clontech)
(1 : 1000), anti-GFP (GenScript) (1 : 5000), anti-SpoVD (Liu et al.,
2010) (1 : 3000) and anti-BdbD (Crow et al., 2009) (1 : 3000). Immunodetection was carried out by chemiluminescence using anti-rabbit
secondary antibodies conjugated to horseradish peroxidase (GE
Healthcare) in 1 : 3000 dilution and SuperSignal West Pico chemiluminescence substrate (Pierce Chemical).
RESULTS
Effect of deletion of the PASTA domain in a GFPSpoVD fusion protein
To analyse the role of the PASTA domain in SpoVD, a gene
encoding a truncated variant of SpoVD lacking the PASTA
domain (residues Lys582–Asp646) was constructed. Fulllength and PASTA-truncated SpoVD were fused to GFP at
their N-terminal end to make it possible to also study the
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subcellular localization of the SpoVD variants. The genes
for GFP-SpoVD and GFP-SpoVDD582–646 were put under a
xylose-inducible promoter and inserted into the amyE
locus of B. subtilis LMD101 (deleted for spoVD), resulting
in strains LMD132B and LMD134, respectively (Fig. 1).
The strains were grown in NSMP in the presence of 0.2 %
xylose for 2 days to complete sporulation and the spores
obtained were tested for heat resistance. Strain LMD132B
produced a WT (i.e. similar to parental strain 1A1) level of
heat-resistant spores (Table 2), indicating that the GFPSpoVD fusion is functional and that addition of 0.2 % xylose
to the medium is sufficient to complement the mutant. In
contrast, strain LMD134 carrying GFP-SpoVDD582–646 was
defective in producing heat-resistant spores (Table 2),
showing that most of the spores produced lack the cortex
layer. Induction of sporulation by using the resuspension
method and growth for 24 h confirmed the production of
defective spores for strain LMD134 (0.04±0.02 % heatresistant spores) and a normal level of heat-resistant spores
for the control strain LMD132B (64±6 %). Immunoblot
analysis of cell lysates of sporulating cells harvested 5 h after
resuspension [the cellular level of SpoVD protein expressed
from the native promoter peaks at this time point, as shown
previously by Liu et al. (2010) and Bukowska-Faniband &
Hederstedt (2013)] revealed a low amount of GFPSpoVDD582–646 (~94 kDa) as compared to that of full-length
GFP-SpoVD (~101 kDa) (Fig. 2a). As expected, both protein variants were found to be membrane bound (data not
shown). Considering that production of GFP-SpoVDD582–646
in LMD134 is dependent on the presence of xylose in the
growth medium, we asked whether an increased concentration of xylose leads to an increased yield of heat-resistant
spores. Addition of 0.5 % xylose to NSMP did not result in a
higher yield of heat-resistant spores (data not shown).
To confirm the difference in the cellular level of SpoVD
fusion protein in strains LMD132B and LMD134, we
carried out microscopy of sporulating cells and compared
the strength of the GFP fluorescence signal from the two
strains. In WT B. subtilis cells, expression of spoVD depends
on the mother-cell-specific sigma factor E (sE). During
sporulation, SpoVD is synthesized in the cytoplasm of the
mother cell and localized to the forespore outer membrane
(Bukowska-Faniband & Hederstedt, 2013; Fay et al., 2010).
We took advantage of the xylose-inducible promoter in our
constructs, which makes it possible to produce SpoVD
during exponential growth (and independently of sporulation) when it becomes inserted into the cytoplasmic
membrane. The localization of SpoVD fusion proteins
during spore development was examined by fluorescence
microscopy, starting with the pre-sporulating cell, through
asymmetrical septation, engulfed spore, up to the stage of
the phase-grey spore (this late time point corresponds to
when the SpoVD-dependent cortex formation takes place).
In sporulating strain LMD134, GFP-SpoVDD582–646 was
enriched at the asymmetrical septum and at the forespore,
resembling the subcellular localization of full-length GFPSpoVD in LMD132B (Fig 2b, c). However, the fluorescence
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333
E. Bukowska-Faniband and L. Hederstedt
Pxyl
LMD132B
GFP
M
DUF
TP
GFP
M
DUF
TP
GFP
M
DUF
TP
P
Pxyl
LMD134
Pxyl
LMD141
LMD104 and
LMD101/pLEB7
LMD142 and
LMD101/pLEB38
P
P
PσE
M
DUF
TP
M
DUF
TP
M
DUF
TP
M
DUF
TP
mCherry
P
PσE
mCherry
PσE
LMD101/pLEB4
P
PσE
LMD101/pLEB38-Stop
mCherry
Fig. 1. Schematic representation of the spoVD gene and fusion-protein variants present in B. subtilis strains used in this study.
Pxyl and PsE indicate a xylose-inducible promoter and a sigma E promoter, respectively. The black triangle indicates a mutation
introducing a translational stop at that position. DUF, Domain of unknown function; M, transmembrane segment; mCherry,
variant of red fluorescent protein; P, PASTA domain; TP, transpeptidase domain.
signal from GFP-SpoVDD582–646 in strain LMD134 was
weaker than that of GFP-SpoVD in LMD132B in all observed
developmental stages (Fig. 3).
The results of the immunoblot and fluorescence microscopy showed that SpoVD with the PASTA domain deleted
was present in the cells at lower amount as compared to the
Table 2. Sporulation efficiencies of B. subtilis strains
Cells were sporulated by growth in NSMP for 2 days at 37 uC. The yield of heat-resistant spores was assayed by incubation at 80 uC for 10 min. The
percentage of heat-resistant spores was calculated as c.f.u. after heating divided by c.f.u. of not-heated culture. The results represent the mean from
three independent experiments±SEM.
Strain
1A1
LMD101
LMD132BD
LMD134D
LMD141D
LMD104
LMD142
LMD101/pLEB7
LMD101/pLEB38
LMD101/pLEB4
LMD101/pLEB38-Stop
Relevant genotype
WT
DspoVD
DspoVD amyE : : Pxyl-gfp-spoVD
DspoVD amyE : : Pxyl-gfp-spoVDD582–646
DspoVD amyE : : Pxyl-spoVDD582–646–pbpB599–716
DspoVD amyE : : PsE-spoVD-mCherry
DspoVD amyE : : PsE-spoVDD582–646-mCherry
DspoVD, pLEB7 (PsE-spoVD-mCherry)
DspoVD, pLEB38 (PsE-spoVDD582–646-mCherry)
DspoVD, pLEB4 (PsE-spoVD)
DspoVD, pLEB38-Stop (PsE-spoVDD582–646)
Yield (%) of heat-resistant spores
67±4
0*
91±5
0.00007±0.0001
26±6
55±2
3±1
76±13
42±4
80±8
74±6
*,661027.
DThe growth medium was supplemented with 0.2 % (w/v) xylose.
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Role of the PASTA domain in SpoVD
LMD132B (GFP-SpoVD)
(b)
(a)
Overlay
Phase
contrast
14
1
LM
D
13
2B
LM
D
LM
D
13
4
Fluorescence
Pre-sporulating
cell
A
100
B
*
70
C
Asymmetric
division
Engulfed
forespore
35
25
BdbD
Phase grey
spore
LMD134 (GFP-SpoVDD582–646)
(c)
Fluorescence
Overlay
Phase
contrast
(d) LMD141 (GFP-SpoVDD582–646–Pbp2b599–716)
Fluorescence
Overlay
Phase
contrast
Pre-sporulating
cell
Asymmetric
division
Engulfed
forespore
Phase grey
spore
Fig. 2. Relative cellular amounts and subcellular localization of full-length, truncated and chimeric GFP-SpoVD (strains
LMD132B, LMD134 and LMD141, respectively). Cells were sporulated by resuspension at 37 6C in the presence of 0.2 %
xylose. (a) Immunoblot of cell lysates of the indicated strains. Samples for analysis were taken at 5 h after resuspension. Cell
amounts were normalized according to the OD600 before lysates were prepared. An equal amount of sample was loaded in
each lane. In the upper panel samples were probed with anti-GFP serum. The position of each GFP fusion protein is indicated
by an arrow. A, GFP-SpoVDD582–646–Pbp2b599–716; B, GFP-SpoVD; C, GFP-SpoVDD582–646. A putative degradation product
of GFP-SpoVDD582–646–Pbp2b599–716 is indicated by an asterisk. In the lower panel samples were probed with anti-BdbD
serum (as a control for equal loading of samples). Molecular mass marker sizes, in kDa, are indicated on the left. (b–d) GFP
fluorescence and phase-contrast images of cells of the indicated strains were taken at hourly intervals after induction of
sporulation by resuspension. Different developmental stages, i.e. pre-sporulating cell (~1 h after resuspension), asymmetric
division (~2–3 h after resuspension), engulfed forespore (~4 h after resuspension) and phase-grey spore (~5 h after
resuspension), were distinguished based on the fluorescence and phase-contrast images. Bars, 2 mm.
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335
E. Bukowska-Faniband and L. Hederstedt
Mean fluorescence intensity (AU)
180
160
Asymmetric division
54
140
120
Engulfed forespore
25
Phase-grey spore
81
100
80
60
with the PASTA domain deleted, yielded about 3 % heatresistant spores (Table 2). This result showed that many
LMD142 developing spores were able to assemble cortex and
thereby acquire heat resistance. These findings indicated that
the PASTA domain is not essential for endospore cortex
synthesis.
Pre-sporulating cell
53
43
48
59
77
10
40
55
47
20
48
0
LMD132B
LMD134
LMD141
Fig. 3. Mean fluorescence intensity of cells of strain LMD132B
(GFP-SpoVD), LMD134 (GFP-SpoVDD582–646) and LMD141
(GFP-SpoVDD582–646–Pbp2b599–716). Cells were sporulated by
resuspension at 37 6C in the presence of 0.2 % xylose and imaged
at hourly intervals after the resuspension. Different developmental
stages, i.e. pre-sporulating cell, asymmetrical division, engulfed
forespore and phase-grey spore, were distinguished based on the
fluorescence and phase-contrast images. The mean fluorescence
of entire cells was measured. Error bars indicate SEM. The number
in each bar shows the number of cells counted. AU, Arbitrary units.
full-length protein. The subcellular localization of GFPSpoVDD582–646 seemed normal, but it has to be noted that
due to the weak fluorescence signal from strain LMD134,
possible signal from the fusion protein in the mother-cell
membrane would not be detected. Therefore, we could not
rule out that lack of cortex, i.e. deficiency in production of
heat-resistant spores, was to some degree a result of
aberrant localization of GFP-SpoVDD582–646.
SpoVDD582–646-mCherry partially complements
SpoVD deficiency
The low amount of GFP-SpoVDD582–646 in strain LMD134
could be an effect of the truncation or altered sequence at
the C-terminal end of SpoVD. In an attempt to compensate
the SpoVD protein for the deleted PASTA domain, we added
the mCherry domain to the C-terminal end of SpoVDD582–646.
From previous work we know that a full-length SpoVDmCherry fusion protein is functional (Bukowska-Faniband
& Hederstedt, 2013). mCherry was used instead of GFP
because SpoVD is inserted in the outer forespore membrane
in such a way that the C-terminus faces the intermembrane
space and mCherry, in contrast to GFP, folds correctly in the
periplasm (Chen et al., 2005).
Genes encoding SpoVD-mCherry and SpoVDD582–646mCherry under the control of the native spoVD promoter
(sE-dependent promoter) were inserted at the amyE locus
of strain LMD101 yielding strains LMD104 and LMD142,
respectively (Fig. 1). Strain LMD104 produced a near WT
level of heat-resistant spores, i.e. 55 %, while strain LMD142,
336
As determined by immunoblot using mCherry antibodies,
LMD142 cells contained SpoVDD582–646-mCherry fusion
protein (91 kDa), but at a low amount compared to fulllength SpoVD-mCherry (99 kDa) in LMD104 (Fig. 4a).
Thus, fusion of mCherry to the C-terminal end of PASTAtruncated SpoVD did not restore the WT level of SpoVD
protein. Due to the very low intracellular level of mCherry
in LMD142 sporulating cells we did not carry out fluorescence
microscopy examination with this strain.
Overproduction of SpoVDD582–646-mCherry
effectively complements SpoVD deficiency
The partial complementation of SpoVD deficiency observed
with strain LMD142 prompted us to increase the amount of
SpoVDD582–646-mCherry in DspoVD cells. We placed the
spoVD-mCherry and spoVDD582–646-mCherry fusions under
the native spoVD promoter in the B. subtilis plasmid pHPSK
yielding plasmids pLEB7 and pLEB38, respectively. B. subtilis
LMD101 was transformed with both plasmids, yielding
LMD101/pLEB7 and LMD101/pLEB38 (Fig. 1). As expected,
strain LMD101/pLEB7 produced a WT level of heat-resistant
spores (Table 2). Strain LMD101/pLEB38 also produced a
relatively high level of heat-resistant spores, 42 % (Table 2).
Immunoblot analysis of lysates of sporulating LMD101/
pLEB38 cells confirmed that SpoVDD582–646-mCherry was
overproduced compared to SpoVD-mCherry in strain
LMD104 (Fig. 4a). These data confirmed that the PASTA
domain of SpoVD is not required for cortex synthesis.
The lower yield of heat-resistant spores obtained with
strain LMD101/pLEB38 (42 %) compared to LMD101/
pLEB7 (76 %) is probably due to the mCherry fusion. This
is supported by the observation that overproduced
SpoVDD582–646, without fusion to mCherry, fully restored
the yield of heat-resistant spores to a WT level. During
construction of pLEB38 we obtained by serendipity one
clone, pLEB38-Stop, with a single nucleotide deletion at
the junction of the spoVDD582–646 and mCherry sequences,
causing a stop codon immediately after the spoVDD582–646
sequence (Fig. 1). B. subtilis LMD101/pLEB38-Stop yielded
74 % heat-resistant spores, which is the same level as that
for B. subtilis LMD101/pLEB4 containing WT SpoVD (Fig.
1, Table 2). Consistent with our findings with the GFP- and
mCherry-fusion proteins, the cellular amount of overproduced SpoVDD582–646 (~64 kDa) was lower than that of
overproduced WT SpoVD (~71 kDa) (Fig. 4b).
Subcellular localization of
SpoVDD582–646-mCherry
To determine whether the subcellular localization of
SpoVDD582–646-mCherry is the same as for the full-length
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Microbiology 161
Role of the PASTA domain in SpoVD
D
LM
LM
D
2
14
L
/p
LM
D
1
10
3
EB
(b)
L
/p
1
10
D
LM
LM
pL D10
EB 1
38 /
-S
top
4
10
8
7
EB
LM
pL D10
EB 1
4 /
(a)
100
A
100
70
B
C
70
35
25
(c)
*
BdbD
Fluorescence
35
25
Overlay
Phase
contrast
LMD101/pLEB7
LMD101/pLEB38
variant, we examined sporulating cells by fluorescence microscopy. The mCherry fluorescence signal of LMD101/pLEB38
cells was weak early in sporulation, preventing conclusions
about localization of SpoVDD582–646-mCherry at this developmental stage. We could determine localization of the fulllength SpoVD-mCherry and its truncated variant at 7 h after
induction of sporulation by resuspension at 30 uC. As shown
in Fig. 4(c), SpoVDD582–646-mCherry was enriched at the
forespore and its subcellular localization resembled that of
full-length SpoVD-mCherry. Thus, the PASTA domain is
not important for targeting SpoVD to the forespore.
Properties of a GFP-SpoVDD582–646–Pbp2b599–716
chimera
As shown above, C-terminal truncation of GFP-SpoVD
affected the cellular level of the fusion protein and this
complicated the interpretation of results. mCherry attached
to the C-terminal end of PASTA-truncated SpoVD did not
restore the WT level of the protein. Therefore, we decided
to attach the twin PASTA domains of B. subtilis Pbp2b
(residues Lys599–Asp716) to the C-terminal end of GFPSpoVDD582–646 (Fig. 1). Pbp2b is a paralogue of SpoVD and
essential for septal peptidoglycan synthesis during vegetative
growth. Both PASTA domains of Pbp2b show sequence
similarity to the PASTA domain of SpoVD (Fig. S1). For this
reason, and since it is possible that folding and/or activity of
these two PASTA domains are interdependent, we fused both
PASTA domains to the C-terminal end of GFP-SpoVDD582–646.
http://mic.sgmjournals.org
D
BdbD
Fig. 4. Relative cellular amounts and subcellular localization of SpoVD-mCherry protein
variants in different B. subtilis strains. (a, b)
Immunoblots of cell lysates. Cells were sporulated by resuspension at 30 6C and samples
were taken at 5 h after resuspension. Cell
amounts were normalized according to OD600
before lysates were prepared. An equal
amount of sample was loaded in each lane.
Samples were probed with: anti-mCherry
serum and anti-BdbD serum (a), and antiSpoVD serum and anti-BdbD serum (b).
The positions of proteins in the gels are indicated by arrows. A, SpoVD-mCherry; B,
SpoVDD582–646-mCherry; C, SpoVD; D,
SpoVDD582–646. An asterisk indicates an
unknown cross-reacting protein independent
of SpoVD. Molecular mass marker sizes, in
kDa, are indicated on the left. (c) Subcellular
localization of overproduced SpoVD-mCherry
(strain LMD101/pLEB7) and SpoVDD582–646mCherry (strain LMD101/pLEB38) in sporulating cells. Images were taken at 7 h after the
start of sporulation by the resuspension
method at 30 6C and cells in different
developmental stages are shown. Bar, 2 mm.
The constructed gene encoding the GFP-SpoVDD582–646–
Pbp2b599–716 chimera under the control of a xyloseinducible promoter was inserted at the amyE locus of B.
subtilis LMD101. The strain obtained, LMD141, produced
near WT levels (26 %) of heat-resistant spores (Table 2).
Immunoblot analysis of total lysates of LMD141 cells
showed that the chimera (~107 kDa) was present at higher
amounts than truncated GFP-SpoVDD582–646, but apparently at lower amounts than full-length GFP-SpoVD (Fig.
2a). This explains the relatively efficient production of heatresistant spores by strain LMD141. Fluorescence microscopy
of LMD141 cells showed a slightly stronger GFP signal
compared to strain LMD134 (Figs 2d and 3), consistent with
the immunoblot data. The fluorescence signal of GFPSpoVDD582–646–Pbp2b599–716 seemed enriched at the asymmetrical septum and at the forespore, similar to the localization of the WT reference protein (GFP-SpoVD), suggesting
normal subcellular localization of the chimeric SpoVD
protein. However, as noted above for GFP-SpoVDD582–646,
we could not exclude that a small amount of GFPSpoVDD582–646–Pbp2b599–716 remained in the mother-cell
membrane.
DISCUSSION
SpoVD is the only sporulation-specific PASTA-domaincontaining PBP in B. subtilis and its transpeptidase activity is
essential for synthesis of the endospore cortex peptidoglycan
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337
E. Bukowska-Faniband and L. Hederstedt
layer (Bukowska-Faniband & Hederstedt, 2013). We took
advantage of the non-essential process of peptidoglycan
synthesis during sporulation to study the effect of deletion of
the PASTA domain in SpoVD. We demonstrated that
SpoVD deleted for the PASTA domain is still able to carry
out its essential role in the cortex synthesis.
SpoVD is a paralogue of Pbp2b, another class B PBP
involved in cell division in B. subtilis. The genes for these
two proteins (spoVD and pbpB) have probably originated
as the result of a tandem gene duplication in the chromosome (Yanouri et al., 1993). Pbp2b contains two PASTA
domains at the C-terminal end. Although no systematic
analysis of the PASTA domains in Pbp2b has been carried
out, a truncation of 84 amino acid residues at the Cterminal end led to the formation of relatively short cell
filaments. This indicated that the second PASTA domain of
Pbp2b is not essential for protein function but the mutant
cells divided at reduced frequency. Carboxy-truncated Pbp2b
seemed either degraded or post-translationally processed in
vegetative cells and the resulting product was present in the
cells at much lower amounts than the full-length protein
(Yanouri et al., 1993). It was recently reported that S.
pneumoniae Pbp2x deleted for both PASTA domains is
subject to proteolytic degradation in the cell (Peters et al.,
2014). Our finding that PASTA-truncated SpoVD is present
in low amounts in the cell is, therefore, in agreement with
the data available for B. subtilis Pbp2b and S. pneumoniae
Pbp2x. Altogether, it appears that PASTA domains of PBPs,
apart from some hitherto unknown function, are important
for protein stability.
In vitro studies on a series of C-terminal-truncated derivatives
of S. pneumoniae Pbp2x showed that the presence of the first
PASTA domain plus the a-helix of the second PASTA
domain appears to be crucial for the binding of b-lactam to
the transpeptidase domain. Deletion of both PASTA
domains dramatically decreased b-lactam binding to below
10 % (Maurer et al., 2012). It is therefore possible that not
only the cellular amount of the protein, but also substrate
binding, is affected when the PASTA domain of SpoVD is
missing. However, our in vivo data unambiguously prove
that the PASTA-truncated SpoVD can bind substrate and
catalyse peptide cross-linking.
Several studies indicate a role of PASTA domains in protein subcellular localization (Beilharz et al., 2012; Fleurie
et al., 2012; Mir et al., 2011; Peters et al., 2014). To study
this we used an N-terminal and a C-terminal fusion of GFP
and mCherry, respectively, to truncated SpoVD. In both
cases the fluorescence signal, although weak, was enriched
at the forespore in a pattern resembling localization of fulllength protein. It was difficult to compare fluorescence
images of cells with different signal strength, i.e. of cells
containing full-length SpoVD versus those with truncated
and chimeric versions. For this reason we cannot unambiguously conclude that all the truncated proteins in the
studied strains are properly targeted and incorporated into
the forespore outer membrane.
338
Localization of SpoVD to the forespore depends on the
presence of SpoVE, a putative lipid II flippase (Fay et al.,
2010). It has been shown that these two membrane proteins directly interact with each other (Fay et al., 2010),
which suggests that subcellular localization of SpoVD
depends on the interaction with SpoVE specifically rather
than on the availability of peptidoglycan precursors transported by SpoVE (which could be sensed by the PASTA
domain). Although specific interaction sites for SpoVD
and SpoVE are unknown, it is unlikely that the PASTA
domain is involved in this interaction. SpoVE has ten
membrane-spanning segments and it probably interacts
with SpoVD through the membrane-spanning domain.
Such interaction has been shown for PBP3 and FtsW in E.
coli (Fraipont et al., 2011). Notably, several bacterial species
encode a single large protein that corresponds to a fusion
of a class B PBP with a putative flippase.
We observed with all tested strains containing GFP-SpoVD
that GFP fluorescence of cells decreased in the late phases
of sporulation (Fig. 3). However, mCherry fluorescence of
cells containing SpoVD-mCherry was visible only at late
stages of sporulation. One possible explanation for this
phenomenon could be a stage-specific fluorescence intensity of GFP and mCherry during sporulation in B. subtilis. It
was previously shown that the mCherry and GFP fluorescence intensity is sensitive to subcellular environmental
changes during spore development. Doherty and co-workers
reported (Doherty et al., 2010) that during sporulation
stages II and III (which correspond to formation of asymmetrical septum and engulfment of the spore by the mother cell)
the mCherry fluorescence is diminished, while the GFP
signal is clearly visible. This fluorescence pattern reverses
when sporulation reaches stages V and VI (i.e. assembly of
the coat layer and spore maturation). Stage V can be distinguished by phase-contrast microscopy, since the spores turn
from phase grey to phase bright. At this stage the GFP
fluorescence signal becomes weak, while mCherry becomes
brighter (Doherty et al., 2010). Another, or additional,
explanation for weak mCherry fluorescence at the early
stages of sporulation could be a long maturation time for the
mCherry fluorophore in B. subtilis sporulating cells, as has
been suggested by de Jong et al. (2010). Finally, one has to
consider the amount of fusion protein present in the cell at
different stages of sporulation. We found that the level of
GFP-SpoVD fusion proteins was slightly lower in the final
stages of sporulation (immunoblot data not shown). In the
case of SpoVD-mCherry fusions, once sE became activated
in the mother-cell (it takes place after asymmetrical
septation), the proteins were produced and their level was
increasing (immunoblot data not shown). sE becomes
inactivated upon completion of engulfment and therefore
synthesis of SpoVD would decrease. We expect that the
mCherry fusion proteins expressed from our constructs
should peak when engulfment is completed. This did not
match the time point of the strongest fluorescence intensity,
which suggests a long maturation time or a stage-specific
fluorescence intensity of mCherry.
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Microbiology 161
Role of the PASTA domain in SpoVD
In summary, the role of the single PASTA domain in
SpoVD remains enigmatic. It is essential neither for the
catalytic activity of the transpeptidase domain nor for the
correct protein localization as demonstrated in this work.
In light of results with other PBPs of class B, the PASTA
domain of SpoVD seems to play a role in protein stability,
and could possibly be of some importance for efficient
binding of the substrate to the active site of the transpeptidase domain.
Eichenberger, P. (2012). Genomics and cellular biology of endospore
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acting proteins required for peptidoglycan synthesis during sporulation. J Mol Biol 399, 547–561.
Fleurie, A., Cluzel, C., Guiral, S., Freton, C., Galisson, F., ZanellaCleon, I., Di Guilmi, A. M. & Grangeasse, C. (2012). Mutational
dissection of the S/T-kinase StkP reveals crucial roles in cell division
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Fortnagel, P. & Freese, E. (1968). Analysis of sporulation mutants. II.
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ACKNOWLEDGEMENTS
This work was supported by grants from the Swedish Research
Council (621-2010-5672) and the Research School in Pharmaceutical
Sciences (FLÄK). We thank Karolina Ravinskaya for performing
preliminary experiments in this project, Elisabeth Barane for expert
assistance in the laboratory, Artur Kozak for writing a program to
calculate the background fluorescence in the microscopy pictures,
Klas Flärdh for assistance and support, and Michael Baureder for
comments on the manuscript.
Fraipont, C., Alexeeva, S., Wolf, B., van der Ploeg, R., Schloesser, M.,
den Blaauwen, T. & Nguyen-Distèche, M. (2011). The integral
membrane FtsW protein and peptidoglycan synthase PBP3 form a
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