Department of Biochemistry, University of
Connecticut Health Center, Farmington, Connecticut
06032,1 and
Department of Biology,
Virginia Polytechnic Institute and State University, Blacksburg,
Virginia 24061-04062
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TEXT |
The polymerization and cross-linking
of peptidoglycan in bacteria is catalyzed by a group of enzymes known
as penicillin-binding proteins (PBPs). In Bacillus subtilis,
a gram-positive bacterium that forms heat-resistant endospores upon
nutrient deprivation, PBPs are required not only for synthesis of
peptidoglycan in vegetative cells but also for synthesis of the
sporulation septum and the spore's primordial germ cell wall and
cortex (7).
Low-molecular-weight PBPs are usually monofunctional
DD-peptidases, which regulate the number of peptide
cross-links formed in the peptidoglycan (11, 12). To
date, three genes from B. subtilis
(dacA, dacB, and dacF) encoding
polypeptides with high sequence homology to low-molecular-weight
DD-peptidases have been cloned and characterized (8,
45, 49). The most well characterized of these is the
PBP5*-encoding gene, dacB, which is transcribed around stage
III of sporulation from a 
E-dependent promoter (8,
37), suggesting a role for PBP5* in spore cortex synthesis.
Indeed, spores of dacB null mutants are heat sensitive
(6, 30), and their cortex has more peptide side chains, a
higher degree of cross-linking, and less muramic acid lactam residues
than that of wild-type spores (4, 29, 30). The gene encoding
PBP5, dacA (45), accounts for most if not all
DD-carboxypeptidase activity in exponentially growing B. subtilis (22, 45, 47) and is present in
lower amounts in stationary-phase and sporulating cells
(39). Inactivation of PBP5 is not lethal for the cell
(5, 45) and also has no effect on spore heat resistance
(6, 30). However, overexpression of Bacillus
stearothermophilus dacA in Escherichia coli results in
cell lysis (10), and attempts to transform E. coli with a plasmid containing B. subtilis dacA
were unsuccessful (45). The dacF gene product has
not yet been identified biochemically, but studies using
dacF-lacZ transcriptional fusions showed that dacF is transcribed in the forespore compartment of the
sporulating cell (49) and that this transcription is
F dependent (36). Disruption of
dacF has no obvious effect on spore formation, spore cortex
structure, or spore properties (4, 29, 49), and thus the
function of this gene is unclear.
Recently, the B. subtilis genome sequencing project
(20, 46) identified the pbp gene (here renamed
dacC) encoding a putative 491-residue low-molecular-weight
PBP with highest sequence homology to E. coli PBP4
(19) and PBP4 from Actinomadura strain R39
(14). In this work we show that dacC
expression is dependent on transcription factor H
and that dacC does indeed encode a new membrane-bound PBP,
which migrates at the position of B. subtilis PBP4*,
between PBP4 and PBP5, on sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE). Therefore, we have named this protein
PBP4a. While a dacC mutation had no phenotypic effect in
B. subtilis, overexpression of dacC was
toxic to E. coli.
Transcriptional regulation of dacC.
To study the
transcriptional regulation of dacC, strain PS2323 carrying a
transcriptional dacC-lacZ fusion at the dacC
locus was constructed (all B. subtilis strains used in
this study are listed in Table 1). PCR
was used to generate a 654-bp fragment containing a part of the
B. subtilis genome starting 162 nucleotides (nt)
upstream and ending 480 nt downstream of the putative dacC translational initiation codon. The primers used for PCR were Y1 and Y2
(Table 2), and the template was
chromosomal DNA from strain PS832. Digestion of the 654-bp PCR
product with BamHI and EcoRI yielded a
649-bp fragment which was ligated into
BamHI/EcoRI-digested plasmid pUC19 to
generate plasmid pTMY1 (Fig. 1A). DNA
sequencing confirmed that the DNA sequence of the insert was correct.
The 649-bp BamHI/EcoRI fragment from pTMY1
was ligated into BamHI/EcoRI-digested plasmid pJF751a (43) to generate plasmid pTMY2 (Fig. 1A),
which was used to transform PS832 to generate strain PS2323, which
contains a transcriptional dacC-lacZ fusion at the
dacC locus. After Southern blot analysis was used to verify
that the chromosome structure of PS2323 was as expected (data not
shown), cells were sporulated at 37°C in 2× SG medium
(24), 1-ml samples were withdrawn at various times, and the
-galactosidase activities of the samples were measured using the
substrate 4-methylumbelliferyl--D-galactoside (27). As shown in Fig. 2A,
dacC-lacZ expression began shortly after the end of
exponential growth and peaked about 2 h into sporulation. However,
no -galactosidase activity was detected in purified spores of PS2323
(results not shown). The level of dacC-lacZ expression
detected was also much lower than for most sporulation genes;
when
o-nitrophenyl--D-galactopyranoside
was used as a substrate, the maximum -galactosidase activity
measured with strain PS2323 (dacC-lacZ) was only 7 to 8 Miller units compared with 2 to 3 Miller units for cells of the
wild-type strain, PS832 (data not shown).
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FIG. 1.
Diagram of the dacC locus, and constructs and
protein variants generated. (A) Map of the dacC locus. (I)
Putative ORFs are indicated by open boxes, potential transcription
terminators are shown as stem-loop structures, and the arrow depicts
the predicted transcription initiation site and direction of
transcription. (II) Fragments used in plasmid constructs for
insertional mutagenesis and for generation of transcriptional
dacC-lacZ fusions. (III) Map of selected restriction
endonuclease cleavage sites. (B) Schematic depiction of PBP4a variants
generated in this work. Amino acids 1 to 29 (gray) constitute a
cleavable signal peptide as described in the text. The three regions
that constitute the penicillin-binding site
(81SSLK84, 328SNN330,
and 440KTG442) were inferred by sequence
alignment of PBP4a with PBP4 from Actinomadura strain R39
(14) and E. coli PBP4 (19) using GCG
software (Wisconsin Package Version 9.1; Genetics Computer Group,
Madison, Wis.). Amino acids 470 to 491 (hatched) were predicted by a
computer analysis (DNA Strider 1.2) to form an amphipathic  -helix,
potentially serving as a membrane anchor. Numbers refer to amino acids
of the PBP4a primary sequence. The figure is not drawn to scale.
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FIG. 2.
Transcriptional regulation of dacC.
B. subtilis strains containing transcriptional
dacC-lacZ fusions at the dacC locus were grown
and sporulated at 37°C in 100 ml of 2× SG medium with no antibiotics
(24), the OD600 values of the cultures were
measured, and 1-ml samples were withdrawn for measurement of
-galactosidase activity as described in the text at the times
indicated (t0 is defined as the end of
exponential phase). Values on the y axis are fluorescence
units per OD600 unit of the cultures. (A) -Galactosidase
activities in strains lacking sigma factors. Symbols and strains
(relevant genotypes) are as follows:  , PS2323 (wild type);  ,
PS2459 (spo0H::Kanr);  , PS2522
( spoIIA::Spr); and  , PS2521
(spoIIGB::Ermr). (B) -Galactosidase
activity in strain PS2629 (Pspac-spo0H) with or
without IPTG (arrow indicates time of IPTG addition). (C)
-Galactosidase activities in transcription factor mutant strains.
Symbols and strains (relevant genotypes) are as follows:  , PS2632
(spo0A::Ermr);  , PS2630
(abrB::Tn917); and , PS2323 (wild type).
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The timing of its expression suggests that dacC may be a
stationary-phase- or early-sporulation-specific gene. To test
this hypothesis, mutations disrupting the genes encoding
H (spo0H::Kanr),
E (spoIIGB::Ermr), or the
F operon (spoIIA::Spr)
were introduced into strain PS2323 and dacC-lacZ
expression was monitored as described above. The
spo0H::Kanr mutation (PS2459) completely
abolished dacC-lacZ expression, while the effects of the
spoIIGB (PS2521) and spoIIA (PS2522) mutations
were minimal (Fig. 2A). To further analyze the dependence of
dacC expression on H, a dacC-lacZ
fusion strain (PS2629) which contains a truncated copy of the
spo0H gene under control of its normal promoter and an
intact copy of spo0H under the control of the
isopropyl--D-thiogalactopyranoside (IPTG)-inducible
promoter Pspac (18, 50) was generated and analyzed. Consistent with the observed lack of dacC-lacZ
expression in the spo0H::Kanr mutant
(PS2459) no significant -galactosidase activity was detected when
strain PS2629 was grown in 2× SG medium without an inducer (Fig. 2B).
In contrast, when 1 mM IPTG was added to an exponentially growing
culture of strain PS2629 at an optical density at 600 nm
(OD600) of 0.5, the -galactosidase activity measured
increased significantly, but only after more than 2 h of
induction, when the cells were well into stationary phase (Fig. 2B). A
likely explanation for this delay is that spo0H expression
is regulated posttranscriptionally so that induction of
spo0H expression does not lead to an immediate rise in
functional H levels (16, 48).
Transcription of spo0H occurs at a very low level during
vegetative growth due to repression of spo0H transcription
by the AbrB repressor (9, 48). This repression is relieved
by the key regulator of sporulation initiation, Spo0A (9,
41), which becomes activated by phosphorylation via a complex
signal transduction pathway when cells are deprived of nutrients
(17). In addition to affecting spo0H
transcription, AbrB also represses transcription of some other
sporulation genes by binding directly to their promoter regions
(34). To analyze the influence of Spo0A and AbrB on dacC transcription, spo0A::Ermr
and abrB::Tn917 mutations were introduced
into PS2323 to generate strains PS2632 and PS2630, respectively, and
the dacC-lacZ expression of these strains was measured.
Neither the spo0A::Ermr nor the
abrB::Tn917 mutation significantly altered
the level of dacC-lacZ expression, although the onset of
expression was slightly earlier in the strain with the abrB
mutation (Fig. 2C), perhaps due to a change in the timing of
spo0H transcription in this strain. The lack of effect of a
spo0A mutation on dacC-lacZ expression is in
contrast to what has been reported for most H-dependent
genes expressed around the onset of sporulation (15). The
lack of dacC-lacZ expression in a spo0A spo0H
double mutant (data not shown) excluded the possibility that the
dacC-lacZ expression observed in the
spo0A::Ermr mutant resulted from the
activation of another promoter in the dacC region. However,
when cells were grown in nutrient sporulation broth (DSM
[35]) instead of 2× SG, both the
spo0A::Ermr and the
abrB::Tn917 mutations abolished
dacC-lacZ expression while in a wild-type background, and
dacC-lacZ expression occurred with a similar timing as in
2× SG but the level of expression was about twofold lower (data not
shown). We have no simple explanation for these findings, but we have
found that omitting glucose from the 2× SG medium had no
effect on dacC-lacZ expression (results not shown). A
mutation in codY, a gene encoding a repressor of other
stationary phase-induced genes (38), also had no obvious effect on dacC-lacZ transcription when cells were grown in
2× SG medium (data not shown).
While H may affect transcription of some genes
indirectly by stimulating transcription of spo0A
(33), the strong dependence of dacC expression on
spo0H transcription suggests that the dacC promoter may be directly recognized by EH
(18). To identify potential regulatory sequences upstream of dacC, plasmid pTMY3 was introduced into PS832 to generate
strain PS2322, which contains a dacC-lacZ fusion at the
amyE locus. Plasmid pTMY3 is a derivative of pDG268
(3) that contains the 649-bp BamHI/EcoRI fragment from pTMY1 (Fig. 1A). No
significant -galactosidase activity was detected in this
strain (results not shown), suggesting that the dacC
promoter is upstream of the 649-bp
BamHI/EcoRI fragment in PS2322. The DNA
sequence of the region upstream of dacC suggests that
dacC may be in a two-gene operon with a gene of unknown
function termed yoxA (20). Strikingly, the region
immediately upstream of the putative yoxA translational
initiation codon contains sequences (5'-TGAAT-3' and
5'-GGAGGAAAT-3') separated by 14 bp that match perfectly the
10 and 35 consensus sequences of H-dependent
promoters (15, 33). To investigate whether this putative
H promoter is functional, a 290-bp fragment containing
the region from 13 nt upstream of the putative yoxA
initiation codon to 153 nt upstream of the putative yoeA
stop codon (yoeA is gene of unknown function located
upstream of yoxA [20]) was PCR amplified
from chromosomal DNA of strain PS832 using primers yoxa-P2a and yoea-3' (Table 2). The PCR product was ligated into pCR2.1 (Invitrogen), generating plasmid pLP1, and the DNA sequence of the insert was confirmed. Digestion of plasmid pLP1 with BamHI and
HindIII yielded a 236-bp fragment, which was cloned into
BamHI/HindIII-digested pDG268
(3), generating plasmid pLP2 (Fig. 1A), which was then introduced into strain PS832, generating strain PS2760, which contains
the yoxA-lacZ fusion at the amyE locus. Southern
blot analysis confirmed that the chromosome structure of strain PS2760 was as expected (results not shown). Measurement of the
-galactosidase activity in a sporulating culture of PS2760 showed
that the timing and level of expression of the yoxA-lacZ
fusion in this strain were identical to those of strain PS2323, which
contains a dacC-lacZ fusion at the dacC locus
(data not shown). This strongly suggests that the promoter
controlling dacC is located within the 236-bp BamHI/HindIII fragment in plasmid pLP2
and further supports the idea that dacC transcription
depends directly on H. However, due to the low level of
dacC transcription, we did not attempt to further
localize the dacC transcription start site.
Generation and analysis of an insertional dacC
mutant.
To begin to study the function of dacC, we
constructed strain PS2324 containing a disrupted dacC gene
by transformation of strain PS832 with plasmid pTMY4 (Fig. 1A). Plasmid
pTMY4 was constructed by digesting plasmid pTMY1 with NsiI
and SalI, which released an ~400-bp fragment from within
the coding region of dacC, and ligating this ~400-bp
fragment into PstI/SalI-digested
plasmid pJL73 (23). Transformation of strain PS832
with plasmid pTMY4 yielded strain PS2324, in which dacC has
been disrupted; Southern blot analysis verified that the genomic
structure of PS2324 was as expected (data not shown).
Membranes from vegetative cells of strains PS2324 (dacC) and
PS832 (wild type) and cells of the same strains harvested 2 h into
sporulation (t2 of sporulation) were purified
and incubated with fluorescein-hexanoic-6-aminopenicillanic acid
(FLU-C6-APA), proteins were separated by SDS-10% PAGE,
and PBPs were visualized with a fluorimager (FluorimagerSI; Vistra) as
described previously (32). Identical PBP profiles were
obtained for the two strains (results not shown), suggesting that PBP4a
is present at levels too low to be detected in B. subtilis and/or has a low affinity for penicillin. Strain PS2324
grew and sporulated, its spores germinated at rates comparable to those
of the wild-type strain, and dacC spores were as heat
resistant as wild-type spores (results not shown). In addition,
analysis of spore cortex structure by reversed-phase high-pressure
liquid chromatography (4, 29) showed no significant
structural differences between the cortices from dacC and
wild-type spores (results not shown). Thus, dacC appears to
be dispensable for B. subtilis under normal growth conditions.
Expression of dacC variants in E. coli.
Given the lack of detection of the dacC gene product in
B. subtilis, we overexpressed dacC in
E. coli in order to determine if it indeed encodes a PBP. We
also decided to overexpress several truncated forms of
dacC-encoded protein to study their function and
localization in E. coli. The four dacC-encoded
variants overexpressed in E. coli are depicted in Fig.
1B. PBP4a corresponds to full-length dacC-encoded
protein (491 residues), PBP4a-C is PBP4a lacking residues 470 to 491, PBP4a-N is PBP4a lacking residues 2 to 29, and PBP4a-NC is PBP4a
lacking both residues 2 to 29 and 470 to 491. Residues 1 to 29 are
found to constitute a cleavable signal peptide (see below), while
residues 470 to 491 are predicted to form an amphipathic
-helix (data not shown) that may constitute a C-terminal
membrane anchor commonly found in low-molecular-weight PBPs
(12). For PCR amplification of the regions encoding the PBP4a variants, primers (Table 2) were as follows: PBP4a, pbpy-5' and pbpy-3'; PBP4a-C, pbpy-5' and pbpy-P6; PBP4a-N, pbpy-P5 and pbpy-3'; and PBP4a-NC, pbpy-P5 and pbpy-P6. PCR products were ligated
into pCR 2.1 (Invitrogen), and the inserts were sequenced to confirm
their identity, removed by digestion with BamHI and NdeI, ligated into
BamHI/NdeI-digested pET11a
(42), and used to transform E. coli
BL21(DE3)/pLysS (42). The resulting E. coli strains were termed PS2599 (PBP4a), PS2690 (PBP4a-C),
2691 (PBP4a-N), and PS2692 (PBP4a-NC).
Recombinant E. coli strains were grown at 37°C to an
OD600 of ~0.5 in 50 ml of 2× YT medium (per liter:
16 g of tryptone, 10 g of yeast extract, 5 g of NaCl)
with chloramphenicol (20 µg/ml) and ampicillin (50 µg/ml), and IPTG
was added to 0.5 mM. After 2 h of further incubation, samples (1 ml) from induced cultures were pelleted by centrifugation, and proteins
were solubilized in 100 µl of SDS-sample buffer (21) and
analyzed by SDS-10% PAGE. A strong 59-kDa band was present in the
lanes containing proteins from induced PS2599 and PS2691, while lysates
of induced PS2690 and PS2692 gave a doublet of 57 and 58 kDa
(57-58-kDa doublet) and a strong 57-kDa band, respectively; these
bands were not present in lysates of induced cells which carried only
the vector (Fig. 3A). In some gels, the
protein whose synthesis was induced in strain PS2599 also migrated as a
59-60-kDa doublet (Fig. 3B, lane 2), suggesting that PBP4a undergoes
posttranslational processing, presumably removal of the signal
sequence, and that the efficiency of processing varies from experiment
to experiment. Although 57 to 59 kDa is larger than the theoretical
molecular mass of PBP4a (52.9 kDa including the N-terminal signal
peptide), the absence of any strong 57- to 59-kDa band in the extract
from strain PS2602, which harbors only the vector (Fig. 3A, lane 1),
strongly suggests that the 57- to 59-kDa bands are the PBP4a variants.
To confirm this, the proteins on a gel run parallel to the one shown in
Fig. 3A were transferred to a polyvinylidene difluoride membrane
and the amino-terminal sequences of the 57- to 59-kDa bands were
determined (28) (for proteins migrating as a doublet, the
lower band was sequenced). The sequence obtained for the major induced
bands for all PBP4a variants was AEKQD, corresponding to residues 30 to
34 of PBP4a, indicating that residues 1 to 29 constitute a signal
peptide. Fractionation of sonicated cells by a high-speed centrifugation method (44) showed that most (>90%)
of PBP4a, PBP4a-N, and PBP4a-C were membrane associated
(presumably in the inner membrane) while PBP4a-NC was present as
inclusion bodies in E. coli (data not shown). Thus, removal
of either the N-terminal signal peptide or the C-terminal putative
membrane anchor did not prevent PBP4a from being membrane associated;
the membrane association of PBP4a-C despite the lack of the putative
membrane anchor could be due to the hydrophobic character of the
protein or to expression of the protein in a heterologous system.
However, removal of both of these regions resulted in loss of
solubility and membrane association. In addition, removal of residues 1 to 29 at the N terminus dramatically increased the amount of PBP4a protein produced in E. coli (Fig. 3A, compare lanes 4 and 5 with lanes 2 and 3), while removal of residues 470 to 491 at the
C terminus of PBP4a had essentially no effect on expression levels (Fig. 3A, compare lanes 2 and 3).
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FIG. 3.
Expression of dacC variants and
penicillin-binding activity of PBP4a. (A) Expression of dacC
variants in E. coli. Recombinant E. coli strains were grown and induced, protein was solubilized as
described in the text, and 7 µl of each sample was analyzed by
SDS-10% PAGE and staining with Coomassie blue. Lanes, corresponding
strains, and proteins they express (parentheses) are as follows: 1, PS2602 (vector alone); 2, PS2599 (PBP4a); 3, PS2690 (PBP4a-C); 4, PS2691 (PBP4a-N); and 5, PS2692 (PBP4a-NC). Lane MW contains molecular
weight markers (molecular masses are in kilodaltons). Asterisks denote
migration positions of the PBP4a variants. (B) Analysis of PBPs in
membranes from induced E. coli strains PS2602 (lane 1;
vector alone) and PS2599 (lane 2; PBP4a). Cells were grown and induced
as for panel A for 90 min, 25 ml of culture was harvested by
centrifugation, membranes were isolated from sonicated cells by
centrifugation (100,000 × g, 1 h) and incubated
for 30 min at 30°C with 100 µM FLU-C6-APA, proteins
(~10 µg) were analyzed by SDS-10% PAGE, and PBPs were visualized
with a FluorimagerSI (Vistra). A lane containing labeled PBPs from
vegetative B. subtilis cells of strain PS832
(32) is shown for comparison (lane 3; the PBPs corresponding
to each band are indicated on the right). Asterisks denote position of
the 59-60-kDa PBP4a doublet.
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The PBP4a signal peptide contains three lysines within the
amino-terminal six residues, a hydrophobic core region of 15 residues that terminates with a proline, and alanine residues at positions 3,
1, and +1 relative to the cleavage site. These features are similar
to those of other B. subtilis signal peptides
(26), suggesting that the PBP4a variants were processed in
E. coli as one might expect them to be processed in
B. subtilis.
PBP4a binds penicillin.
To analyze whether recombinant PBP4a
binds penicillin, membranes from cells of induced strain PS2599 or
PS2602 were incubated with FLU-C6-APA, proteins were
separated by SDS-10% PAGE, and bands were visualized by fluorimaging
(Fig. 3B). A labeled 59-60-kDa doublet was present in membranes from
strain PS2599 (Fig. 3B, lane 2) but not in labeled membranes from
strain PS2602 harboring only the vector (Fig. 3B, lane 1), suggesting
that the 59-60-kDa doublet is PBP4a. Comparison with
FLU-C6-APA-labeled membranes from vegetative cells of
strain PS832 or from cells of the same strain harvested at
t2 of sporulation showed that recombinant PBP4a
from E. coli membranes migrated at the same position as B. subtilis PBP4*, between PBP4 and PBP5 (Fig. 3B,
lane 3, and data not shown). However, we could not detect PBP4a in
membranes isolated from sporulating cells of strain PS1805, which lacks PBP4* (data not shown).
PBP4a variants affect growth and viability of E. coli.
Overexpression of dacA from B. subtilis or B. stearothermophilus appears to
be toxic for E. coli (10, 45). To
investigate whether this was also the case for dacC,
cultures of strains PS2599, PS2690, PS2691, PS2692, and the control
strain PS2602 were grown to an OD600 of ~0.5 and induced
with 0.5 mM IPTG and the OD600 was monitored. All cultures
continued to grow for 30 min after induction, but after 60 min, the
OD600 of cells expressing PBP4a, PBP4a-C, and PBP4a-N
stopped increasing, while the culture expressing PBP4a-NC continued to
grow at the same rate as the strain carrying the vector alone (Fig.
4A). Consistent with these observations, determination of the number of viable cells in the induced cultures showed decreased viability of strains PS2599, PS2690, and PS2691 30 min
after induction while the number of viable cells in induced cultures of
strains PS2602 and PS2692 increased throughout the 120-min induction
period (Fig. 4B). Thus, expression of PBP4a, PBP4a-C, and PBP4a-N is
toxic for E. coli, while expression of PBP4a-NC is not.
The lack of toxicity of PBP4a-NC may be related to its presence as
inclusion bodies in E. coli.
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FIG. 4.
Effects of PBP4a variants on growth and viability of
E. coli. Recombinant E. coli strains
were grown and induced with IPTG as described in the text, and the
OD600 (A) and viability (B) were measured after induction.
The viability was measured by plating dilutions on 2× YT agar plates
containing chloramphenicol (20 µg ml 1) and ampicillin
(50 µg ml1). Symbols, strains, and proteins they
express (parentheses) are as follows:  , PS2602 (vector); , PS2599
(PBP4a); , PS2690 (PBP4a-C); , PS2691 (PBP4a-N); and , PS2692
(PBP4a-NC).
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Enzymatic activity of PBP4a?
During the course of growth and
induction of recombinant E. coli, microscopic
examination of cultures expressing PBP4a, PBP4a-C, or PBP4a-N revealed
the presence of lysed cells, and in cultures expressing PBP4a or
PBP4a-C, spherical cells were occasionally observed (results not
shown). Lysed and/or spherical cells have previously been observed in
recombinant E. coli cells that overexpress proteins
with DD-carboxypeptidase activity (10,
25), suggesting that PBP4a may have a similar enzymatic
activity. Preliminary efforts to determine the enzymatic activity of
PBP4a by reversed-phase high-pressure liquid chromatography analysis of
purified cell walls from induced recombinant E. coli
(13) or by using PBP4a-NC purified from inclusion bodies
from strain PS2692 in in vitro assays (1) were unsuccessful.
Thus, the enzymatic activity of PBP4a, if any, remains to be
determined.
In summary, we have shown that (i) dacC transcription
depends strongly on transcription factor H and appears
to be initiated from a promoter immediately upstream of the
yoxA gene; (ii) disruption of dacC has no
dramatic effects on B. subtilis growth,
sporulation, and spore properties; and (iii) dacC encodes a
membrane-bound PBP which is toxic when overexpressed in E. coli.
We are grateful to A. D. Grossman, P. Stragier, A. L. Sonenshein, R. Losick, and S. Meyer for B. subtilis strains.
This work was supported by a grant from the National Institutes of
Health to P.S. (GM19698) and a postdoctoral fellowship from the Danish
Natural Science Research Council to L.B.P. (9601026).
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