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Journal of Bacteriology, January 1999, p. 126-132, Vol. 181, No. 1
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Roles of Low-Molecular-Weight Penicillin-Binding
Proteins in Bacillus subtilis Spore Peptidoglycan
Synthesis and Spore Properties
David L.
Popham,1,*
Meghan E.
Gilmore,1 and
Peter
Setlow2
Department of Biology, Virginia Tech,
Blacksburg, Virginia 24061,1 and
Department of Biochemistry, University of Connecticut
Health Center, Farmington, Connecticut 06030-33052
Received 2 September 1998/Accepted 28 October 1998
 |
ABSTRACT |
The peptidoglycan cortex of endospores of Bacillus
species is required for maintenance of spore dehydration and dormancy, and the structure of the cortex may also allow it to function in
attainment of spore core dehydration. A significant difference between spore and growing cell peptidoglycan structure is the low
degree of peptide cross-linking in cortical peptidoglycan; regulation
of the degree of this cross-linking is exerted by
D,D-carboxypeptidases. We report here the
construction of mutant B. subtilis strains lacking all
combinations of two and three of the four apparent D,D-carboxypeptidases encoded within the genome
and the analysis of spore phenotypic properties and peptidoglycan
structure for these strains. The data indicate that while the
dacA and dacC products have no significant role
in spore peptidoglycan formation, the dacB and
dacF products both function in regulating the degree of
cross-linking of spore peptidoglycan. The spore peptidoglycan of a
dacB dacF double mutant was very highly cross-linked, and this structural modification resulted in a failure to achieve normal
spore core dehydration and a decrease in spore heat resistance. A model
for the specific roles of DacB and DacF in spore peptidoglycan synthesis is proposed.
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INTRODUCTION |
Peptidoglycan (PG) is the structural
element of the bacterial cell wall which determines cell shape and
which resists the turgor pressure within the cell. The bacterial
endospores produced by species of Bacillus,
Clostridium, and several other bacterial genera are modified
cells that are able to survive long periods and extreme conditions in a
dormant, relatively dehydrated state. The PG wall within the endospore
is required for maintenance of the dehydrated state (10,
11), which is the major determinant of spore heat
resistance (2, 17, 22). Spore PG appears to be comprised of
two distinct though contiguous layers. The thin inner layer, the germ
cell wall, appears to have a structure similar to that of the
vegetative wall and serves as the initial cell wall of the germinated
spore (1, 20, 21, 31). The thicker outer layer, the spore
cortex, has a modified structure which may determine its ability to
carry out roles specific to the spore, and is rapidly degraded during
spore germination (1, 20, 35, 37). The most dramatic of the
cortex structural modifications results in partial cleavage or complete
removal of ~75% of the peptide side chains from the glycan strands.
Loss of these peptides limits the cross-linking potential of the PG and
results in the formation of only one peptide cross-link per 35 disaccharide units in the spore PG, compared to one peptide cross-link
per 2.3 to 2.9 disaccharide units in the vegetative PG (1, 20,
36). This low degree of cross-linking has been predicted to give
spore PG a flexibility that allows it to have a role in attainment of
spore core dehydration (14, 34) in addition to its clear
role in maintenance of dehydration. We are studying the structure and
mechanism of synthesis of spore PG in an attempt to discern the roles
of this structure and its individual components in determining spore properties.
A family of proteins called the penicillin-binding proteins
(PBPs) polymerizes PG on the external surface of the cell
membrane (reviewed in reference 7). The
high-molecular-weight (high-MW) members of this family (generally
60
kDa) carry the transglycosylase and transpeptidase activities involved
in polymerization and cross-linking of the glycan strands.
The low-MW PBPs have commonly been found to possess
D,D-carboxypeptidase activity. This activity
can remove the terminal D-alanine of the peptide side
chains and thereby prevent the side chain from serving as a donor in
the formation of a peptide cross-link. Analysis of the B. subtilis genome reveals six low-MW PBP-encoding genes:
dacA (33), dacB (4),
dacC (19), dacF (38),
pbpE (23), and pbpX (accession no.
Z99112). The four dac gene products exhibit very high
sequence similarity to proven
D,D-carboxypeptidases, and this activity has
been demonstrated in vitro for the dacA and dacB
products, PBP5 (12) and PBP5* (32), respectively.
The sequences of the pbpE and pbpX products are
more distantly related, and no activity has yet been established or
ruled out for them.
PBP5 is the major penicillin-binding and
D,D-carboxypeptidase activity found in
vegetative cells (12). Although dacA
expression declines significantly during sporulation, a significant
amount of PBP5 remains during the time of spore PG synthesis
(29). A dacA-null mutation results in no obvious
effects on vegetative growth, sporulation, spore characteristics, or
spore germination (3, 33). However, loss of PBP5 does
result in a reduction of cleavage of peptide side chains from
the tetrapeptide to the tripeptide form in the spore PG
(20). PBP5* is expressed only during sporulation and only in
the mother cell compartment of the sporangium, under the control of the
RNA polymerase
E subunit (4, 5, 28, 29). A
dacB-null mutation leading to loss of this
D,D-carboxypeptidase results in a fourfold
increase in the effective cross-linking of the spore PG (1, 20,
22). This structural change is accompanied by only slight
decreases in spore core dehydration and heat resistance (3,
22). The suspected D,D-carboxypeptidase
activities of the products of the dacC and dacF
genes have not been demonstrated. The latter two genes are expressed
only during the postexponential growth phase: dacC is
expressed during early stationary phase under the control of
H (19) and dacF is expressed only
within the forespore under the control of
F (27,
38). Null mutations effecting either gene result in no
obvious phenotype and no change in spore PG structure (19, 38).
The multiplicity of these proteins in sporulating cells and the lack of
effect of loss of some of them suggested redundancy of function among
these proteins, a situation observed previously with PBPs of a high-MW
class (25, 30, 39). In order to examine this possibility we
have constructed mutants lacking multiple low-MW PBPs and have examined
their sporulation efficiency, spore PG structure, spore heat resistance
and wet density, and spore germination and outgrowth. The present study
demonstrates a role for the dacF gene product in synthesis
of spore PG, and we also present a model for the roles of the
dacB and dacF gene products in spore PG formation.
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MATERIALS AND METHODS |
Bacterial strains, growth, sporulation, spore germination, and
spore properties.
All B. subtilis strains described in
Table 1 are derivatives of strain 168. Growth rates were determined and sporulation was carried out in 2×SG
medium containing no antibiotics (13) at 37°C. Spores were
purified by washing with water as previously described (18)
and were stored in H2O at 4°C. Microscopic examination indicated that all spore preparations were >98% free of vegetative cells, sporulating cells, or germinated spores. Dipicolinic acid (DPA)
was measured as described previously (18). Spore viability was assayed by plating dilutions of untreated spores on Luria broth
plates (16). Spore chloroform resistance was assayed by vortex mixing of spores in a 10% (vol/vol) suspension of chloroform for 1 min prior to dilution and plating. For determination of spore
heat resistance, identical samples of purified spores were heated in
H2O at 85°C for various times, and survivors on Luria broth plates were enumerated (16). For determination of
germination rates, spores were heat activated in H2O at
70°C for 30 min prior to germination in 2× YT medium (16 g of
tryptone, 10 g of yeast extract, and 5 g of NaCl per liter)
containing 4 mM L-alanine at 37°C. Spore protoplast wet
density was determined by using metrizoic acid or Nycodenz (Sigma)
gradients as previously described (15, 22). Dormant spores
were permeabilized (incubation in 50 mM Tris HCl [pH 8.0], 8 M urea,
1% sodium dodecyl sulfate, 50 mM dithiothreitol for 60 min at 37°C)
and washed five times in H2O prior to density
determinations such that the gradient material could permeate the spore
coats and cortex (15, 22).
Strain construction.
For construction of new dacA
mutations, primers complementary to positions 80806 to 80829 and 82494 to 82516 in the dacA sequence (accession no. D26185) were
used to PCR amplify the gene. The 1,710-bp PCR product was cut with
StuI and SacI at sites which occur upstream and
downstream of the gene, respectively, and a 1,654-bp fragment was
inserted into HincII-SacI-digested pUC19 to
produce pDPV51. A Spr cassette, the 1,148-bp
PstI fragment of pDG1726 (9), was inserted into a
unique NsiI site at codon 31 of dacA in pDPV51 to
produce pDPV54. B. subtilis PS832 was transformed with
ScaI-linearized pDPV54 with selection for Spr.
Recombination of the Spr cassette into the genomic copy of
dacA by a double-crossover event to produce strain DPVB6 was
verified by Southern blotting. A deletion removing codons 7 to 159 of
dacA (resulting mutant termed
dacA) was
constructed by digesting pDPV51 with HincII and ligating to
produce pDPV53. A 1,200-bp EcoRI-SphI fragment from pDPV53 containing
dacA was inserted into
EcoRI-SphI-digested pJH101 (6) to
produce pDPV55. This plasmid was transformed into PS832, and selection
for Cmr gave transformants in which the plasmid had
inserted into the dacA locus by a single crossover event.
These Cmr transformants were screened by Southern blotting
to identify one, DPVB9, in which a gene conversion event resulted in
both copies of dacA having the internal deletion. DPVB9 was
grown nonselectively through ~40 generations and then plated
nonselectively for single colonies. The resulting colonies were
screened for chloramphenicol sensitivity. One chloramphenicol-sensitive
isolate, DPVB10, was demonstrated by Southern blotting to have lost the
inserted plasmid and to have a single copy of the dacA with
the internal deletion in its chromosome.
Spore PG structure determination.
Spore PG was extracted and
digested with a muramidase (Mutanolysin; Sigma) and muropeptides were
analyzed by reversed-phase high-performance liquid chromatography
(HPLC) as described previously (1, 20). For some samples the
cross-linking of Mutanolysin-solubilized muropeptides was determined by
amino acid analysis as described previously (24). The amount
of diaminopimelic acid (Dpm) detected prior to and following
1-fluoro-2,4-dinitrobenzene (FDNB) treatment was normalized to the
amount of glutamic acid detected in each sample. Amino acid analysis
was carried out as previously described (8). The HPLC system
used for PG structure and amino acid analyses consisted of a Waters
600E controller and pump, a Waters 486 UV detector, and a Powerchrom
hardware and software system (ADInstruments Inc.) on an Apple
PowerMacintosh 5400 computer, used for signal integration.
 |
RESULTS |
Growth and sporulation of dac mutant strains.
The
growth rates and sporulation efficiencies of each of the single
dac mutant strains have been previously reported (19, 22, 33, 38). In all cases the vegetative growth rates and the
sporulation efficiencies are indistinguishable from those of the
wild-type strain. Since dacA is the only one of the four genes expressed during vegetative growth, it was expected that in no
case would loss of additional dac gene products have an effect on the growth rate, and this was indeed the case for all the
strains analyzed (data not shown). When cultures were assayed for the
production of chloroform-resistant spores 24 h after the initiation of sporulation, it was found that all mutant strains, except
those lacking both dacB and dacF, sporulated as
efficiently as the wild type, producing approximately 109
spores/ml (data not shown). Double and triple mutants lacking both
dacB and dacF appeared to produce as many spores
as the wild type within the first 12 h of sporulation (>90% of
cells produced spores based upon microscopic observation). However,
these spores lost viability during further incubation, and only 10 to
35% of the spores of these mutants were able to produce colonies after 24 h at 37°C. During the time that these spores were purified by
repeated washing in H2O at 4°C they retained this level
of viability; a suspension of dacB dacF spores at a
particular optical density produced only 10% as many colonies as a
similar suspension of wild-type spores.
Heat resistance and wet density of dac mutant
spores.
Purified spores produced by the wild type and each of the
mutant strains were also tested for their heat resistance (Table 2). As observed previously, the only
single dac mutation that resulted in an alteration in spore
heat resistance was in dacB (3, 19, 22, 38). No
combination of dac mutations that did not include
dacB, even the dacA dacC dacF triple mutant, had any effect on spore heat resistance. Among dacB mutants, the
additional loss of dacA, dacC, or both produced
no change in spore heat resistance. However, disruption of
dacF in a dacB mutant did result in a more dramatic loss of heat resistance. Once again, the further loss of
dacA or dacC produced no additional change in
spore heat resistance.
The relative dehydration of the spore core has previously been shown to
be directly related to spore heat resistance (
2,
17,
22).
Spore core dehydration was measured as spore wet
density by equilibrium
density gradient centrifugation. Prior
to density determination the
spores were chemically permeabilized
to allow penetration of the
gradient matrix through the spore
coats and peptidoglycan layers. This
allows specific assay of
the density of the spore core and has no
effect on the heat resistance
of the spores (
15,
22). In no
case did loss of
dacA,
dacC,
or both have any
significant effect on spore core dehydration
(Table
2, changes of

0.004 g/ml). The
dacF mutation also had
no large effect on
spore core dehydration, even in a
dacA dacC dacF triple
mutant, until it was combined with a
dacB mutation
(Table
2). As we found previously (
22), the reduced heat resistance
of
dacB spores is accompanied by little or no change in
spore
core dehydration (Table
2). However, the reduced heat resistance
of these spores is explained by the fact that they begin to take
up
water during heating, with an accompanying loss of heat resistance
(
22). The
dacB dacF double and triple mutant
spores did have
a significantly reduced spore core wet density (Table
2) (changes
of >0.01 g/ml), even in the absence of
heating.
PG structure in dac mutant spores.
Previous
work has shown that changes in spore heat resistance are often
accompanied by changes in spore PG structure (1, 20).
Consequently, PG was extracted from purified spores of the wild-type
strain and the various dac mutants and was digested with a
muramidase, and the resulting muropeptides were analyzed by
reversed-phase HPLC (Fig. 1; Table
3). As observed previously, single
mutations in dacC and dacF produced no
significant alteration in the observed muropeptide profiles, while a
mutation in dacA resulted in a twofold decrease in the
number of tripeptide side chains (1, 19, 20) (Table 3). A
dacB mutation produces the largest effects, a nearly twofold
increase in the number of tetrapeptide side chains with a corresponding
decrease in L-Ala side chains and a two- to threefold
increase in the percentage of side chains that are cross-linked
(1, 20) (Fig. 1B; Table 3). Strains containing combinations
of these mutations in most cases produced muropeptide profiles
resembling a combination of those produced by the parent strains.
Strains with combinations of dacA, dacC, and
dacF mutations showed only the reduction of tripeptide side
chains characteristic of the dacA mutation, and dacA
dacB double mutants had an exact combination of the muropeptide alterations produced by the two single mutations (Table 3).

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FIG. 1.
HPLC separation of spore PG muropeptides. Muropeptides
produced by muramidase digestion of purified PG from spores of PS832
(wild-type) (A), PS2066 ( dacB) (B), and PS2421
( dacB dacF::Cm) (C) were separated
by using a methanol gradient system (20). Peaks labeled B
are buffer components seen in control samples. Peaks are numbered as
previously described (20). Peaks 1, 2, and 3 are
disaccharides with tripeptide (DS-TriP), alanine, and tetrapeptide
(DS-TP) side chains, respectively. Peaks 10 and 13 are tetrasaccharides
with tetrapeptide (TS-TP) and alanine side chains, respectively. Peaks
18 and 19 are hexasaccharides with tetrapeptide (HS-TP) and alanine
side chains, respectively. Peaks 8, 9, 14, 17, and 20 are cross-linked
DS-TriP-DS-TP, DS-TP-DS-TP, DS-TP-TS-TP, TS-TP-TS-TP, and
TS-TP-HS-TP, respectively. Peaks 6, 7, 11, and 12 are reduction
products of peaks 13, 10, 14, and 17, respectively. Peaks labeled X are
also reduction products of muramic- -lactam-containing muropeptides;
corrections for this reduction were performed in calculation of PG
structural parameters (20).
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|
The exceptions to this trend were
dacB dacF strains (Fig.
1C; Table
3), as loss of
dacF produced a dramatic change in
the
spore PG structure above that produced by the
dacB
mutation. Most
notable was the appearance of a large number of novel
muropeptide
peaks eluting in the later part of the gradient (Fig.
1C
[60 to
110 min]), in the region where high-mass muropeptides elute
(note
also the elevated baseline in Fig.
1C compared to those in Fig.
1A and B). These novel peaks and the elevated baseline were observed
in
several independent muropeptide preparations from
dacB dacF spores as well as from spores of the triple mutants lacking these
two
genes. We calculated some spore PG structural parameters for
these
strains by using data for only the identifiable peaks (Table
3).
However, it is important that these calculations do not take
into
account a large percentage of the muropeptides present in
unidentified
peaks late in the gradient. In general the peaks
eluting in the latter
half of the gradient include muropeptides
containing multiple
muramic-

-lactam residues, hexasaccharides,
and cross-linked
compounds (
1,
20). Reduction of a muropeptide
containing two
muramic-

-lactam residues can give rise to eight
different products
(there are two possible reduction products
for each lactam residue) and
possibly eight different peaks (
1,
20,
37). Our
interpretation of the
dacB dacF double-mutant
muropeptide
chromatogram is that there was a very high level of
peptide
cross-linking, giving rise to trimer and possibly tetramer
muropeptides. The very large number of reduction products for
such
compounds results in a large number of small peaks eluting
late in the
HPLC gradient and thus the elevated baseline seen
in Fig.
1C. To verify
this postulated high level of cross-linking
we used FDNB modification
of Dpm residues in the peptide side
chains, followed by amino acid
analysis to estimate cross-linking
(
24). This method is not
as accurate as the HPLC muropeptide
analysis (
1,
20,
24),
but it does provide values useful
for comparison of the relative
cross-linking between samples.
The cross-linking values obtained for
dacB dacF double- and triple-mutant
spore PG were in most
cases significantly higher than those produced
by the
dacB
single-mutant spore PG (Table
3). We attribute the
slightly lower
cross-linking value for the
dacA dacB dacF triple
mutant
(Table
3) to the instability of these spores and potential
degradation
of some of the spore
PG.
Germination and outgrowth of dac mutant spores.
Modification of cortical PG structure can result in alteration of not
only spore heat resistance but also spore germination properties
(1, 20, 24). Consequently, we tested each of the purified
spore preparations for their germination and outgrowth (data not
shown). All of the spore preparations were able to initiate spore
germination, as evidenced by a decrease in optical density of spore
suspensions, with kinetics similar to those of wild-type spores.
Spores of strains with combinations of mutations in dacA, dacC, and dacF also commenced outgrowth with
kinetics indistinguishable from those of the wild type, while
dacB spores were delayed in outgrowth, as observed
previously (22). All the double and triple dac
mutant spores that contained a dacB mutation exhibited a
similar delay in outgrowth. For mutants lacking both
dacB and dacF the delay in spore outgrowth was in
some cases even greater; this is likely due to the low viability of
these spores.
 |
DISCUSSION |
Analysis of the phenotype and PG structure of spores produced by
strains containing multiple dac mutations has allowed us to
determine the roles and potential redundancies of the multiple carboxypeptidases present during B. subtilis sporulation. As
observed previously, a dacA mutation had only a very slight
effect on spore PG structure but no effect on spore phenotype (3,
20, 33). This change in PG structure was also seen in spores of
multiple dac mutants lacking dacA but was never
correlated with any obvious phenotype. A mutation in dacC
also had no effect on spore phenotype and no effect on spore PG
structure (19). Even in combination with mutations in
dacF or dacB, no change in spore PG structure or
phenotype due to a dacC mutation could be observed.
The effects of a dacB mutation include a large increase in
PG cross-linking (1, 20, 22) which is associated with an inability to maintain spore core dehydration upon heating and therefore
a slight loss in spore heat resistance (22). No significant effect of a dacF mutation was previously found on spore PG
structure (1, 20, 22), dehydration (22), or heat
resistance (22, 38). Apparently, DacF is at least
partially redundant with DacB, such that it is only in the
absence of DacB that a function for DacF can be observed. Spores of
dacB dacF mutant strains exhibited a variety of phenotypes
different from those of dacB spores, including lower
viability, higher PG cross-linking, higher core water content, and
decreased heat resistance. It is likely that all of these differences
are the result of the loss of DacF carboxypeptidase activity, which
would lead to an increase in the number of cortical PG side chains
available for cross-link formation above that in the dacB
strain. If the increased cross-linking in dacB dacF spores results in the inability to achieve or maintain normal spore core dehydration, reduced spore heat resistance would be a direct result. Loss of viability, even at a low temperature, may also be a result of
increased spore core water content, as increased hydration of spore
core macromolecules may result in a decrease in their stability and/or
an increased rate of spontaneous damage.
Interestingly, spm mutant spores have a similar defect in
spore core dehydration, yet they do not lose viability as fast or to as
great a degree as the dacB dacF double-mutant spores
(22). This may be due to the type of defect that results in
the increased spore water content. The spm mutant spores
have a relatively normal spore PG structure (22), while the
altered structure of the dacB dacF spore PG may render the
spore more susceptible to loss of some additional protective factor.
This additional factor was not DPA, as the double-mutant spores
retained wild-type levels of DPA throughout their purification (data
not shown). The spore germination machinery must be relatively
insensitive to the dacB dacF mutant spores' loss of
viability and altered PG structure, as they retained the ability to
initiate germination and apparently to degrade cortex PG. However, the
delay in outgrowth of dacB and dacB dacF mutant
spores could be related to a slowed degradation of the highly
cross-linked spore PG (22).
The significant sequence similarity and apparent partial
functional redundancy of the dacB and
dacF products suggests that they both have
D,D-carboxypeptidase activities that are
involved in regulating spore PG cross-linking. Why is there no obvious effect of the dacF mutation alone and what might be the
purpose of these redundant activities? The lack of phenotypic change
associated with a dacF mutation could be partially due to a
significant difference in the abundance of the DacB and DacF proteins.
While studies directly comparing the expression of these two
dac genes have not been carried out, two lines of evidence
suggest that dacB is more strongly expressed than
dacF. Data on the expression of transcriptional and
translational fusions of the two genes to lacZ indicate that
dacB-lacZ fusions are expressed at a level 10- to 50-fold
higher than the level of endogenous B. subtilis
-galactosidase activity, while dacF-lacZ expression is
only 2- to 5-fold higher than the endogenous activity (22,
38). However, interpretation of these results is complicated by
differences in growth media and strain backgrounds. The second line of
evidence suggesting greater expression of dacB than
dacF involves detection of the proteins with labeled
-lactams. DacB is easily detected by standard procedures using
radio- or fluorescence-labeled
-lactams (3, 26,
29), while multiple attempts to clearly identify DacF by
these procedures have failed (26). However, the inability to
visualize DacF by these methods could be due to poor binding of the
-lactams by DacF, rapid decay of the complex, or poor recovery of
the inner forespore membrane.
It is interesting that dacB is expressed only in the mother
cell compartment of the developing sporangium (4, 28) while dacF is expressed only in the forespore compartment
(27). Most of the spore PG is believed to be synthesized
from the mother cell side of the developing spore wall (31).
If the dacB and dacF products are each
transported across the membrane into the intermembrane space in which
the spore PG is synthesized, then they may have greatly different
opportunities for acting upon the nascent spore PG. DacF may only have
access to the limited amount of PG synthesized from the forespore side.
This portion of the spore PG is generally thought to represent the germ
cell wall PG (31). In contrast, the dacB product,
PBP5*, may have access to the much greater amount of PG synthesized
from the mother cell side, generally thought to represent the spore
cortex. This model would explain the large effect on spore PG structure
produced by a dacB mutation and the insignificant effect of
a dacF mutation. However, the structure of the PG of
dacB dacF spores suggests that this model is overly
simplistic. If DacF acted only on the germ cell wall then it should
affect only muropeptides which do not contain muramic-
-lactam, as
this modification appears to be confined to the cortical PG (1,
20, 21). However, in the dacB dacF mutant spores there
is clearly an increase in cross-linking of
muramic-
-lactam-containing muropeptides over that seen in dacB spores, indicating that DacF can indeed participate in
cortical PG synthesis.
We propose another model for the effects of
D,D-carboxypeptidases on spore PG
structure, in which a gradient of peptide cross-linking is produced
during cortex synthesis (Fig. 2). A
cortex in which the innermost layers are loosely cross-linked and the
outer layers are much more highly cross-linked might have the
mechanical properties suggested by the anisotropically expansive cortex
model of spore core dehydration proposed by Warth (34). The
presence of both PBP5* and DacF at the initiation of spore PG
synthesis would result in low cross-linking in the innermost layers of
the spore cortex, closest to the inner forespore
membrane. As cortex synthesis progresses outward, the effect of DacF
diminishes due to its association with the inner forespore
membrane or a decrease in its enzymatic activity through protein
instability and/or decreased expression. At a certain point only PBP5*
would be determining the degree of PG cross-linking. If the
gradient of PG cross-linking were to continue after this point, then
the relative effect of PBP5* would have to decrease over time. This may
be a function of reduced expression of dacB during later
sporulation, as transcription of dacB begins and peaks early
during sporulation and decreases dramatically around the time of
initiation of cortex synthesis (28). The activity of PBP5*
may therefore decrease relative to the rate of cortex PG synthesis,
through protein instability or through expansion of the surface area
over which cortex synthesis is taking place, resulting in a gradient of
cross-linking independent of DacF.

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FIG. 2.
Schematic representation of a model for spore PG
synthesis and spore core dehydration. Only the developing forespore is
shown; the forespore is contained within the mother cell cytoplasm
during sporulation. (A) DacB is expressed in the mother cell and
appears on the outer forespore membrane while DacF is expressed in the
forespore and appears on the inner forespore membrane; DacB is
expressed at a level higher than is DacF. Both of these
D,D-carboxypeptidases act on the first layers
of spore PG synthesized, resulting in a very low level of peptide
cross-linking. (B and C) Additional layers of PG are synthesized from
the mother cell side (adjacent to the outer forespore membrane). DacB
specific activity progressively decreases due to protein degradation
and/or expansion of the surface area for PG synthesis. DacF is too
distant to affect the cross-linking of these layers. DacF specific
activity may also decrease, but this is not a necessary feature of the
model. PG cross-linking increases as
D,D-carboxypeptidase activity decreases,
resulting in a gradient of cross-linking across the span of the spore
cortex PG. (D) The cortex PG swells due to its overall low level of
cross-linking. The swelling is predominantly inward due to the gradient
of cross-linking, resulting in a decrease in the volume and water
content of the spore core. Shaded arcs and short lines connecting arcs
represent newly synthesized PG strands and peptide cross-links,
respectively. Solid arcs and connecting lines represent PG and
cross-links produced during the earlier stages of synthesis.
Abbreviations: B, DacB molecule; F, DacF molecule.
|
|
This model can explain the lack of effect of a dacF mutation
alone on spore PG structure. In a dacF mutant the innermost
layers of the cortex might be slightly more cross-linked (such a slight increase was observed previously [20]), but PBP5* by
itself may be able to produce a gradient of cross-linking which is
sufficient to allow normal spore formation. In a dacB mutant
the activity of DacF could produce a gradient of cross-linking within
the innermost layers of the cortex, but the remainder of the cortex
would have the high cross-linking observed (1, 20, 22)
(Table 3). This slight cross-linking gradient may allow the attainment
of normal spore core dehydration but might be insufficient to
allow maintenance of core dehydration upon heating (22). The
potential difference in the expression levels for dacB and
dacF has no effect on this model, since the important
factors for generation of a gradient of cross-linking are the
spatial separation of the two gene products and some decay of
PBP5* activity over time. The dacB dacF double
mutant's spores would have a high degree of cross-linking throughout the cortex, essentially nullifying the cortex's ability to participate in spore core dehydration. The remaining degree of core
dehydration observed in the double mutant spores may be attributable to another spore dehydration mechanism(s). In order to
obtain definitive evidence for this model of cortex PG synthesis, a
gradient of PG cross-linking within the spore cortex PG needs to be
demonstrated directly. This will require analysis of spore PG structure
throughout its synthesis, and these analyses are in progress.
 |
ACKNOWLEDGMENTS |
This work was supported by grants GM19698 (to P.S.)
and GM56695 (to D.L.P) from the National Institutes of Health and
by funds from Virginia Tech.
We acknowledge the technical assistance of Laura Koller in the
completion of this work. We thank R. Schuch, P. J. Piggot, T. Murray, and L. B. Pedersen for supplying strains.
 |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biology, 2119 Derring Hall MC0406, Virginia Tech, Blacksburg, VA
24061. Phone: (540) 231-2529. Fax: (540) 231-9307. E-mail:
dpopham{at}vt.edu.
 |
REFERENCES |
| 1.
|
Atrih, A.,
P. Zöllner,
G. Allmaier, and S. J. Foster.
1996.
Structural analysis of Bacillus subtilis 168 endospore peptidoglycan and its role during differentiation.
J. Bacteriol.
178:6173-6183[Abstract/Free Full Text].
|
| 2.
|
Beaman, T. C., and P. Gerhardt.
1986.
Heat resistance of bacterial spores correlated with protoplast dehydration, mineralization, and thermal adaptation.
Appl. Environ. Microbiol.
52:1242-1246[Abstract/Free Full Text].
|
| 3.
|
Buchanan, C. E., and A. Gustafson.
1992.
Mutagenesis and mapping of the gene for a sporulation-specific penicillin-binding protein in Bacillus subtilis.
J. Bacteriol.
174:5430-5435[Abstract/Free Full Text].
|
| 4.
|
Buchanan, C. E., and M.-L. Ling.
1992.
Isolation and sequence analysis of dacB, which encodes a sporulation-specific penicillin-binding protein in Bacillus subtilis.
J. Bacteriol.
174:1717-1725[Abstract/Free Full Text].
|
| 5.
|
Buchanan, C. E., and J. L. Strominger.
1976.
Altered penicillin-binding components in penicillin-resistant mutants of Bacillus subtilis.
Proc. Natl. Acad. Sci. USA
73:1816-1820[Abstract/Free Full Text].
|
| 6.
|
Ferrari, F. A.,
A. Nguyen,
D. Lang, and J. A. Hoch.
1983.
Construction and properties of an integrable plasmid for Bacillus subtilis.
J. Bacteriol.
154:1513-1515[Abstract/Free Full Text].
|
| 7.
|
Ghuysen, J.-M.
1991.
Serine -lactamases and penicillin-binding proteins.
Annu. Rev. Microbiol.
45:37-67[Medline].
|
| 8.
|
González-Castro, M. J.,
J. López-Hernández,
J. Simal-Lozano, and M. J. Oruña-Concha.
1997.
Determination of amino acids in green beans by derivitization with phenylisothiocyanate and high-performance liquid chromatography with ultraviolet detection.
J. Chromatogr. Sci.
35:181-185.
|
| 9.
|
Guérout-Fleury, A.-M.,
K. Shazand,
N. Frandsen, and P. Stragier.
1995.
Antibiotic-resistance cassettes for Bacillus subtilis.
Gene
167:335-337[Medline].
|
| 10.
|
Imae, Y., and J. L. Strominger.
1976.
Relationship between cortex content and properties of Bacillus sphaericus spores.
J. Bacteriol.
126:907-913[Abstract/Free Full Text].
|
| 11.
|
Koshikawa, T.,
T. C. Beaman,
H. S. Pankratz,
S. Nakashio,
T. R. Corner, and P. Gerhardt.
1984.
Resistance, germination, and permeability correlates of Bacillus megaterium spores successively divested of integument layers.
J. Bacteriol.
159:624-632[Abstract/Free Full Text].
|
| 12.
|
Lawrence, P. J., and J. L. Strominger.
1970.
Biosynthesis of the peptidoglycan of bacterial cell walls. The reversible fixation of radioactive penicillin G to the D-alanine carboxypeptidase of Bacillus subtilis.
J. Biol. Chem.
245:3660-3666[Abstract/Free Full Text].
|
| 13.
|
Leighton, T. J., and R. H. Doi.
1971.
The stability of messenger ribonucleic acid during sporulation in Bacillus subtilis.
J. Biol. Chem.
254:3189-3195.
|
| 14.
|
Lewis, J. C.,
N. S. Snell, and H. K. Burr.
1960.
Water permeability of bacterial spores and the concept of a contractile cortex.
Science
132:544-545[Abstract/Free Full Text].
|
| 15.
|
Lindsay, J. A.,
T. C. Beaman, and P. Gerhardt.
1985.
Protoplast water content of bacterial spores determined by bouyant density sedimentation.
J. Bacteriol.
163:735-737[Abstract/Free Full Text].
|
| 16.
|
Maniatis, T.,
E. F. Fritsch, and J. Sambrook.
1982.
Molecular cloning: a laboratory manual.
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
|
| 17.
|
Nakashio, S., and P. Gerhardt.
1985.
Protoplast dehydration correlated with heat resistance of bacterial spores.
J. Bacteriol.
162:571-578[Abstract/Free Full Text].
|
| 18.
|
Nicholson, W. L., and P. Setlow.
1990.
Sporulation, germination, and outgrowth, p. 391-450.
In
C. R. Harwood, and S. M. Cutting (ed.), Molecular biological methods for Bacillus. John Wiley & Sons Ltd., Chichester, England.
|
| 19.
|
Pedersen, L. B.,
T. Murray,
D. L. Popham, and P. Setlow.
1998.
Characterization of dacC, which encodes a new low-molecular-weight penicillin-binding protein in Bacillus subtilis.
J. Bacteriol.
180:4967-4973[Abstract/Free Full Text].
|
| 20.
|
Popham, D. L.,
J. Helin,
C. E. Costello, and P. Setlow.
1996.
Analysis of the peptidoglycan structure of Bacillus subtilis endospores.
J. Bacteriol.
178:6451-6458[Abstract/Free Full Text].
|
| 21.
|
Popham, D. L.,
J. Helin,
C. E. Costello, and P. Setlow.
1996.
Muramic lactam in peptidoglycan of Bacillus subtilis spores is required for spore outgrowth but not for spore dehydration or heat resistance.
Proc. Natl. Acad. Sci. USA
93:15405-15410[Abstract/Free Full Text].
|
| 22.
|
Popham, D. L.,
B. Illades-Aguiar, and P. Setlow.
1995.
The Bacillus subtilis dacB gene, encoding penicillin-binding protein 5*, is part of a three-gene operon required for proper spore cortex synthesis and spore core dehydration.
J. Bacteriol.
177:4721-4729[Abstract/Free Full Text].
|
| 23.
|
Popham, D. L., and P. Setlow.
1993.
Cloning, nucleotide sequence, and regulation of the Bacillus subtilis pbpE operon, which codes for penicillin-binding protein 4* and an apparent amino acid racemase.
J. Bacteriol.
175:2917-2925[Abstract/Free Full Text].
|
| 24.
|
Popham, D. L., and P. Setlow.
1993.
The cortical peptidoglycan from spores of Bacillus megaterium and Bacillus subtilis is not highly cross-linked.
J. Bacteriol.
175:2767-2769[Abstract/Free Full Text].
|
| 25.
|
Popham, D. L., and P. Setlow.
1996.
Phenotypes of Bacillus subtilis mutants lacking multiple class A high-molecular-weight penicillin-binding proteins.
J. Bacteriol.
178:2079-2085[Abstract/Free Full Text].
|
| 26.
| Popham, D. L., and P. Setlow. Unpublished
data.
|
| 27.
|
Schuch, R., and P. J. Piggot.
1994.
The dacF-spoIIA operon of Bacillus subtilis, encoding F, is autoregulated.
J. Bacteriol.
176:4104-4110[Abstract/Free Full Text].
|
| 28.
|
Simpson, E. B.,
T. W. Hancock, and C. E. Buchanan.
1994.
Transcriptional control of dacB, which encodes a major sporulation-specific penicillin-binding protein.
J. Bacteriol.
176:7767-7769[Abstract/Free Full Text].
|
| 29.
|
Sowell, M. O., and C. E. Buchanan.
1983.
Changes in penicillin-binding proteins during sporulation of Bacillus subtilis.
J. Bacteriol.
153:1331-1337[Abstract/Free Full Text].
|
| 30.
|
Suzuki, H.,
Y. Nishimura, and Y. Hirota.
1978.
On the process of cellular division in Escherichia coli: a series of mutants of E. coli altered in the penicillin-binding proteins.
Proc. Natl. Acad. Sci. USA
75:664-668[Abstract/Free Full Text].
|
| 31.
|
Tipper, D. J., and P. E. Linnet.
1976.
Distribution of peptidoglycan synthetase activities between sporangia and forespores in sporulating cells of Bacillus sphaericus.
J. Bacteriol.
126:213-221[Abstract/Free Full Text].
|
| 32.
|
Todd, J. A.,
E. J. Bone, and D. J. Ellar.
1985.
The sporulation-specific penicillin-binding protein 5a from Bacillus subtilis is a DD-carboxypeptidase in vitro.
Biochem. J.
230:825-828[Medline].
|
| 33.
|
Todd, J. A.,
A. N. Roberts,
K. Johnstone,
P. J. Piggot,
G. Winter, and D. J. Ellar.
1986.
Reduced heat resistance of mutant spores after cloning and mutagenesis of the Bacillus subtilis gene encoding penicillin-binding protein 5.
J. Bacteriol.
167:257-264[Abstract/Free Full Text].
|
| 34.
|
Warth, A. D.
1985.
Mechanisms of heat resistance, p. 209-225.
In
G. J. Dring, D. J. Ellar, and G. W. Gould (ed.), Fundamental and applied aspects of bacterial spores. Academic Press, Inc., London, England.
|
| 35.
|
Warth, A. D., and J. L. Strominger.
1972.
Structure of the peptidoglycan from spores of Bacillus subtilis.
Biochemistry
11:1389-1396[Medline].
|
| 36.
|
Warth, A. D., and J. L. Strominger.
1971.
Structure of the peptidoglycan from vegetative cell walls of Bacillus subtilis.
Biochemistry
10:4349-4358[Medline].
|
| 37.
|
Warth, A. D., and J. L. Strominger.
1969.
Structure of the peptidoglycan of bacterial spores: occurrence of the lactam of muramic acid.
Proc. Natl. Acad. Sci. USA
64:528-535[Abstract/Free Full Text].
|
| 38.
|
Wu, J.-J.,
R. Schuch, and P. J. Piggot.
1992.
Characterization of a Bacillus subtilis operon that includes genes for an RNA polymerase factor and for a putative DD-carboxypeptidase.
J. Bacteriol.
174:4885-4892[Abstract/Free Full Text].
|
| 39.
|
Yousif, S. Y.,
J. K. Broome-Smith, and B. G. Spratt.
1985.
Lysis of Escherichia coli by -lactam antibiotics: deletion analysis of the role of penicillin-binding proteins 1A and 1B.
J. Gen. Microbiol.
131:2839-2845[Abstract/Free Full Text].
|
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