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Journal of Bacteriology, November 2001, p. 6688-6693, Vol. 183, No. 22
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.22.6688-6693.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Precise Deletion of tagD and Controlled Depletion
of Its Product, Glycerol 3-Phosphate Cytidylyltransferase, Leads to
Irregular Morphology and Lysis of Bacillus subtilis
Grown at Physiological Temperature
Amit P.
Bhavsar,1
Terry J.
Beveridge,2 and
Eric D.
Brown1,*
Antimicrobial Research Centre, Department of
Biochemistry, McMaster University, Hamilton, Ontario, Canada L8N
3Z5,1 and Department of Microbiology and
Canadian Bacterial Disease Network, University of Guelph, Guelph,
Ontario, Canada N1G 2W12
Received 25 June 2001/Accepted 28 August 2001
 |
ABSTRACT |
Using a previously reported conditional expression system
for use in Bacillus subtilis (A. P. Bhavsar,
X. Zhao, and E. D. Brown, Appl. Environ. Microbiol.
67:403-410, 2001), we report the first precise deletion of
a teichoic acid biosynthesis (tag) gene, tagD,
in B. subtilis. This teichoic acid mutant showed a lethal
phenotype when characterized at a physiological temperature and in
a defined genetic background. This tagD mutant was subject to full phenotypic rescue upon expression of the complementing copy of
tagD. Depletion of the tagD gene
product (glycerol 3-phosphate cytidylyltransferase) via modulated
expression of tagD from the amyE locus revealed
structural defects centered on shape, septation, and division.
Thickening of the wall and ultimately lysis followed these events.
| |
TEXT |
Cell wall teichoic acids are a
diverse group of phosphate-rich polymers that are covalently linked to
peptidoglycan and can constitute a substantial portion of the cell wall
of gram-positive bacteria. Of the organisms so far characterized, a
large number produce predominantly either poly(glycerol phosphate) or
poly(ribitol phosphate) wall teichoic acids (1). In
Bacillus subtilis 168, the predominant wall teichoic acid is
a 1,3-linked poly(glycerol 3-phosphate) that is abundantly glucosylated
at the 2 position of glycerol (19).
A considerable body of work points to an essential role for teichoic
acid in B. subtilis 168 and has outlined steps in
poly(glycerol phosphate) teichoic acid synthesis (4, 11, 13,
14, 17, 18). Temperature-sensitive mutations have been localized
to a number of genes in the poly(glycerol phosphate) teichoic acid biosynthesis gene cluster (tag) of this organism
(13), most notably tagB, tagD
(coding for glycerol 3-phosphate cytidylyltransferase) (15), and tagF [putatively assigned to
poly(glycerol phosphate) polymerase] (18). Attempts at
insertional mutagenesis of these genes or their homologues in B. subtilis and Staphylococcus epidermidis have proven
unsuccessful (9, 11, 13, 14). This raises the prospect
that the essential nature of teichoic acids may extend to other
gram-positive bacteria, including pathogens.
Nevertheless, considerable ambiguity surrounds the apparently essential
role of this polymer. For example, two other wall polymers,
poly(glucose N-acetylgalactosamine phosphate) and
teichuronic acid, are also produced by B. subtilis 168 and
are capable of at least partially substituting for the predominant
polymer (7, 8). Therefore, while teichoic acid
biosynthesis may have great potential as a therapeutic drug target in
gram-positive physiology, a clear resolution of the question of
dispensability for this polymer, even in the model organism B. subtilis 168, remains a puzzle worthy of further study.
The most compelling evidence to date for the indispensability of wall
teichoic acid comes from the isolation of temperature-sensitive mutants
created through chemical mutagenesis (3, 4). While these
mutations were ultimately mapped to the tag genes of
B. subtilis 168 (13), their genetic
background remains somewhat unclear due to the nature of their
construction. Only recently was an unequivocal role demonstrated for
tagD in the temperature sensitivity of one such mutant,
tag-12 (2). In that work, we showed in
trans complementation of the tag-12 mutant at the
restrictive temperature with tagD under control of the
xylose promoter at the amyE locus. Another ambiguity
surrounding temperature-sensitive defects in teichoic acid biosynthesis
is rooted in the possibility that lethality in these mutants is
dependent on unusual cell physiology at the high temperatures (45 to
47°C) used for growth. Regarding growth temperature, it is noteworthy
that the minor teichoic acid polymer poly(glucose
N-acetylgalactosamine phosphate) is not synthesized at the
restrictive temperatures previously used in studies with temperature-sensitive mutants (3, 10).
One of the most intriguing aspects of the temperature-sensitive
teichoic acid mutants has been the observation that several of these
undergo a transition from rod shape to irregular spheres upon shift to
the nonpermissive temperature. Detailed electron microscopic
ultrastructural analyses of this transition were last described more
than 20 years ago with a variety of temperature-sensitive teichoic acid
mutants (3, 5, 21-23, 25), some of which would later be
characterized as tagB- and tagF-defective
B. subtilis mutants (13). Those studies
indicated that the loss of teichoic acid drastically altered wall
ultrastructure, with concomitant effects on cell division (septation)
and overall cell shape.
In the work reported here, we have revisited the dispensability of a
teichoic acid biosynthesis gene, tagD, at a physiological temperature (30°C) and in a defined genetic background. We have targeted tagD in this work, since it is postulated to have a
central role in teichoic acid biogenesis in B. subtilis,
providing activated glycerol phosphate for both linkage unit and
polymer synthesis (19). As such, TagD is hypothesized to
function in the formation of both the minor [poly(glucose
N-acetylgalactosamine phosphate)] and major [poly(glycerol
phosphate)] wall teichoic acid polymers of B. subtilis
strain 168 (10). We have used a xylose-based conditional
expression system to facilitate the construction of a precise deletion
of tagD in B. subtilis 168 with a complementing copy of the gene present under tight transcriptional control of the
xylose regulon (2).
To incorporate a complementing copy of tagD at
amyE, linearized pSWEET-tagD was used to
transform wild-type B. subtilis 168 (EB6) by established
methods (6). Positive transformants (strain EB124) were
selected for on Luria-Bertani (LB) solid medium (24) supplemented with 10 µg of chloramphenicol (CHL) per ml. The
disruption of amyE was verified by the absence of
"halos" in a starch utilization assay (6). To
precisely replace tagD at the tag locus, 500 bp
of sequence flanking either side of tagD was amplified via an asymmetric method (12) by using primer pairs AB10/AB11
and AB12/AB13 for sequence upstream and downstream of tagD,
respectively (Tables 1 and
2). The flanking sequence, reamplified
with primers AB10/AB13 to generate a single product, was cloned into
pBluescript SKII+, generating pBS-tagDflank. The
spectinomycin (SPC) resistance cassette, amplified from pUS19
with primers AB14/AB15, was subsequently incorporated between the
tagD flanking sequence (at the SrfI site) to
generate pBS-tagDflankspec. Replacement of
tagD at the native locus was accomplished by transformation
of linearized pBS-tagDflankspec into strain
EB124, and transformants (EB240) were selected on LB medium
supplemented with 10 µg of CHL per ml, 100 µg of SPC per ml, and
2% xylose. We took care to include the putative promoter for the SPC
resistance cassette, but not the transcriptional terminator to ensure
expression of downstream genes, particularly tagF. To verify
replacement of tagD at the tag locus, we used a
PCR-based analysis that showed an insert corresponding to the size of
the resistance cassette at the tag locus in strain EB240,
but not in strain EB124 (data not shown). Furthermore, amplification
with primers that annealed to the SPC resistance cassette and the
tagD flanking sequence gave product only when EB240 was used
as a template (data not shown).
The tagD deletion strain is conditionally
complemented at physiological temperature.
Strains EB124 and EB240
were examined after growth overnight at 30°C on LB agar plates
supplemented with 10 µg of CHL per ml under both inducing (2%
xylose) and noninducing (no xylose) conditions. As seen in Fig.
1, the latter condition gave rise to
robust growth of EB124 with a wild-type colony morphology. In stark
contrast, EB240 showed no discernible growth in the absence of xylose.
The presence of the inducer (xylose) completely rescued the lethal
phenotype of the tagD mutant strain. Thus, at a
physiological temperature, the growth of the tagD deletion
strain was exquisitely dependent upon the induction of tagD
under xyl control at amyE. We believe this
indicates that the altered phenotype of this mutant is not the result
of a polar effect as a consequence of gene replacement with the SPC
resistance cassette. Instead the defect is clearly centered on the
depletion of TagD.
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FIG. 1.
Xylose dependence of tagD deletion strain
EB240. Strains EB124 and EB240 were plated on LB-CHL medium in the
presence or absence of 2% xylose. Strains were grown overnight at
30°C.
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TagD-depleted cells show altered cell morphology and lysis.
Although it was clear that EB240 had a lethal phenotype in the absence
of xylose, it was possible to grow TagD-depleted cells with a very
heavy inoculum yielding detectable growth in smears of high cell
density (i.e., isolated colonies were absent). Interestingly, prolonged
incubation of these cells resulted in visible clearing of the colonies,
indicative of a lytic phenotype (data not shown). While the lytic
phenotype of the tagD-null mutant reported here has not been
commonly documented with temperature-sensitive teichoic acid mutants,
we also observed a significant decrease in cell density of the
tag-12 temperature-sensitive mutant (ascribed to tagD) when temperature was increased from 30 to 47°C in
late exponential growth (2). It has previously been
suggested that ultrastructural abnormalities evident in the cell walls
of teichoic acid mutants may be the result of malfunctioning autolysins
impacted by the loss of teichoic acid as a site of localization
(22).
To further explore the phenotype of cells depleted of TagD, the
deletion strain EB240 was examined by microscopy after growth
overnight
at 30°C on LB solid medium supplemented with 10 µg of
CHL and 100 µg of SPC per ml in the absence of xylose (Fig.
2).
Strain EB124 was also examined for
comparison after similar growth
on LB solid medium supplemented with 10 µg of CHL per ml. Cells
from both strains were gently resuspended in
sterile saline, pelleted,
and resuspended in sterile saline containing
15% (vol/vol) glycerol.
Differential interference microscopy was
performed with bright-field
illumination. As seen in Fig.
2A, TagD
depletion resulted in gross
morphological changes as the rod-shaped
cells progressed towards
irregularly shaped spheres with diameters
close to that of the
length of wild-type EB124. This apparent swelling
in the mutant
was accompanied by a disposition towards clumping of
three or
more cells, which was not seen with EB124. EB124 and EB240
were
grown under similar conditions and prepared for transmission
electron
microscopy. Examination of EB124 (control strain)
revealed ultrastructure
typical of wild-type
B. subtilis, with well-defined cell wall
and chromosome. In cases in
which dividing cells were observed,
septa appeared normal (data not
shown). In contrast, sections
of TagD-depleted cells (EB240)
revealed an irregular shape, multiple
cytoplasmic compartments, uneven
and thickened cell walls, and
curved septa (Fig.
2B). The unusual
septation was particularly
striking, because initiation sites were
found at multiple and
asymmetric locations along the cell length, in
contrast to the
regular septal pattern in wild-type cells. Also
remarkable was
the finding that in many of these cells, septa were only
partially
formed, emanating from only one side of a cell and ending in
the
cytoplasm (data not shown). Scanning electron microscopy was used
to examine the detailed wall structure of both the
tagD
deletion
strain and the
tag+ strain. Cells
were originally grown as described above and prepared
for scanning
electron microscopy. Micrographs highlighted the
unusual shape of the
TagD-depleted cells (Fig.
2C). Whereas wild-type
B. subtilis
revealed a typical rod-shaped morphology, the teichoic
acid mutant was
almost spherical, with conspicuous furrows that
may correspond to the
aberrant septa noted above. Also noteworthy
was the absence of any
distinctive flagella on the mutant cells
(Fig.
2C).
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FIG. 2.
Characterization of a tagD deletion
strain by microscopy. EB240 (TagD depleted) and EB124 (TagD wild type)
were visualized by differential interference contrast microscopy (A)
and transmission electron microscopy (B). EB240 and EB124 were also
examined by scanning electron microscopy (C). Size bars are 500 nm.
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TagD depletion leads to a stepwise transition from cell rounding to
cell lysis.
Since the xylose-based conditional expression system
showed a particular capacity for modulated expression (2),
we wanted to examine the effects of controlled depletion of TagD in
B. subtilis. EB124 and EB240
(tag+ and tagD deletion mutant,
respectively) were grown as described above on solid medium in the
absence of inducer, resuspended in saline, and diluted to similar
optical density at 600 nm (OD600) values. Fresh
LB medium supplemented with 10 µg of CHL per ml and 2% xylose was
inoculated 1:100 with EB124. LB medium supplemented with 10 µg of CHL
per ml, 100 µg of SPC per ml, and various amounts of xylose (2, 0.2, 0.06, and 0.02% or none) was inoculated 1:100 with EB240. Growth at
30°C with shaking at 250 rpm was monitored via
OD600 for 1,140 min at regular intervals. Cell
density measurements (Fig. 3, graph)
revealed that, at 2% xylose, the mutant had similar growth kinetics to
the wild type. At lower levels of inducer, however, the growth kinetics
deviated slightly from those of the wild type, with very little growth
in cultures in which xylose was absent. Interestingly, although our
data suggested very tight transcriptional control of the complementing
tagD copy when assessed on solid medium, we nevertheless
observed detectable, albeit minor levels of growth from EB240 in the
absence of xylose in liquid culture. For cells grown in liquid culture,
we cannot rule out a compensatory mutation that might facilitate the
growth of a small subset of the population (e.g., mutation in the
xylose-based regulation system that would confer inducer insensitivity
to these cells). Nevertheless, we did not observe cells with wild-type morphology in micrographs obtained from this sample (Fig. 3F) (data not
shown). A similar growth assay was performed for EB240 in LB medium
supplemented with 10 µg of CHL per ml, 100 µg of SPC per ml,
various amounts of xylose (2, 0.2, 0.06, and 0.02% or none), and 1 mM
glycerol enriched with [2-3H]glycerol (6.3 µCi/ml). Cells were fractionated into cell wall and protoplast as
previously reported (20) to monitor the
incorporation of [2-3H] glycerol
into the cell wall fraction of B. subtilis cells subjected to controlled depletion of TagD. Whereas the amount of labeled glycerol
incorporated into the pelleted protoplast fraction did not vary
significantly over the range of xylose used in the experiment, incorporation of label into the solubilized wall fraction was reduced
approximately 88% in the TagD-depleted strain (EB240, no xylose)
compared to the fully complemented strain (EB240, 2% xylose).
Interestingly, we did not notice a significant deviation in the amount
of labeled glycerol incorporated into cell walls of the tagD
deletion mutant grown at intermediate xylose concentrations (2, 0.2, 0.06, and 0.02%).
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FIG. 3.
Growth curve and morphology of TagD depletion. (Top)
EB240 was inoculated into LB-CHL-SPC medium with 2% xylose (B), 0.2%
xylose (C), 0.06% xylose (D), 0.02% xylose (E), and no xylose (F).
EB124 was also inoculated into LB-CHL medium with 2% xylose (A).
Culture growth was monitored for 19 h, and samples were
immediately prepared for transmission electron microscopy. The size bar
(bottom right) is 500 nm.
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To observe the effects of TagD depletion on the morphology of
B. subtilis, cells were harvested at the final time point of
the
growth curve shown in Fig.
3 and prepared for transmission
electron
microscopy. Panels A to F in Fig.
3 depict the ultrastructural
details
of the lethal transition resulting from controlled depletion
of TagD in
B. subtilis. Panels A and B (Fig.
3) show that there
are no
detectable differences between the wild-type and teichoic
acid mutant
cells grown in medium supplemented with 2% xylose,
respectively. At
0.2% xylose (Fig.
3C), the cells began to lose
the integrity of their
rod shape, but had no other obvious defects.
At 0.06% xylose (Fig.
3D), three morphologically distinct populations
of cells began to
emerge. A minor population consisted of large
irregularly shaped cells
that were substantially enlarged with
respect to the wild type. These
showed a diminished intensity
of cytoplasmic staining, suggesting that
lysis had occurred. A
second dominant population was comprised of
smaller cells that
were also irregular but roughly spherical. A third
and minor subpopulation
of cells at this xylose concentration (0.06%)
resembled wild-type
rods. Multicompartmentalized cells with irregularly
formed and
localized septa were first apparent at a xylose
concentration
of 0.02% (Fig.
3E). At this concentration, enlarged,
lysed, and
rounded cells were typical, while cells with wild-type
characteristics
were rarely observed. The catastrophic effect of the
complete
removal of inducer is shown in Fig.
3F. Septal abnormalities,
including mislocalization, curvature, and multiplicity (several
septa
formed within a single cell), were typical of almost all
of the cells
grown in the absence of inducer. Cell lysis was also
representative of
this growth condition, since cell remnants were
clearly visible in
these micrographs. Interestingly, only in the
absence of xylose did we
observe thickening of the peptidoglycan
layer (Fig.
3F, top, and 2B).
These experiments indicated the
following step-by-step progression upon
depletion of TagD: (i)
deviations from rod to curved shape, (ii)
enlargement to irregular,
bloated spheres, (iii) aberrant cell division
evident in malformed
septa, and (iv) thickened peptidoglycan and cell
lysis. It has
previously been suggested that loss of teichoic acid may
lead
to pleiotropic effects related to a lack of rod cylinder extension
(
16,
19). Our analysis of the transition from rods through
to lysis is consistent with such a hypothesis. Our results also
point
to a role for autolysins; however, it remains doubtful that
cell wall
autolysis is the primary and catastrophic defect associated
with loss
of teichoic acid. Cell lysis, in this work, required
an extended
incubation of heavily inoculated plates, and ultrastructural
analysis
indicated that wall thickening and widespread lysis were
delayed events
in the rod-to-lysis transition. These later events,
therefore, may well
be consequential to a defect routed principally
in cell wall extension
in the growing rod, although the role of
wall teichoic acid in this
process remains
elusive.
In summary, the experiments reported here substantiate the
indispensable role of teichoic acid in
B. subtilis 168 in a
defined
genetic background and at temperatures conventionally used for
growth of this organism. Our studies of the transition from rod
to
sphere to lysis underscore the complexity of catastrophic events
resulting from depletion of a critical enzyme in teichoic acid
synthesis and provide a basis for further exploration of the
therapeutic
potential of this pathway in gram-positive
physiology.
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ACKNOWLEDGMENTS |
We thank Petra Levin (Washington University) for pUS19 and Tamara
O'Connor for assistance with the Olympus BX-51 microscope.
This work was supported by an operating grant and scholarship from the
Medical Research Council of Canada to E.D.B., an operating grant from
the Canadian Bacterial Diseases Network to T.J.B., and a postgraduate
scholarship to A.P.B. from the Natural Sciences and Engineering
Research Council of Canada.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Antimicrobial
Research Centre, Department of Biochemistry, McMaster University, 1200 Main St. West, Hamilton, Ontario L8N 3Z5, Canada. Phone: (905) 525-9140, ext. 22392. Fax: (905) 522-9033. E-mail:
ebrown{at}mcmaster.ca.
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Journal of Bacteriology, November 2001, p. 6688-6693, Vol. 183, No. 22
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.22.6688-6693.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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