Department of Microbiology and Immunology,
School of Medicine, University of North Dakota, Grand Forks, North
Dakota 58202-9037
Although general physiological functions have been ascribed to the
high-molecular-weight penicillin binding proteins (PBPs) of
Escherichia coli, the low-molecular-weight PBPs have no
well-defined biological roles. When we examined the morphology of a set
of E. coli mutants lacking multiple PBPs, we observed that
strains expressing active PBP 5 produced cells of normal shape,
while mutants lacking PBP 5 produced cells with altered diameters,
contours, and topological features. These morphological effects were
visible in untreated cells, but the defects were exacerbated in cells forced to filament by inactivation of PBP 3 or FtsZ. After
filamentation, cellular diameter varied erratically along the length of
individual filaments and many filaments exhibited extensive branching.
Also, in general, the mean diameter of cells lacking PBP 5 was
significantly increased compared to that of cells from isogenic strains
expressing active PBP 5. Expression of cloned PBP 5 reversed the
effects observed in 
dacA mutants. Although deletion of
PBP 5 was required for these phenotypes, the absence of additional PBPs
magnified the effects. The greatest morphological alterations required
that at least three PBPs in addition to PBP 5 be deleted from a single strain. In the extreme cases in which six or seven PBPs were deleted from a single mutant, cells and cell filaments expressing PBP 5 retained a normal morphology but cells and filaments lacking PBP 5 were
aberrant. In no case did mutation of another PBP produce the same
drastic morphological effects. We conclude that among the
low-molecular-weight PBPs, PBP 5 plays a principle role in determining
cell diameter, surface uniformity, and overall topology of the
peptidoglycan sacculus.
 |
INTRODUCTION |
Of the 12 known penicillin binding
proteins (PBPs) of Escherichia coli, the only enzymes with
known physiological roles are four of the five high-molecular-weight
PBPs: 1a, 1b, 2, and 3 (10, 21). These four proteins
polymerize new peptidoglycan strands and incorporate them into the
existing sacculus, which forms the rigid layer of the cell envelope. In
contrast, the seven low-molecular-weight (LMW) PBPs have no
well-defined physiological functions, even though the enzymological
knowledge of these proteins is well advanced.
Among the LMW proteins, PBP 5 has been studied in the most depth, but
its biological function remains mysterious. PBP 5 is the major cellular
DD-carboxypeptidase, an enzyme that removes the terminal
D-alanine residue from the pentapeptide side chain of
peptidoglycan (10, 12), thereby altering the types of
cross-linking that can occur between glycan chains. Even though PBP 5 is the most numerous of the PBPs, its loss is not lethal (11,
20). One possible reason that PBP 5 is dispensable is that one or
more other LMW PBPs might compensate for its absence. However, E. coli survives in the absence of all known
DD-carboxypeptidases (3-5) and even in the
absence of all seven LMW PBPs (4). This suggests that PBP 5 has no important physiological role or that its role is invisible in
the laboratory environment. On the other hand, overproduction of PBP 5 is lethal, causing E. coli to grow as spheres before dying
(9, 22), suggesting that PBP 5 may have a morphological
effect on growth of the cell wall.
Recently, we constructed a set of E. coli mutants lacking
every possible combination of eight PBPs (4). Among these
mutants are gene combinations in which PBP 5 is present or absent in
genetic backgrounds that differ only in the complement of other active PBPs. Some of these mutants exhibited severe morphological defects, especially when forced to filament by inactivating PBP 3 (4, 8). The PBP patterns of these defective mutants led us to ask which of the PBPs plays the dominant role in maintaining normal bacterial shape. We report here that, in the absence of multiple LMW
PBPs, deletion of PBP 5 creates E. coli mutants with cell walls of abnormal diameter and contour. Furthermore, when cells lacking
PBP 5 are forced to filament, the overall topology of the bacterial
cell is severely affected, resulting in gross abnormalities and
branching. Therefore, PBP 5 plays an important role in the genesis
and/or maintenance of bacterial shape.
| |
MATERIALS AND METHODS |
Strains and growth conditions.
The E. coli
strains used in this work are listed in Table
1. pFAD38, a plasmid carrying
sulA (sfiA) under araP control, was provided by J. Lutkenhaus. pBAD24-Amp and pBAD18-Cam were provided by
J. Beckwith. Strains were maintained on Luria-Bertani (LB) broth or
agar plates (13) with appropriate antibiotic selection, as
follows: chloramphenicol (20 µg/ml) or ampicillin (100 µg/ml). When
necessary, glucose (0.1%) was added to inhibit expression from the
pBAD promoter. Overnight cultures were diluted 1:200 into 50 ml of
fresh LB medium and were incubated with vigorous shaking at 37°C
until reaching an A550 of approximately 0.2. Cells were filamented by adding 10 µg of aztreonam (Leo
Pharmaceuticals, Ballerup, Denmark) per ml or 2 µg of mitomycin C per
ml. All chemicals were purchased from Sigma Chemical Co. (St. Louis,
Mo.), unless otherwise noted.
Molecular techniques.
Plasmid preparations, restriction
digests, and ligations were performed as described previously
(17). Competent strains were prepared by the calcium
chloride technique and transformed by heat shock (17). CS109
chromosomal DNA was prepared by boiling 200 µl of overnight culture
with 800 µl of distilled water for 10 min, followed by centrifugation
at 14,000 × g for 1 min and collection of the
supernatant. Oligonucleotide primers P1
(5'-GATCGAGAATTCGTCATGAATACCATTTTTCCGC-3') and P2
(5'-GCATGCAAGCTTCTAGATTTTTAACCAAACCAGTGATG-3') were
purchased from Gibco Life Sciences (Grand Island, N.Y.) and were used
to amplify the wild-type dacA gene from CS109 chromosomal
DNA by PCR. An EcoRI-HindIII fragment of the
dacA PCR product was ligated into pBAD24-Amp (7),
creating the intermediate plasmid pPJ5A. An
NheI-HindIII fragment was excised from pPJ5A
and ligated into pBAD18-Cam (7), creating the
dacA expression vector pPJ5C. The nucleotide sequence of the
dacA gene in pPJ5C was confirmed by Sanger dideoxynucleotide
sequencing (18). PBP 5 expression from pPJ5C was quantified
by 125I-penicillin-X labeling followed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis, as described previously
(8). Restriction endonucleases, T4 ligase, and other
molecular reagents were purchased from New England Biolabs (Beverly,
Mass.).
Photography and data analysis.
Poly-L-lysine
slides were prepared by briefly immersing microscope slides in
poly-L-lysine (0.1% [wt/vol]; Sigma Chemical Co.) and
letting them dry overnight. Bacteria were grown to exponential phase
(A550 
0.2), and samples were prepared for
microscopy by placing 3.5 µl of broth culture on
poly-L-lysine-treated slides and covering each sample with
a coverslip. Samples were viewed with a Nikon EFD-3 microscope with a
100× oil immersion objective and photographed with an attached SenSys
charge-coupled device camera and capture software (Photometrics Ltd.,
Tucson, Ariz.) at 1,000× total magnification. Cell measurements and
analyses were performed with Image Pro Plus version 3.01 software
(Media Cybernetics, Silver Spring, Md.). Cell diameters of individual cells were calculated by measuring the cross-sectional area and dividing that value by the cell length. The reference measurement bar
included on each photograph was calibrated using a slide-mounted 0.01-mm micrometer (Fisher Scientific, Chicago, Ill.). Statistical and
graphical analyses were performed with SigmaPlot (SSPS, Chicago, Ill.)
and Microsoft Excel (Microsoft Inc., Redmond, Wash.).
| |
RESULTS |
Previously, we reported that some E. coli mutants
produced cells with abnormal diameters and uneven contours and cells
which were prone to topological oddities such as branching
(7). Therefore, we investigated several mutants lacking
various PBPs to determine more specifically which of these proteins
contributes most strongly to these morphological alterations.
Morphological aberrations in a mutant lacking seven PBPs.
When
E. coli CS109 was incubated in the presence of aztreonam
(which inhibits the septum-specific enzyme PBP 3), the resulting filamentous cells had a constant diameter equal to that of the original
cell, producing regularly shaped elongated rods with a smooth outer
contour (Fig. 1B). Somewhat surprisingly,
aztreonam-induced filaments of strain CS604-2 were very similar to
those of the wild-type parent even though the mutant lacked six PBPs
(the deleted PBPs were 1a, 4, 6, 7, AmpC, and AmpH) (4)
(Fig. 1D). In contrast, filaments of E. coli CS701-1
were grossly abnormal (Fig. 1F). The single difference between CS604-2
and CS701-1 is the deletion from the latter of the gene encoding PBP 5 (4).
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FIG. 1.
Morphology of PBP mutants before and after
filamentation by aztreonam. Bacteria were grown in LB broth at 37°C
until reaching an A550 of approximately 0.2, at
which point 10 µg of aztreonam per ml was added. Samples were
prepared for microscopy immediately before and 45 min after addition of
the antibiotic. (A) CS109; (B) CS109 plus aztreonam; (C) CS604-2; (D)
CS604-2 plus aztreonam; (E) CS701-1; (F) CS701-1 plus aztreonam; (G)
CS601-3; (H) CS601-3 plus aztreonam; (I) CS612-1; (J) CS612-1 plus
aztreonam.
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|
Aztreonam-induced filaments of E. coli CS701-1 exhibited
three types of morphological aberrations. First, filaments exhibited large cell-to-cell variations in diameter. Second, the diameter of an
individual filament fluctuated along its length to a degree that was
often extraordinary (Fig. 1F). We refer to this irregular diameter
within an individual cell as a deviation in cell contour, to
distinguish the phenotype from variations between different cells. The
third aberration involved the overall shape of individual filaments.
Many filaments produced one or more knobs, branches, or various
outgrowths that were distinctly different from simple alterations in
diameter (Fig. 1F). We refer to these types of morphological deviations
as differences in cell topology. Untreated cells of CS701-1 (Fig. 1E)
were also morphologically diverse compared to cells of the parent CS109
(Fig. 1A) and the mutant CS604-2 (Fig. 1C). Even though the differences
were not as visually remarkable as those observed in filaments, it was
apparent that CS701-1 cells had different diameters, uneven contours,
and a variety of inappropriate shapes (Fig. 1E).
Severe morphological aberrations in mutants lacking PBP 5.
Because the only difference between strains CS604-2 and CS701-1 was the
absence of PBP 5 from CS701-1, it was possible that this PBP was
responsible for the observed morphological defects. To test this, we
chose seven strains from which different sets of six PBPs were deleted.
Each mutant was isogenic with CS701-1 and expressed one of the PBPs
missing from CS701-1 (Table 1) (4).
When mutants lacking six PBPs were filamented in the presence of
aztreonam, the only strain that produced filaments of relatively normal
length, diameter, contour, and shape was the mutant that expressed
active PBP 5, CS604-2 (Fig. 1D). The five mutants that expressed one of
the other LMW PBPs (PBPs 4, 6, 7, AmpC, and AmpH) produced filaments
with aberrant morphology. The same general effect was observed in
untreated cells: mutants lacking PBP 5 and any combination of five
other PBPs were much more variable in size and shape. For example,
strain CS601-3 (expressing AmpC) exhibited morphological irregularities
in untreated cells (Fig. 1G) and in aztreonam-induced filaments (Fig.
1H). Similar results were observed in untreated cells of other sextuple
mutants lacking PBP 5, as follows: CS605-4 (expressing PBP 4), CS606-1
(expressing PBP 7), and CS602-1 (expressing AmpH) (data not shown).
Thus, an active PBP 5 gene reversed the morphological oddities seen in
E. coli CS701-1, whereas expression of any one of the other LMW PBPs had little or no effect.
The same PBP 5-dependent morphological characteristics were observed in
mutants having fewer than six PBPs deleted. For example, strains
producing PBP 5, such as CS502-2 (lacking PBPs 1a, 4, 7, AmpC, and
AmpH) (Fig. 2A) and CS508-1 (lacking PBPs
1a, 4, 6, 7, and AmpC) (Fig. 2B), produced cells that looked quite
normal, though some were longer than usual. On the other hand, strains lacking PBP 5, such as CS506-1 (lacking PBPs 1a, 4, 5, 6, and AmpC)
(Fig. 2D) and CS509-3 (lacking PBPs 1a, 4, 5, 7, and AmpH) (Fig. 2E),
produced a high percentage of cells whose morphology was extremely
unusual. Once again, the morphological differences were reversed in
isogenic mutants expressing PBP 5. For example, CS403-3 (lacking PBPs
1a, 4, 7, and AmpH) is an isogenic relative of CS509-3 that produces
active PBP 5 (4), and this one difference returned the cells
to normal size and shape (Fig. 2C). The same result was observed for
the isogenic partner of CS506-1, strain CS408-1, which expresses active
PBP 5 (data not shown). (Note: the examples shown in Fig. 2 were
selected from photographs of many cells and are not meant to imply that
every cell was equally aberrant. Nonetheless, the photographs do
reflect the high degree of morphological variation among cells of these
mutants.)
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FIG. 2.
Morphology of untreated PBP mutants. Bacteria
were grown in LB broth at 37°C until reaching log phase and then
photographed. (A) CS502-1; (B) CS508-1; (C) CS403-3; (D) CS506-1; (E)
CS509-3.
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|
Although deletion of PBP 5 was associated with significant changes in
cell morphology, the degree of morphological variation increased in
frequency and extent as the total number of LMW PBPs was reduced. For
example, in the mutant in which PBP 5 was the only gene deleted (strain
CS12-7) there were only very slight morphological changes visible in
filaments formed after treatment with aztreonam (data not shown). This
was also true for all but one of the mutants in which two PBPs were
deleted (data not shown). The exception to this rule was the double
mutant CS211-2 (PBPs 5 and 6 deleted) in which a few irregularities,
such as minor branching at the ends of fewer than 1% of the filaments,
appeared (data not shown). Deletion of PBP 5 produced the greatest
morphological defects in mutants that were missing a total of three or
more PBPs.
The morphological effects of losing PBP 5 did not depend on the
simultaneous absence of PBP 1a. For example, PBP 1a was active in the
5 mutant, CS612-1, but untreated cells and filaments of this strain
still exhibited abnormal morphologies (Fig. 1I and J). In addition, we
examined five quintuple mutants related to CS535-1, each of which
contained a deletion of PBP 5 in place of one of the five genes missing
from CS535-1 (strains CS531-3, CS533-1, CS534-1, CS536-1, and CS539-1).
In these five isogenic mutants, filaments formed in the presence of
aztreonam were significantly abnormal, indicating that no one of the
other five LMW PBPs could substitute completely for the loss of PBP 5 (data not shown). Because PBP 1a was expressed in these five quintuple
mutants, the alterations in morphology did not depend on the
simultaneous absence of PBPs 1a and 5.
Loss of PBP 5 increases the average diameter of E. coli.
The morphological results of deleting PBP 5 were visible by qualitative
microscopic inspection. In an effort to obtain a more quantitative idea
of the effect of PBP 5 on cell morphology, photographs of cells were
subjected to image analysis to measure cell length, cross-sectional
area, and derived diameter (Materials and Methods).
The derived diameters of 92% of untreated wild-type E. coli
CS109 cells clustered between 1.0 and 1.1 µm (Fig.
3A), and only 2% of the cells had
diameters greater than 1.2 µm (Table
2). In strain CS604-2, which lacks six
PBPs but in which PBP 5 is active, 68% of the cells were 1.1 to 1.2 µm in diameter (Fig. 3A) and 23% had diameters that exceeded 1.2 µm (Table 2). Thus, even in the presence of active PBP 5, the average
diameter increased slightly in this mutant. However, what is not
reflected in these figures is cell shape. As stated above, cells
containing active PBP 5 were unbranched and uniform in width along the
length of individual cells (i.e., they had a uniform topology and
contour). Untreated CS701-1 cells (which lack PBP 5) had much larger
derived diameters than did CS604-2 cells (Fig. 3A and Table 2). The
diameters of CS701-1 were spread over a greater range, reaching as high as 2.3 µm (Fig. 3A), and 65% of the cells had diameters greater than
normal (>1.2 µm) (Table 2). When CS109, CS604-2, and CS701-1 were
filamented in the presence of aztreonam for 45 min, the resulting filaments were long enough to exhibit visible differences in morphology but short enough so that each cell could be photographed in a single
plane. The same relationships were observed among the diameters of
these filaments (Table 2 and data not shown).
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FIG. 3.
Differences in cellular diameter of strains isogenic for
dacA. Strains were grown in LB broth at 37°C until
reaching an A550 of approximately 0.2, at which
point samples were prepared for microscopy. "Frequency" indicates
the proportion of the population with a particular diameter. n, the
number of cells measured for each population.
|
|
The effect of PBP 5 was also evident in untreated cells from which PBP
1b had been deleted instead of PBP 1a. In CS610-1 (lacking PBP 1b and
five LMW PBPs), the derived diameters of cells were virtually
indistinguishable from those of the wild-type strain CS109 (Fig. 3B and
Table 2). However, in CS702-1, from which PBP 5 is deleted in addition
to those PBPs absent in CS610-1, the derived diameters increased (Fig.
3B and Table 2).
Cloned PBP 5 reverses the morphological defects in dacA
deletion mutants.
The PBP 5 gene, dacA, was placed
under control of the arabinose promoter, creating plasmid pPJ5C
(Materials and Methods). When this plasmid was transformed into
CS701-1, a small amount of PBP 5 was produced by cells grown in LB
medium in the absence of inducer, protein expression rose with
increasing concentrations of arabinose, and no PBP 5 was produced when
glucose was present (data not shown). Complementation studies were
performed in the absence of arabinose or by inducing PBP 5 by adding
0.005% arabinose.
Expression of cloned PBP 5 complemented the major morphological
defects in E. coli CS701-1. pPJ5C and the control vector
pBAD18-Cam were introduced into E. coli strains CS701-1
and CS604-2, and the cells were filamented by addition of aztreonam.
CS701-1 cells containing the vector pBAD18-Cam had diameters and
morphologies as abnormal as those exhibited by CS701-1 without the
plasmid (Table 2 and data not shown). However, aztreonam-induced
filaments of CS701-1 pPJ5C returned to a uniform size and shape (Fig.
4A). Cloned PBP 5 complemented the
morphological defects in CS701-1 even if arabinose was omitted from the
medium, indicating that the low level of basal expression from the
arabinose promoter produced sufficient PBP 5 to counteract the
dacA deletion (data not shown). When PBP 5 expression was
inhibited by adding glucose to the medium, CS701-1(pPJ5C) cells had the
same types of aberrant diameters and shapes as the mutant without the
plasmid (Fig. 4B). Thus, visual inspection established that controlled
expression of PBP 5 eliminated the gross morphological defects
associated with deletion of the dacA gene. In addition, PBP
5 expression in CS701-1(pPJ5C) reduced the range of untreated cell
diameters to that exhibited by strain CS604-2(pBAD18), the isogenic
strain in which the dacA gene was intact (Table 2).
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FIG. 4.
Restoration of normal morphology by cloned PBP 5. E. coli CS701-1 pPJ5C was grown at 37°C in LB broth
supplemented with 20 µg of chloramphenicol and either 0.005%
arabinose or 0.1% glucose until reaching an
A550 of 0.2, at which point 10 µg of aztreonam
per ml was added. Samples were prepared for microscopy 45 min
after addition of the antibiotic. (A) PBP 5 expression
induced by 0.005% arabinose; (B) inhibition of PBP 5 expression
by 0.1% glucose.
|
|
Loss of PBP 5 produces morphological defects in SulA-induced
filaments.
In the preceding experiments the most obvious
morphological defects were produced in filaments induced by the
addition of aztreonam, which binds to PBP 3 and inhibits septation. It
was therefore possible that some of the morphological changes
associated with the loss of PBP 5 might require the simultaneous
inactivation of PBP 3. To test this, cells were filamented by
controlled expression of the SulA protein from plasmid pFAD38
(1). Because SulA inhibits the activity of FtsZ and halts
septation at its earliest identified stage (14, 24), this
method allowed us to produce filamentous cells without directly
inactivating PBP 3.
E. coli CS604-2 pFAD38 exhibited nearly wild-type morphology
both before and after induction of SulA with arabinose, with only a
small fraction of cells having minor terminal branching (not shown). In
contrast, the isogenic mutant lacking PBP 5, CS701-1(pFAD38), exhibited
extremely distorted contours along the length of individual filaments
(data not shown). In addition, the derived diameters of these
populations were increased in cells lacking PBP 5. For CS701-1(pFAD38)
(lacking PBP 5), 79% of the filaments had diameters greater than 1.2 µm, whereas for CS604-2 pFAD38 (with active PBP 5), only 11% of the
filaments were larger than 1.2 µm in diameter (Table 2). Because the
morphological effects were similar to those observed in filaments
produced by addition of aztreonam, the effect of PBP 5 does not appear
to require the simultaneous inactivation of PBP 3. However, we cannot
exclude the possibility that disruption of FtsZ activity and the
cytokinetic ring may have affected the activity of PBP 3 indirectly by
interfering with normal septal structure.
| |
DISCUSSION |
There are two major difficulties in determining the biological
roles of the LMW PBPs. First, because multiple PBPs have similar enzymatic activities there is always the question of whether or not the
loss of one protein might be compensated for by the presence of another
PBP. The second problem is that no one LMW PBP nor any combination of
LMW PBPs is essential for normal laboratory growth of E. coli (4). Thus, if one of these proteins plays a
significant physiological role, then that role either is subtle or is
expressed only under specific circumstances. Previously, we addressed
the first of these problems by creating a set of mutants that
encompasses all possible combinations of deletion mutations among eight
PBPs (4). The premise was that the function of one or more
PBPs might become visible in genetic backgrounds from which
collaborating PBPs had been removed. By examining a subset of these
multiple mutants, we now report that PBP 5 plays an important role in
determining the diameter, topology, and proper proportions of the cell
wall in E. coli.
PBP 5 and shape control.
The deletion of PBP 5 was the major
determining factor in producing cells with abnormal morphology. In
fact, mutants from which all LMW PBPs were deleted still formed cells
with virtually wild-type morphology as long as PBP 5 remained active.
No other individual PBP could substitute for PBP 5 in this regard.
However, even in the absence of PBP 5, major morphological alterations did not appear until at least three PBPs had been deleted from a single
strain. Therefore, it is likely that some combination of
high-molecular-weight and/or LMW PBPs can substitute for the relevant
activity of PBP 5. As yet, we do not know the exact combination of PBPs
that can do this, and it is possible that several different PBP
combinations may be able to obscure the loss of PBP 5. This would
explain why the morphological role of PBP 5 was not discovered until now.
In addition to producing cells with branching and gross morphological
alterations, a more general effect of the loss of PBP 5 was to increase
the overall diameter of cells in a mutant population. However, although
PBP 5 clearly affects diameter, the protein does not appear to
determine diameter, at least not by itself. In the absence of PBP 5 the
cells were still rod-like, and filamentous versions of PBP 5 mutants
remained within a fairly narrow range of increased diameters. Even so,
the contour of single cells or filaments was not uniform. Thus,
although some mechanism imparts to E. coli a rough
cylindrical form, the uniformity of individual cells requires the
operation of PBP 5.
Recently, Gullbrand et al. reported that an E. coli strain
with an altered form of chromosomal replication forms branches at a
fairly high rate in a minimal medium (6; see also
reference 2). Of particular interest is that the
branching frequency increased in the presence of 
-lactam antibiotics
that bound multiple PBPs but was not affected by -lactams specific
for one PBP, nor did it depend on defects in the cell division proteins
PBP 3 or FtsZ (6). These results are consistent with those
reported here: branching and other morphological aberrations were
enhanced in cells lacking multiple PBPs, and filamentation by aztreonam or SulA expression demonstrated that neither PBP 3 nor FtsZ was required for the effect. What is not apparent in the previous studies
is the proximal cause of branching. In light of our current results,
the simplest extrapolation is that the reported medium- and
strain-dependent morphologies are mediated indirectly by an effect on
the level or activity of PBP 5.
Phenotypes associated with loss of PBP 5 homologues in other
bacteria.
Unfortunately, no other gram-negative PBP 5 homologues
have been studied to any degree. However, an extensive set of studies has been performed in the gram-positive organism Bacillus
subtilis (15, 16). Even though numerous mutants have
been constructed from which multiple PBPs were deleted, there is no
reported phenotype in vegetative cells that accompanies the loss of on
of the PBP 5 homologues, either alone or in combination with one or two
other LMW PBPs (15, 23). Therefore, either such a PBP does
not exert a major influence on the morphology of B. subtilis or other proteins continue to substitute for that role in
these multiple mutants.
Two gram-positive cocci, Streptococcus pneumoniae
and Staphylococcus aureus, each contain only one known
LMW PBP that acts as a D,D-carboxypeptidase (19,
25). Each species is viable in the absence of its single LMW PBP,
but morphological effects have been observed. Cells of a mutant
S. pneumoniae lacking the LMW PBP are irregularly shaped
with septa at inappropriate positions or at multiple sites, the
daughter cells have difficulty separating, and the peptidoglycan
surface is laid down in peculiar fashion and is of variable thickness
(19). Although the results were not reported in detail, a
similar PBP mutant of S. aureus may exhibit a similar
phenotype in that the strain grows as enlarged spheres in certain media
(25). Both of these proteins are homologous to PBP 5 from
E. coli, though neither is as strongly similar as the PBPs
from B. subtilis (data not shown). In short, homologues of
E. coli PBP 5 may influence cell morphology in bacteria
other than E. coli.
Summary.
The genesis of specific bacterial shapes remains
mysterious. From the data presented here, we cannot derive a detailed
picture of how E. coli determines its diameter or shape
except to say that PBP 5 is required for constructing a uniform
peptidoglycan cylinder. Because PBP 5 is a carboxypeptidase, the
evidence suggests that the length of the peptide side chains may play a
significant role in how these morphological parameters are determined,
although the exact mechanism is left undecided for now.
We especially thank Tom Henderson, Fran Sailer, and Bernadette
Meberg for technical advice, Joe Lutkenhaus for pFAD38, and Gayle
Streier for graphics.
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Abstract
Introduction
