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Scientific
Publications - Work Done by Microbiology Reader Free Online Full-text Article Journal of Bacteriology, May 2001, p. 3055-3064, Vol. 183, No. 10 Contributions of PBP 5 and DD-Carboxypeptidase Penicillin Binding Proteins to Maintenance of Cell Shape in Escherichia coli
Department of Microbiology and Immunology, University of North Dakota School of Medicine and Health Sciences, Grand Forks, North Dakota 58202-9037 Received 13 November 2000/Accepted 22 February 2001
Escherichia coli has 12 recognized penicillin binding proteins (PBPs),
four of which (PBPs 4, 5, and 6 and DacD) have DD-carboxypeptidase
activity. Although the enzymology of the DD-carboxypeptidases
has been studied extensively, the in vivo functions of these proteins
are poorly understood. To explain why E. coli maintains four
independent loci encoding enzymes of considerable sequence identity
and comparable in vitro activity, it has been proposed that the
DD-carboxypeptidases may substitute for one
another in vivo. We tested the validity of this equivalent
substitution hypothesis by investigating the effects of these
proteins on the aberrant morphology of
To synthesize or modify its peptidoglycan cell wall, Escherichia coli maintains 12 penicillin binding proteins (PBPs), most of which have no demonstrable physiological purpose. Although each of these enzymes mediates one or more known biochemical reactions, most are not essential because E. coli survives without them (8). Traditionally, these proteins are separated into two subfamilies, only one of which contributes to cell survival. The high-molecular-weight (HMW) PBPs (PBPs 1a, 1b, 1c, 2, and 3) are bifunctional transglycosylase/transpeptidases or monofunctional transpeptidases which synthesize and incorporate individual peptidoglycan strands into the murein sacculus (9, 10, 19, 26). Each of these HMW PBPs except PBP 1c has a defined physiological function (19, 26, 27, 29, 30). In contrast, the functions of the seven low-molecular-weight (LMW) PBPs remain enigmatic (3, 8, 13). The LMW PBPs (PBPs 4, 5, 6, and 7, DacD, AmpC, and AmpH) are subdivided into
four enzymatic classes: three monofunctional DD-carboxypeptidases,
PBP 5, PBP 6, and DacD; one bifunctional DD-carboxypeptidase/DD-endopeptidase,
PBP 4; one DD-endopeptidase, PBP 7; and two
class C
These similarities and genetic data have led to the widespread assumption that the DD-carboxypeptidases act as functional equivalents in vivo, so that these multiple carboxypeptidases may compensate for one another in situations where one is lost or inactivated (3, 5). Unfortunately, this "equivalent substitution" hypothesis has been difficult to test because these proteins are not essential for bacterial survival. For example, a quadruple mutant grows normally even though it lacks PBPs 4, 5, and 6 and DacD (3, 8), and mutants lacking all seven LMW PBPs grow well (8). However, there is a small amount of evidence for differences among the LMW PBPs. As discussed in more detail below, morphological defects accompany the loss of PBP 5 but of no other PBP (22). Also, deletion of the dacA gene (encoding PBP 5) substantially reverses the filamentation phenotype of a temperature-sensitive ftsK allele, whereas deletion of dacB (encoding PBP 4) or dacC (encoding PBP 6) does not (4). In addition, overexpression of either PBP 5 or PBP 6, but not PBP 4, reverses the effects of a specific temperature-sensitive allele of PBP 3 (5), implying that PBPs 5 and 6 (but not 4) might perform similar functions in vivo. Finally, it has been reported that although overexpression of PBP 5 causes E. coli to become spherical and eventually lyse (18), PBP 4, PBP 6, and DacD are nonlethal (3, 17, 33), suggesting that these latter PBPs might differ from PBP 5. Because so few of these types of observations have been made, the idea of universal interchangeability among these proteins persists in the literature and minds of many workers, and none of the observations has brought us closer to understanding what these PBPs are doing. In short, the paucity of testable phenotypes has limited experimental approaches to the question of the biological function of the LMW PBPs. Recently, we reported that PBP 5 helps maintain normal cell wall morphology
and diameter in E. coli (22). By comparing isogenic
mutants, we determined that no other LMW PBP could substitute
for PBP 5 to correct these defects in vivo at wild-type expression
levels. In this report we compare additional isogenic mutant strains
to determine the minimum complement of PBP deletions necessary to
generate the morphological defects. We also show that no other
DD-carboxypeptidase can complement the
Bacterial strains and growth conditions. The E. coli
strains used in this work are listed in Table 1. Strains
carrying the dacD insertion-deletion mutation were constructed
by moving dacD::Kan536-2 from CS18-2K (8) into the
indicated strains by P1 transduction (20). Plasmid
vectors pBAD18-Cam and pBAD24-Amp were provided by J. Beckwith (11).
Bacterial strains were maintained on Luria-Bertani (LB) broth or agar
plates, supplemented when appropriate with chloramphenicol (20 µg/ml)
or ampicillin (50 µg/ml) to maintain plasmids or with glucose
(0.1%) to inhibit expression from pBAD promoters. Overnight cultures
for morphological determinations were diluted 1:500 into fresh LB
broth supplemented with 0.005 to 0.1% (wt/vol) arabinose and were
incubated at 37°C with shaking until reaching an A600 of
approximately 0.2. When required, aztreonam (Bristol Meyers-Squibb,
New York, N.Y.) was added to a final concentration of 10 µg/ml.
Other chemicals were purchased from Sigma Chemical Co. (St. Louis,
Mo.) unless otherwise noted. Growth curves for determining lysis
after overexpression of PBPs and for comparisons of growth rates were
performed in flask assays and by growth in a Bioscreen C Microbiology
Reader (Labsystems, Helsinki, Finland).
Microscopic evaluation of morphological aberration. To compare the relative morphologies of the different E. coli strains, cultures were diluted to an A600 of 0.005 and grown in LB at 37°C, and samples were removed during early logarithmic growth phase (A600 = 0.2 ± 0.02). Photographs were collected from random microscopic fields as described previously (22), and cell populations were evaluated by two to three individuals for the degree of morphological aberration. In our earlier work we could measure and quantify alterations in cell diameter (22). However, the morphological differences that we observed in this study did not lend themselves to straightforward quantification. Therefore, the results are expressed as a subjective measure of relative morphological aberration based on differences in the numbers of cells affected and the degree to which they exhibited increased diameters, uneven contours, squaring of the poles, lateral branching, and filamentation. The extent of these differences is reported on a relative scale of 0 (no observable differences from the parental strain) to 4 (numerous and extreme shape differences). Molecular and biochemical techniques. Plasmid preparations and DNA isolation from agarose gels were performed with commercially available kits from Qiagen (Valencia, Calif.). Competent strains were prepared and transformed by using a Bio-Rad (Hercules, Calif.) Gene Pulser electroporation apparatus according to the manufacturer's instructions. Chromosomal DNA was isolated as described previously (22). Oligonucleotides for PCR were purchased from Gibco Life Sciences (Grand Island, N.Y.). PBP expression was confirmed by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by silver staining (25) or by labeling the PBPs with 125I-penicillin X as described previously (13). The amounts of PBPs produced were quantified by imaging SDS-PAGE gels with a Molecular Dynamics 445SI PhosphorImager (Amersham Pharmacia, Uppsala, Sweden) and measuring band intensities by using ImageQuant software (Amersham Pharmacia, Uppsala, Sweden). Enzymes and reagents were purchased from New England Biolabs (Beverly, Mass.). Placing PBP genes under control of the arabinose promoter. Primers B1 (5'-CTCTCTCTGAATTCTTATGCGATTTTCCAG-3') and B2 (5'-CTCTCTCTCTAGACTTTTTGACTAATTGTTCTG-3') were used to PCR amplify the dacB (PBP 4) gene from the CS109 chromosome. The EcoRI-XbaI fragment of this PCR product was ligated into pBAD24-Amp to create plasmid pPJ4-Amp. Afterwards, the NheI-XbaI fragment was excised from pPJ4-Amp and ligated into pBAD18-Cam, creating the PBP 4 expression vector, pPJ4. Primer pairs C1 (5'-CTCTTTTGCTAGCAGGAGGAATTCACCATGACGCAATACTCCTCTC-3') plus C2 (5'-CTCTCTCTCTAGAAGAAGAATTAGAGAACCAGCTGC-3') and D1 (5'-CTCTCTCGCTAGCAGGAGGAATTCACCATGCTGTTGAAACGCCGTC-3') plus D2 (5'-CCCCCCCTCTCAGATCTGCAAAAGAAAGGTCAGGCCTTATG-3') were used to PCR amplify the wild-type genes dacC and dacD, respectively, from the CS109 chromosome. The NheI-XbaI fragments of the dacC and dacD PCR products were ligated into pBAD18-Cam to create the PBP expression vectors pPJ6 and pPJDacD, respectively. Primers E1 (5'-CGCGGCGGCTAGCAGGAGGAATTCGTCATGAATACCATTTTTTCCGC-3') and E2 (5'-CCAAGGATTCTAGAAGCTTTTTTTAGTTACCTTCCGGGATTTC-3') were used to PCR amplify a truncated fragment of the dacA gene from the PBP 5 expression vector pPJ5C (22). This fragment encoded the full-length PBP 5 protein minus the 54 3'-terminal nucleotides of the dacA coding sequence, so that the C-terminal amphipathic anchor of PBP 5 was deleted. The NheI-XbaI fragment of this PCR product was ligated into pBAD18-Cam, creating plasmid pPJ5D. pPJ5S was constructed by using a Stratagene QuickChange site-directed mutagenesis kit and primers F1 (5'-CGCCGCGATCCTGCCGGCCTGACCAAAATGATG-3') and F2 (5'-CATCATTTTGGTCAGGCCGGCAGGATCGCGGCG-3'), with pPJ5C as the template (Stratagene Co., La Jolla, Calif.). All clones were sequenced commercially at Colorado State University using the ABI Prism Terminator Cycle Sequencing technique (Fort Collins, Colo.).
Previously, we began to determine which PBPs were necessary to maintain the normal morphology of E. coli by comparing isogenic strains that retained only one or two active LMW PBPs, in mutants from which six to seven PBP genes had been deleted (22). Although loss of PBP 5 was the primary determinant of aberrant morphology, deletion of dacA alone had little effect on the wild-type strain (22). Thus, it was possible that an unknown combination of different PBPs could take the place of PBP 5. Therefore, to further clarify the role of PBP 5, and to determine the minimum complement of deletions necessary to yield the morphological phenotype, we examined the morphology of 41 single, double, and triple PBP mutants from our comprehensive PBP mutant set (8). Morphological defects are associated with deletion of dacA.
In agreement with previous findings (22), no significant
morphological defects were associated with loss of a single PBP (data
not shown). In particular, the dacA mutant, CS12-7, showed no
significant differences from the wild-type strain, CS109 (Table
2; Fig. 1A and B), and though a few
aztreonam-induced filaments of CS12-7 were slightly rounded or
branched, the numbers were too small to quantify (data not shown). Of
special importance was that no double or triple mutant in which PBP
5 remained active exhibited significant morphological oddities (data
not shown), once again implicating PBP 5 as the dominant determinant
for this phenotype.
Although deletion of PBP 5 alone had little effect on cellular morphology, observable defects were augmented by loss of additional PBPs. The differences among mutants lacking PBP 5 were assessed subjectively by inspection of multiple photographs of cell cultures, and the strains were organized according to their relative degree of morphological aberration (Table 2). Because of their subjective nature, these rankings should be considered as a rough guide to the degree of morphological differences between strains. Nonetheless, large differences in scoring (>2 units) do depict significant differences in the frequency and/or degree of cell shape aberrations.
The mutants exhibiting the most dramatic morphological defects were those
lacking PBPs 5 and 6 and either PBP 4 or PBP 7 (strains CS322-1 and
CS331-1, respectively) (Table 2; Fig. 1C and
D). On the other hand, the morphology of strain CS316-1, which is
missing PBPs 4, 6, and 7 (described as
The data reinforced the primary role for PBP 5 in creating morphologically
uniform cells. For example, strain CS362-1 (
Only PBP 5 complements the morphological defects of dacA mutants. The preceding experiments addressed the question of whether wild-type levels of different LMW PBPs could substitute for PBP 5. It remained possible that these other PBPs might complement the loss of PBP 5 if they were expressed in higher quantities. Therefore, to determine which of E. coli's DD-carboxypeptidases could complement the morphological defects of dacA mutants, we cloned the dacB, dacC, and dacD genes of wild-type strain CS109 into the arabinose-inducible expression vector pBAD18-Cam (11). The PBP subclones were transformed into CS109 and into the septuple PBP mutant CS701-1, and the viability of each strain was evaluated across a range of arabinose concentrations (0.1 to 0.0001%) using a Bioscreen C Microbiology Reader to determine the most effective inducing conditions (data not shown). Final arabinose concentrations for gene induction and complementation studies were selected by comparing the growth rates of cells harboring the PBP expression vectors to those of identical strains containing the control vector pBAD18-Cam, supplemented by subjective visual assessment of morphology. Cells were photographed during log-phase growth and again 45 min after
treatment with aztreonam (10 µg/ml) to enhance any morphological
aberrations by filamenting the cells. As reported previously (22),
E. coli CS604-2(pBAD18-Cam), which lacks six PBPs but retains
wild-type dacA, was morphologically similar to wild-type
E. coli (Fig. 2A). In contrast, CS701-1(pBAD18-Cam),
which is isogenic to CS604-2(pBAD18-Cam) but lacks the dacA gene,
exhibited drastic alterations in morphology both before and after
filamentation induced by aztreonam (Fig. 2B). Regulated
expression of PBP 5 in CS701-1(pPJ5C) reversed these defects, and the
complemented strain was visually indistinguishable from
CS604-2(pBAD18) (Fig. 2C). In contrast, expression
of cloned PBP 4 (Fig. 2D), PBP 6 (Fig.
2E), or DacD (Fig. 2F) did not complement
the morphological defects of CS701-1 at any level of expression. In
fact, in most cases the severity of the morphological defects in
CS701-1 was exacerbated by expression of these non-PBP 5 DD-carboxypeptidases
(Fig. 2D to F).
Both the active site and membrane anchoring terminus of PBP 5 are required for complementation. PBP 5 has three known domains: one containing the DD-carboxypeptidase activity, one responsible for localizing PBP 5 to the outer face of the cytoplasmic membrane, and a third domain of unknown function between these other two (7, 15, 23, 34). To determine if the enzymatic and membrane-binding activities were necessary to complement the morphological phenotype of dacA mutants, we constructed vectors to express mutant PBP 5 proteins in which one or the other of these two domains was inactivated. Plasmid pPJ5D expressed a truncated mutant protein lacking the carboxy-terminal 18 amino acids that anchor PBP 5 to the membrane. Plasmid pPJ5S expressed a full-length PBP 5 in which a Ser44-to-Gly44 mutation eliminated the protein's DD-carboxypeptidase activity (34). Truncated PBP 5 expressed from pPJ5D was unable to complement the morphological defects of CS701-1, either before or after treatment with aztreonam (Fig. 2G). In fact, even low-level expression of the truncated protein was lethal or actually augmented the morphological abnormalities observed in these cells, indicating that membrane association of PBP 5 is important for its proper functioning in vivo. Similarly, enzymatically inactive PBP 5 encoded by pPJ5S was unable to complement the defects in CS701-1 (Fig. 2H), indicating that the DD-carboxypeptidase activity was essential for creating morphologically normal cells.
All DD-carboxypeptidases are lytic
when overexpressed early in the growth phase. While performing
complementation studies, we noted that prolonged expression of each of the
DD-carboxypeptidases decreased the viability
of fast-growing cultures, but that this effect diminished in
slow-growing or stationary-phase cells. However, previous reports
suggested that only PBP 5 induced lysis when overexpressed. Therefore,
to clarify the differences between our observations and previous
experiments, we overexpressed each of the DD-carboxypeptidases
in E. coli CS109. All active DD-carboxypeptidases
lysed CS109 within 2 h of gene induction in flask assays (Fig.
3A), and viable cell counts in these cultures
dropped 2 logs within 2 h of protein induction (data not shown). The
onset of lysis in cultures grown in the Bioscreen C Microbiology
Reader was delayed and the rate of lysis was reduced, but the results
were similar in that all of the DD-carboxypeptidases
lysed E. coli (Fig. 3B). In each case, the
microscopic effects of overexpressing the carboxypeptidases were
similar: cells grew normally for a brief period then rapidly became
spherical and lysed shortly thereafter (data not shown). This is
consistent with the effects previously described for PBP 5 (28).
Lysis, but not lethality, was inhibited by addition of 15% sucrose to
osmotically stabilize the labile spheroplasts, and lysis did not
occur if protein synthesis was interrupted by adding tetracycline
(12.5 µg/ml) 30 min after gene induction (though lysis occurred
normally if tetracycline was added 60 min after induction) (data not
shown).
Because these lysis phenotypes were at odds with those reported previously,
we examined possible reasons for the discrepancy. In the earlier
studies in which DD-carboxypeptidase overexpression
failed to lyse E. coli, the proteins were not expressed until
cultures reached the mid or late logarithmic growth phase. Therefore,
to determine if lysis sensitivity was influenced by growth phase,
we induced the carboxypeptidases in cultures at different times
in the growth cycle. Growth was monitored in the Bioscreen C Microbiology
Reader as before, but the genes were induced by adding arabinose
at time points corresponding to early, mid, and late log phases.
As cells progressed from early to late log phase, induction of
the various PBPs had a decreasing lytic effect (Fig. 4). This
was not because PBP expression was reduced; indeed, in each case,
high levels of PBPs were expressed at these times, as determined
by labeling the PBPs and visualizing them by SDS-PAGE (data not
shown). Thus, each of the DD-carboxypeptidases was lytic
when overexpressed, but cell cultures were more sensitive to lysis
during early logarithmic growth and became increasingly resistant
as they approached stationary phase.
Removal of the C-terminal membrane anchor increases lethality
of PBP 5. That the membrane anchoring domain of PBP 5 may serve a
physiological purpose was suggested by the observation that the
carboxy-truncated form of PBP 5 did not complement CS701-1 and,
instead, apparently exacerbated morphological defects in the strain.
In light of these results, we wished to determine if the membrane
anchor affected the lytic activity of overexpressed PBP 5. Lysis of
CS109 by wild-type PBP 5 (from pPJ5) and lysis by carboxy-truncated
PBP 5 (from pPJ5D) were compared in flask assays (data not shown) and
in the Bioscreen C Microbiology Reader, using a gradient of arabinose
concentrations to induce different amounts of each cloned protein
(Fig. 5). Although overexpression of truncated PBP
5 did not appreciably reduce the time of lysis onset, lysis required
a substantially lower concentration of truncated PBP 5 than wild-type
PBP 5. A 7-fold overexpression of truncated PBP 5 lysed CS109 with
kinetics that were comparable to a 65-fold overexpression of the
full-length protein (Fig. 5). Even a fourfold
overexpression of truncated PBP 5 was rapidly lethal (Fig.
5). Roughly speaking, compared to the overexpression
of wild-type PBP 5, only 1/10 of the amount of truncated PBP 5 was
required to induce a comparable amount and extent of lysis.
Despite 25 years of work, little is known about the biologically relevant roles of the DD-carboxypeptidase PBPs in E. coli. Previously, we noted that deletion of PBP 5 altered the antibiotic resistance and morphology of several PBP mutants (22), representing the first phenotype associated with the loss of one of the DD-carboxypeptidases in an otherwise wild-type background. These observations enabled us to perform a direct test of the idea that several of these proteins have similar or identical functions, an idea that we call the equivalent substitution hypothesis. To date, little or no evidence contradicts this assertion of interchangeability (with the possible exception of two reports describing the effects of DD-carboxypeptidases on non-PBP mutants [4, 5]). Our findings indicate that PBP 5 has a unique role among the DD-carboxypeptidases in the development of normal bacterial shape and that no other PBP can completely substitute for PBP 5 in this regard. PBP mutant combinations and abnormal morphology. Although
all strains with morphological abnormalities lacked PBP 5, the extent of
deviation in diameter, contour, branching, and other defects varied
greatly among mutants from which PBP 5 was deleted. The most dramatic
aberrations appeared in cells lacking at least two additional PBPs
and were correlated with two prominent combinations of mutations:
deletion of PBP 5 and one of the endopeptidases (PBP 4 or 7), or
deletion of PBP 5 and one of the class C
An important observation was that the mutational patterns associated with
altered morphology were strongly correlated with the classical
groupings of PBPs based on sequence similarity and biochemical
activity; that is, the DD-carboxypeptidases,
endopeptidases, and class C
Possible model for PBP 5 action. How might PBP 5 contribute to the construction of a normally shaped cell? Theoretically, PBP 5 could contribute a specific and necessary enzymatic activity or the protein could be a structural component of a larger morphological complex (although the two possibilities are not mutually exclusive). We approached this question by creating two PBP 5 derivatives: one enzymatically inactive, and the other truncated to remove its membrane anchor. Neither derivative complemented dacA mutants, suggesting that PBP 5 must be both enzymatically active and properly localized to create the normal morphological form of E. coli. Although the data do not address whether PBP 5 acts in concert with other proteins, they do diminish the possibility that PBP 5 acts solely as a structural protein. This is the first demonstration that membrane localization of a LMW PBP has physiological significance, because removal of the membrane anchor prevented truncated PBP 5 from performing its normal morphological function. An alternate explanation is that removing the carboxyl terminus altered the enzymatic activity of PBP 5 so that losing the membrane anchor had only an indirect effect. However, two pieces of evidence argue against this interpretation. First, truncated PBP 5 retained the ability to bind radiolabeled penicillin, a property of the active site and an indication that the enzymatic activity was unchanged. Second, the shortened protein remained lethal when overexpressed, a characteristic that also requires a functional DD-carboxypeptidase. In fact, the lethality of anchorless PBP 5 was increased approximately 10-fold over its wild-type counterpart, suggesting not only that membrane localization of PBP 5 is necessary for proper morphological control but that such localization may regulate the numbers or types of substrates on which PBP 5 can act.
Given these results, there are at least two ways by which PBP 5 may function. First, the carboxy-terminal membrane anchor may play a relatively nonspecific role by restricting access of PBP 5 to its peptidoglycan substrate; second, the membrane anchor may play a more active role by interacting with proteins or specific regions of the inner membrane to confine PBP 5's activity to localized sites. Since there is no evidence for the second possibility, we will discuss only the first possibility at more length.
The recent determination of the crystal structure of a truncated form of PBP
5 (7) allows us to visualize a simple, speculative
model by which the activity of PBP 5 might be constrained (Fig.
6). As reported by Davies et al. PBP 5 is composed of a
globular domain, which contains the DD-carboxypeptidase
active site on its outer face, and a domain of unknown function,
which is composed almost entirely of
This speculative model suggests a mechanism by which PBP 5 might maintain the smooth rod shape of the cell. In the absence of PBP 5, peptide side chains extending toward the inner membrane may remain available for inappropriate cross-linking with newly synthesized glycan chains, giving rise to misaligned sections of cell wall which manifest themselves as altered shapes of the peptidoglycan sacculus. If present, an endopeptidase (PBP 4 or 7) might remove these irregular cross-links, thus reversing or masking the morphological effects of losing PBP 5. Such a two-step pathway would be consistent with the fact that the most severely affected mutants lack PBP 5 and at least one of the endopeptidases. Of course, we do not know enough to explain why one endopeptidase cannot fulfill the role of the other, nor do we know enough about AmpC or AmpH to fit them into this scheme. Although the specifics of this putative morphological pathway must await further experimentation, the results do reinforce the argument that bacterial shape is not determined simply by general physical forces but that, instead, cell shape is monitored and manipulated by specific cellular proteins.
Overexpression of any DD-carboxypeptidase lyses E. coli. As discussed above, excessive DD-carboxypeptidase activity may inhibit formation of a normal sacculus by interfering with the cross-linking of peptide side chains. Previous work supports this hypothesis for PBP 5 because overexpression of the protein causes E. coli to assume a rounded morphology, blocks cell division, and eventually leads to lysis after the cells grow to greatly increased diameters (17). On the other hand, overexpression of PBPs 4 and 6 was reported to be nonlethal (17, 18), and Baquero et al. reported no lysis after overexpression of DacD (3). However, with the exception of a single report calling the activity of PBP 6 into question (33), it has been consistently reported that PBPs 4, 5, and 6 and DacD are active DD-carboxypeptidases (1-3). Therefore, why should only PBP 5 induce cell lysis? In contrast to earlier reports, we observed that overexpression of each of
these other DD-carboxypeptidases was lethal and
lytic. The difference between our results and those reported by
other laboratories is most easily explained by the fact that in
previous experiments PBP 4, PBP 6, and DacD were expressed late in
logarithmic growth or near the beginning of stationary phase.
However, we observed that these PBPs lysed cells only if the proteins
were overexpressed in the early stages of logarithmic growth. This
behavior is reminiscent of the lytic effects of
Biological relevance of the DD-carboxypeptidases. The DD-carboxypeptidases are not essential for bacterial viability (8), nor do they appear to completely substitute for one another. Therefore, ignoring the possibility that novel DD-carboxypeptidases remain undiscovered in E. coli, we entertain two scenarios which might explain the difficulty of ascribing functions to these proteins. First, a DD-carboxypeptidase activity may be essential in E. coli's normal ecological niche in the digestive tract of vertebrates but unnecessary in the zoo-like environment of laboratory culture. Thus, the phenotype could be subtle or undetectable except under special conditions. Second, the enzymes might contribute an incremental growth advantage to E. coli in the wild. In this case, the significance of defects caused by inactivation of one or more DD-carboxypeptidases might be overlooked. For example, there are small, but definite, differences in growth rates among our collection of multiple PBP mutants (unpublished results). Although a 1% difference in growth rate between two strains grown in flask culture would probably be inconsequential in the laboratory, the long-term selective advantage of such a difference in the wild can be significant.
We thank Avery Paulson for help in evaluating the extent of morphological abnormalities among the mutants. This work was supported by grant GM61019 from the National Institutes of Health. D. Nelson was supported by a North Dakota EPSCoR doctoral fellowship from the National Science Foundation.
* Corresponding author. Mailing address: Department of Microbiology & Immunology, University of North Dakota School of Medicine and Health Sciences, Grand Forks, ND 58202-9037. Phone: (701) 777-2624. Fax: (701) 777-2054. E-mail: kyoung@medicine.nodak.edu.
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dacA
mutants, which produce no PBP 5. Although cloned PBP 5 complemented
the morphological phenotype of a
-lactamases,
AmpC and AmpH (
;
CS109(pPJ4),
;
CS109(pPJ5C),
;
CS109(pPJ6),
;
CS109(pPJDacD),
.
.
;
PBP 5 (12× expression),
;
carboxy-truncated PBP 5 (7× expression),
-helical
head portion of the molecule. The
