Modulation of cell wall synthesis by DNA replication in Escherichia coli during initiation of cell growth.

Resting cells of Escherichia coli are able to initiate growth and murein biosynthesis in the presence of beta-lactam antibiotics binding to penicillin-binding proteins (PBPs) 1a and 1b (E. J. de la Rosa, M. A. de Pedro, and D. Vázquez, Proc. Natl. Acad. Sci. USA 82:5632-5635, 1985). Under these conditions, cells elongate normally until they approach the first doubling in mass, the time at which cell lysis starts. Assuming that coupling between DNA replication and cell division both in cells starting growth and in growing cells is essentially similar, triggering of the lytic response in the beta-lactam-treated cells coincides with the termination of the first round of DNA replication. This coincidence suggests that both events are interrelated. We investigated this possibility by studying the initiation of growth in cultures of wild-type strains and in cell division mutants treated with beta-lactams inhibiting PBPs 1a and 1b and with the DNA replication inhibitor nalidixic acid. Addition of nalidixic acid, even late in the first cell cycle, prevented the lytic response of the cells to the blockade of PBPs 1a and 1b. The effect of nalidixic acid is more likely due to its action on DNA replication itself than to its indirect inhibitory effect on cell division or to its ability to induce the SOS system of the cell. These observations favor the idea that the cell wall biosynthetic machinery might be modulated by DNA replication at precise periods during cell growth.     

Lysis of Escherichia coli by beta-lactams which bind penicillin-binding proteins 1a and 1b: inhibition by heat shock proteins.

The heat shock proteins (HSPs) of Escherichia coli were artificially induced in cells containing the wild-type rpoH+ gene under control of a tac promoter. At 30 degrees C, expression of HSPs produced cells that were resistant to lysis by cephaloridine and cefsulodin, antibiotics that bind penicillin-binding proteins (PBPs) 1a and 1b. This resistance could be reversed by the simultaneous addition of mecillinam, a beta-lactam that binds PBP 2. However, even in the presence of mecillinam, cells induced to produce HSPs were resistant to lysis by ampicillin, which binds all the major PBPs. Lysis of cells induced to produce HSPs could also be effected by imipenem, a beta-lactam known to lyse nongrowing cells. These effects suggest the existence of at least two pathways for beta-lactam-dependent lysis, one inhibited by HSPs and one not. HSP-mediated lysis resistance was abolished by a mutation in any one of five heat shock genes (dnaK, dnaJ, grpE, GroES, or groEL). Thus, resistance appeared to depend on the expression of the complete heat shock response rather than on any single HSP. Resistance to lysis was significant in the absence of the RelA protein, implying that resistance could not be explained by activation of the stringent response. Since many environmental stresses promote the expression of HSPs, it is possible that their presence contributes an additional mechanism toward development in bacteria of phenotypic tolerance to beta-lactam antibiotics.     

Interaction of beta-lactam antibiotics with penicillin-binding proteins from Bacillus megaterium

The binding properties of 25 beta-lactam antibiotics to Bacillus megaterium membranes have been studied. The affinities of the antibiotics for the penicillin-binding proteins (PBPs) are also reported. We found that PBP 4 has the highest affinity for nearly all the antibiotics studied whereas PBP 5 has the lowest affinity. Both PBP 4 and PBP 5 appear to be dispensable for the maintenance of bacterial growth and survival and appear to be DD-carboxypeptidases. Only the beta-lactam cefmetazol bound preferentially to PBP 5 and has been used to study the inhibition of DD-carboxypeptidase. Comparative studies with beta-lactam that simultaneously result in (a) binding to PBPs 1 and 3, (b) inhibition of cell growth and (c) lysis, stressed the importance of PBPs 1 and 3 for cell growth and survival.     

Expression and characterization of the ponA (ORF I) gene of Haemophilus influenzae: functional complementation in a heterologous system.

The coding sequence of the Haemophilus influenzae ORF I gene was amplified by PCR and cloned into different Escherichia coli expression vectors. The ORF I-encoded protein was approximately 90 kDa and bound 3H-benzyl-penicillin and 125I-cephradine. This high-molecular-weight penicillin-binding protein (PBP) was also shown to possess transglycosylase activity, indicating that the ORF I product is a bifunctional PBP. The ORF I protein was capable of maintaining the viability of E. coli delta ponA ponB::spcr cells in transcomplementation experiments, establishing the functional relevance of the significant amino acid homology seen between E. coli PBP 1A and 1B and the H. influenzae ORF I product. In addition, the physiological functioning of the H. influenzae ORF I (PBP 1A) product in a heterologous species established the ability of the enzyme not only to recognize the E. coli substrate but also to interact with heterologous cell division proteins. The affinity of the ORF I product for 3H-benzylpenicillin and 125I-cephradine, the MIC of beta-lactams for E. coli delta ponA ponB::spcr expressing the ORF I gene, and the amino acid alignment of the PBP 1 family of high-molecular-weight PBPs group the ORF I protein into the PBP 1A family of high-molecular-weight PBPs.     

Inhibition of the binding of penicillin to the pneumococcal penicillin-binding proteins (PBPs) by exogenous cell wall peptides

Incubation of pneumococci with D-alanine-containing peptides naturally occurring in peptidoglycan protected cells against lysis and killing by beta-lactam antibiotics near MIC. Such peptides caused decreased binding of the antibiotic to penicillin-binding proteins (PBPs), primarily PBP 2B. This provides direct evidence in vivo for the hypothesis that beta-lactams act as substrate analogues and identifies PBP 2B as a killing target in pneumococci.     

Interaction of non-lytic beta-lactams with penicillin-binding proteins in Streptococcus pneumoniae

The monobactam aztreonam and the cephalosporin ceftazidime, beta-lactam antibiotics that possess the same side chain R1, showed unusual effects on exponentially growing pneumococci compared to other beta-lactams. Both antibiotics did not induce lysis even at concentrations up to 2 mg ml-1, values well above the respective MICs. However, morphological alterations and growth inhibition of the cells were observed at much lower concentrations. Binding to penicillin-binding proteins (PBPs) in vitro could be monitored directly by using anti-aztreonam antiserum and the Western blot technique. Both antibiotics showed high affinity for PBP 3, but had an extremely low affinity for PBP 2b. It is suggested that the failure to bind to PBP 2b is responsible for the failure to induce lysis in pneumococci.     

Unstable Escherichia coli L forms revisited: growth requires peptidoglycan synthesis

Growing bacterial L forms are reputed to lack peptidoglycan, although cell division is normally inseparable from septal peptidoglycan synthesis. To explore which cell division functions L forms use, we established a protocol for quantitatively converting a culture of a wild-type Escherichia coli K-12 strain overnight to a growing L-form-like state by use of the beta-lactam cefsulodin, a specific inhibitor of penicillin-binding proteins (PBPs) 1A and 1B. In rich hypertonic medium containing cefsulodin, all cells are spherical and osmosensitive, like classical L forms. Surprisingly, however, mutant studies showed that colony formation requires d-glutamate, diaminopimelate, and MurA activity, all of which are specific to peptidoglycan synthesis. High-performance liquid chromatography analysis confirmed that these L-form-like cells contain peptidoglycan, with 7% of the normal amount. Moreover, the beta-lactam piperacillin, a specific inhibitor of the cell division protein PBP 3, rapidly blocks the cell division of these L-form-like cells. Similarly, penicillin-induced L-form-like cells, which grow only within the agar layers of rich hypertonic plates, also require d-glutamate, diaminopimelate, and MurA activity. These results strongly suggest that cefsulodin- and penicillin-induced L-form-like cells of E. coli-and possibly all L forms-have residual peptidoglycan synthesis which is essential for their growth, probably being required for cell division.     

Differential effect of mutational impairment of penicillin-binding proteins 1A and 1B on Escherichia coli strains harboring thermosensitive mutations in the cell division genes ftsA, ftsQ, ftsZ, and pbpB.

To study the functional differences between penicillin-binding proteins (PBPs) 1A and 1B, as well as their recently postulated involvement in the septation process (F. García del Portillo, M. A. de Pedro, D. Joseleau-Petit, and R. D'Ari, J. Bacteriol. 171:4217-4221, 1989), a series of isogenic strains with mutations in the genes coding for PBP 1A (ponA) or PBP 1B (ponB) or in the cell division-specific genes ftsA, ftsQ, pbpB, and ftsZ was constructed and used as the start point to produce double mutants combining the ponA or ponB characters with mutations in cell division genes. PBP 1A seemed to be unable to preserve cell integrity by itself, requiring the additional activities of PBP 2, PBP 3, and FtsQ. PBP 1B was apparently endowed with a more versatile biosynthetic potential that permitted a substantial enlargement of PBP 1A-deficient cells when PBP 2 or 3 was inhibited or when FtsQ was inactive. beta-Lactams binding to PBP 2 (mecillinam) or 3 (furazlocillin) caused rapid lysis in a ponB background. The lytic effect of furazlocillin to ponB cell division double mutants was suppressed at the restrictive temperature irrespective of the identity of the mutated cell division gene. These results indicate that PBPs 1A and 1B play distinct roles in cell wall synthesis and support the idea of a relevant involvement of PBP 1B in peptidoglycan synthesis at the time of septation.     

Structural basis for the beta lactam resistance of PBP2a from methicillin-resistant Staphylococcus aureus

The multiple antibiotic resistance of methicillin-resistant strains of Staphylococcus aureus (MRSA) has become a major clinical problem worldwide. The key determinant of the broad-spectrum beta-lactam resistance in MRSA strains is the penicillin-binding protein 2a (PBP2a). Because of its low affinity for beta-lactams, PBP2a provides transpeptidase activity to allow cell wall synthesis at beta-lactam concentrations that inhibit the beta-lactam-sensitive PBPs normally produced by S. aureus. The crystal structure of a soluble derivative of PBP2a has been determined to 1.8 A resolution and provides the highest resolution structure for a high molecular mass PBP. Additionally, structures of the acyl-PBP complexes of PBP2a with nitrocefin, penicillin G and methicillin allow, for the first time, a comparison of an apo and acylated resistant PBP. An analysis of the PBP2a active site in these forms reveals the structural basis of its resistance and identifies features in newly developed beta-lactams that are likely important for high affinity binding.     

Nucleotide sequences of the pbpX genes encoding the penicillin-binding proteins 2x from Streptococcus pneumoniae R6 and a cefotaxime-resistant mutant, C506

Development of penicillin resistance in Streptococcus pneumoniae is due to successive mutations in penicillin-binding proteins (PBPs) which reduce their affinity for beta-lactam antibiotics. PBP2x is one of the high-Mr PBPs which appears to be altered both in resistant clinical isolates, and in cefotaxime-resistant laboratory mutants. In this study, we have sequenced a 2564 base-pair chromosomal fragment from the penicillin-sensitive S. pneumoniae strain R6, which contains the PBP2x gene. Within this fragment, a 2250 base-pair open reading frame was found which coded for a protein having an Mr of 82.35kD, a value which is in good agreement with the Mr of 80-85 kD measured by SDS-gel electrophoresis of the PBP2x protein itself. The N-terminal region resembled an unprocessed signal peptide and was followed by a hydrophobic sequence that may be responsible for membrane attachment of PBP2x. The corresponding nucleotide sequence of the PBP2x gene from C504, a cefotaxime-resistant laboratory mutant obtained after five selection steps, contained three nucleotide substitutions, causing three amino acid alterations within the beta-lactam binding domain of the PBP2x protein. Alterations affecting similar regions of Escherichia coli PBP3 and Neisseria gonorrhoeae PBP2 from beta-lactam-resistant strains are known. The penicillin-binding domain of PBP2x shows highest homology with these two PBPs and S. pneumoniae PBP2b. In contrast, the N-terminal extension of PBP2x has the highest homology with E. coli PBP2 and methicillin-resistant Staphylococcus aureus PBP2'. No significant homology was detected with PBP1a or PBP1b of Escherichia coli, or with the low-Mr PBPs.     

Increase of the deacylation rate of PBP2x from Streptococcus pneumoniae by single point mutations mimicking the class A beta-lactamases.

The class A beta-lactamases and the transpeptidase domain of the penicillin-binding proteins (PBPs) share the same topology and conserved active-site residues. They both react with beta-lactams to form acylenzymes. The stability of the PBP acylenzymes results in the inhibition of the transpeptidase function and the antibiotic activity of the beta-lactams. In contrast, the deacylation of the beta-lactamases is extremely fast, resulting in a high turnover of beta-lactam hydrolysis, which confers resistance to these antibiotics. In TEM-1 beta-lactamase from Escherichia coli, Glu166 is required for the fast deacylation and occupies the same spatial location as Phe450 in PBP2x from Streptococcus pneumoniae. To gain insight into the deacylation mechanism of both enzymes, Phe450 of PBP2x was replaced by various residues. The introduction of ionizable side chains increased the deacylation rate, in a pH-dependent manner, for the acidic residues. The aspartic acid-containing variant had a 110-fold faster deacylation at pH 8. The magnitude of this effect is similar to that observed in a naturally occurring variant of PBP2x, which confers increased resistance to cephalosporins.     

Role of penicillin-binding protein 1b in competitive stationary-phase survival of Escherichia coli

The penicillin-binding proteins (PBPs) catalyze the synthesis and modification of bacterial cell wall peptidoglycan. Although the biochemical activities of these proteins have been determined in Escherichia coli, the physiological roles of many PBPs remain enigmatic. Previous studies have cast doubt on the individual importance of the majority of PBPs during log phase growth. We show here that PBP1b is vital for competitive survival of E. coli during extended stationary phase, but the other nine PBPs studied are dispensable. Loss of PBP1b leads to the stationary phase-specific competition defective phenotype and causes cells to become more sensitive to osmotic stress. Additionally, we present evidence that this protein, as well as AmpC, may assist in cellular resistance to beta-lactam antibiotics.     

Crystal structure of penicillin-binding protein 1a (PBP1a) reveals a mutational hotspot implicated in beta-lactam resistance in Streptococcus pneumoniae

Streptococcus pneumoniae is a major human pathogen whose infections have been treated with beta-lactam antibiotics for over 60 years, but the proliferation of strains that are highly resistant to such drugs is a problem of worldwide concern. Beta-lactams target penicillin-binding proteins (PBPs), membrane-associated enzymes that play essential roles in the peptidoglycan biosynthetic process. Bifunctional PBPs catalyze both the polymerization of glycan chains (glycosyltransfer) and the cross-linking of adjacent pentapeptides (transpeptidation), while monofunctional enzymes catalyze only the latter reaction. Although S. pneumoniae has six PBPs, only three (PBP1a, PBP2x, PBP2b) are major resistance determinants, with PBP1a being the only bifunctional enzyme. PBP1a plays a key role in septum formation during the cell division cycle and its modification is essential for the development of high-level resistance to penicillins and cephalosporins. The crystal structure of a soluble form of pneumococcal PBP1a (PBP1a*) has been solved to 2.6A and reveals that it folds into three domains. The N terminus contains a peptide from the glycosyltransfer domain bound to an interdomain linker region, followed by a central, transpeptidase domain, and a small C-terminal unit. An analysis of PBP1a sequences from drug-resistant clinical strains in light of the structure reveals the existence of a mutational hotspot at the entrance of the catalytic cleft that leads to the modification of the polarity and accessibility of the mutated PBP1a active site. The presence of this hotspot in all variants sequenced to date is of key relevance for the development of novel antibiotherapies for the treatment of beta-lactam-resistant pneumococcal strains.     

Escherichia coli low-molecular-weight penicillin-binding proteins help orient septal FtsZ, and their absence leads to asymmetric cell division and branching

Escherichia coli cells lacking low-molecular-weight penicillin-binding proteins (LMW PBPs) exhibit morphological alterations that also appear when the septal protein FtsZ is mislocalized, suggesting that peptidoglycan modification and division may work together to produce cell shape. We found that in strains lacking PBP5 and other LMW PBPs, higher FtsZ concentrations increased the frequency of branched cells and incorrectly oriented Z rings by 10- to 15-fold. Invagination of these rings produced improperly oriented septa, which in turn gave rise to asymmetric cell poles that eventually elongated into branches. Branches always originated from the remnants of abnormal septation events, cementing the relationship between aberrant cell division and branch formation. In the absence of PBP5, PBP6 and DacD localized to nascent septa, suggesting that these PBPs can partially substitute for the loss of PBP5. We propose that branching begins when mislocalized FtsZ triggers the insertion of inert peptidoglycan at unusual positions during cell division. Only later, after normal cell wall elongation separates the patches, do branches become visible. Thus, a relationship between the LMW PBPs and cytoplasmic FtsZ ultimately affects cell division and overall shape.     

The ponA gene of Enterococcus faecalis JH2-2 codes for a low-affinity class A penicillin-binding protein

A soluble derivative of the Enterococcus faecalis JH2-2 class A PBP1 (*PBP1) was overproduced and purified. It exhibited a glycosyltransferase activity on the Escherichia coli 14C-labeled lipid II precursor. As a DD- peptidase, it could hydrolyze thiolester substrates with efficiencies similar to those of other class A penicillin-binding proteins (PBPs) and bind beta-lactams, but with k2/K (a parameter accounting for the acylation step efficiency) values characteristic of penicillin-resistant PBPs.     

State of penicillin-binding proteins and requirements for their bactericidal interaction with beta-lactam antibiotics in Serratia marcescens highly resistant to extended-spectrum beta-lactams

The quantities of penicillin-binding proteins (PBPs), and sensitivity to extended-spectrum beta-lactams, were measured in isogenic strains of Serratia marcescens with high (HR) and low (LR) resistance to extended-spectrum beta-lactam antibiotics and with constitutively overproduced chromosomal beta-lactamase in the periplasm. The binding of structurally different beta-lactams to PBPs in growing resistant bacteria was determined quantitatively. In S. marcescens HR, the amounts of PBPs 3 and 6 were, respectively, 1.5 and 2 times those in strain LR and in sensitive reference strains. Sensitivities of the essential PBPs in S. marcescens LR and HR to the tested beta-lactams were identical. Only a single target, PBP 3, was highly sensitive to cefotaxime, ceftazidime and aztreonam. In contrast, three PBPs (2, 1A and 3) were highly sensitive to imipenem. In growing S. marcescens HR and LR, all antibiotics, even at fractions of their minimal growth inhibitory concentrations (MICs), bound extensively to those PBPs which were highly sensitive to them. Thus, overproduced beta-lactamase did not prevent PBP-beta-lactam interaction. Only at or above their (high) MICs did cefotaxime, ceftazidime and aztreonam bind to multiple targets. Growth inhibition of the otherwise highly resistant S. marcescens HR at the lower MIC of imipenem was correlated with the binding of this antibiotic to multiple, highly sensitive targets in the bacteria. Killing of the bacteria by inactivation of multiple targets was suggested. This assumption was supported by the synergistic killing of HR bacteria by combinations of the PBP-2-specific mecillinam with PBP-3-specific beta-lactams.     

Glycosyltransferase domain of penicillin-binding protein 2a from Streptococcus pneumoniae is membrane associated

Penicillin-binding proteins (PBPs) are bacterial cytoplasmic membrane proteins that catalyze the final steps of the peptidoglycan synthesis. Resistance to beta-lactams in Streptococcus pneumoniae is caused by low-affinity PBPs. S. pneumoniae PBP 2a belongs to the class A high-molecular-mass PBPs having both glycosyltransferase (GT) and transpeptide (TP) activities. Structural and functional studies of both domains are required to unravel the mechanisms of resistance, a prerequisite for the development of novel antibiotics. The extracellular region of S. pneumoniae PBP 2a has been expressed (PBP 2a*) in Escherichia coli as a glutathione S-transferase fusion protein. The acylation kinetic parameters of PBP 2a* for beta-lactams were determined by stopped-flow fluorometry. The acylation efficiency toward benzylpenicillin was much lower than that toward cefotaxime, a result suggesting that PBP 2a participates in resistance to cefotaxime and other beta-lactams, but not in resistance to benzylpenicillin. The TP domain was purified following limited proteolysis. PBP 2a* required detergents for solubility and interacted with lipid vesicles, while the TP domain was water soluble. We propose that PBP 2a* interacts with the cytoplasmic membrane in a region distinct from its transmembrane anchor region, which is located between Lys 78 and Ser 156 of the GT domain.     

Deacylation kinetics analysis of Streptococcus pneumoniae penicillin-binding protein 2x mutants resistant to beta-lactam antibiotics using electrospray ionization- mass spectrometry

Penicillin-binding proteins (PBPs) catalyze the transpeptidase reaction involved in peptidoglycan synthesis and are covalently inhibited by the beta-lactam antibiotics. In a previous work we have focused on acylation efficiency measurements of various Streptococcus pneumoniae PBP2x* mutants to study the molecular determinants of resistance to beta-lactams. In the present paper we have developed a method to improve an accurate determination of the deacylation rate constant using electrospray ionization-mass spectrometry. This method is adaptable to the analysis of deacylation of any beta-lactam. Compared to the fluorographic technique, the ESI-MS method is insensitive to variations in the concentration of functional proteins and is therefore more reliable. We have established that the resistance of PBPs to beta-lactams is mostly due to a decrease of the acylation efficiency with only marginal effects on the deacylation rates.     

Temperature-Sensitive Cell Division Mutants of Escherichia coli with Thermolabile Penicillin-Binding Proteins

The thermostability of the penicillin-binding proteins (PBPs) of 31 temperature-sensitive cell division mutants of Escherichia coli has been examined. Two independent cell division mutants have been found that have highly thermolabile PBP3. Binding of [(14)C]benzylpenicillin to PBP3 (measured in envelopes prepared from cells grown at the permissive temperature) was about 30% of the normal level at 30 degrees C, and the ability to bind [(14)C]benzylpenicillin was rapidly lost on incubation at 42 degrees C. The other PBPs were normal in both mutants. At 30 degrees C both mutants were slightly longer than their parents and on shifting to 42 degrees C they ceased dividing, but cell mass and deoxyribonucleic acid synthesis continued and long filaments were formed. At 42 degrees C division slowly recommenced, but at 44 degrees C this did not occur. The inhibition of division at 42 degrees C was suppressed by 0.35 M sucrose, and in one of the mutants it was partially suppressed by 10 mM MgCl(2). PBP3 was not stabilized in vitro at 42 degrees C by these concentrations of sucrose or MgCl(2). Revertants that grew as normal rods at 42 degrees C regained both the normal level and the normal thermostability of PBP3. The results provide extremely strong evidence that the inactivation of PBP3 at 42 degrees C in the mutants is the cause of the inhibition of cell division at this temperature and identify PBP3 as an essential component of the process of cell division in E. coli. It is the inactivation of this protein by penicillins and cephalosporins that results in the inhibition of division characteristic of low concentrations of many of these antibiotics.     

Cell cycle-independent lysis of Escherichia coli by cefsulodin, an inhibitor of penicillin-binding proteins 1a and 1b.

Cefsulodin lyses actively growing Escherichia coli by binding specifically to penicillin-binding proteins (PBPs) 1a and 1b. Recent findings (F. García del Portillo, M. A. de Pedro, D. Joseleau-Petit, and R. D'Ari, J. Bacteriol. 171:4217-4221, 1989) have linked cefsulodin-induced lysis to septation during the first division cycle after a nutritional shift-up or chromosome replication realignment. We synchronized cells by membrane filtration to determine whether cefsulodin-induced lysis depended on septation in normally growing cells. Populations of newly divided cells were allowed to grow for variable lengths of time. Cefsulodin was added to these synchronous cultures, which represented points in two to three rounds of the cell cycle. Since the cell numbers were small, a new lysis assay was developed that was based on the release of DNA measured by fluorometry. Lysis occurred at a constant time after addition of the antibiotic, regardless of the time in the cell cycle at which the addition was made. Thus, cefsulodin-induced lysis is not linked to septation or to any other cell cycle-related event.