Daughter cell separation by penicillin-binding proteins and peptidoglycan amidases in Escherichia coli

As one of the final steps in the bacterial growth cycle, daughter cells must be released from one another by cutting the shared peptidoglycan wall that separates them. In Escherichia coli, this delicate operation is performed by several peptidoglycan hydrolases, consisting of multiple amidases, lytic transglycosylases, and endopeptidases. The interactions among these enzymes and the molecular mechanics of how separation occurs without lysis are unknown. We show here that deleting the endopeptidase PBP 4 from strains lacking AmiC produces long chains of unseparated cells, indicating that PBP 4 collaborates with the major peptidoglycan amidases during cell separation. Another endopeptidase, PBP 7, fulfills a secondary role. These functions may be responsible for the contributions of PBPs 4 and 7 to the generation of regular cell shape and the production of normal biofilms. In addition, we find that the E. coli peptidoglycan amidases may have different substrate preferences. When the dd-carboxypeptidase PBP 5 was deleted, thereby producing cells with higher levels of pentapeptides, mutants carrying only AmiC produced a higher percentage of cells in chains, while mutants with active AmiA or AmiB were unaffected. The results suggest that AmiC prefers to remove tetrapeptides from peptidoglycan and that AmiA and AmiB either have no preference or prefer pentapeptides. Muropeptide compositions of the mutants corroborated this latter conclusion. Unexpectedly, amidase mutants lacking PBP 5 grew in long twisted chains instead of straight filaments, indicating that overall septal morphology was also defective in these strains.     

Possible role of Escherichia coli penicillin-binding protein 6 in stabilization of stationary-phase peptidoglycan.

Plasmids for high-level expression of penicillin-binding protein 6 (PBP6) were constructed, giving rise to overproduction of PBP6 under the control of the lambda pR promoter in either the periplasmic or the cytoplasmic space. In contrast to penicillin-binding protein 5 (PBP5), the presence of high amounts of PBP6 in the periplasm as well as in the cytoplasm did not result in growth as spherical cells or in lysis. Deletion of the C-terminal membrane anchor of PBP6 resulted in a soluble form of the protein (PBP6s350). Electron micrographs of thin sections of cells overexpressing both native membrane-bound and soluble PBP6 in the periplasm revealed a polar retraction of the cytoplasmic membrane. Cytoplasmic overexpression of native PBP6 gave rise to the formation of membrane vesicles, whereas the soluble PBP6 formed inclusion bodies in the cytoplasm. Both the membrane-bound and the soluble forms of PBP6 were purified to homogeneity by using the immobilized dye Procion rubine MX-B. Purified preparations of PBP6 and PBP6s350 formed a 14[C]penicillin-protein complex at a 1:1 stoichiometry. The half-lives of the complexes were 8.5 and 6 min, respectively. In contrast to PBP5, no DD-carboxypeptidase activity could be detected for PBP6 by using bisacetyl-L-Lys-D-Ala-D-Ala and several other substrates. These findings led us to conclude that PBP6 has a biological function clearly distinct from that of PBP5 and to suggest a role for PBP6 in the stabilization of the peptidoglycan during stationary phase.     

Autolysis-resistant peptidoglycan of anomalous composition in amino-acid-starved Escherichia coli.

Nongrowing Escherichia coli deprived of an essential amino acid continued to produce peptidoglycan at a rate approximately 30% of that of growing cells. The composition of this peptidoglycan was very different from that of growing cells and resembled that of peptidoglycan left undegraded during partial autolysis of the bacteria. Synthesis of this peptidoglycan of anomalous composition began at once upon the removal of the amino acid from the medium. Fifteen minutes of amino acid deprivation was sufficient to virtually completely prevent penicillin-induced autolytic wall degradation in vivo. During this time, although the specific activities of soluble and membrane-bound hydrolytic transglycosylases and endopeptidases remained high, the peptidoglycan produced showed decreased sensitivity to degradation in vitro. After more extensive (2-h) starvation, triggering of autolysis by chaotropic agents was also blocked. Autolysis in growing cells may be selective for peptidoglycan representing the cylindrical portion of the sacculus. It is suggested that at least part of the mechanism of the well-known lysis resistance of nongrowing E. coli is related to the deposition of structurally anomalous and relatively autolysin-resistant peptidoglycan at some strategically located sites on the bacterial surface.     

Topographical distribution of penicillin-binding proteins in the Escherichia coli membrane.

The penicillin-binding proteins (PBPs) found in the membranes of Escherichia coli X925 minicells (primarily cell ends or septa) were compared with those found in rod-shaped cells (primarily sidewalls) in an effort to determine whether certain PBPs are unevenly distributed over the bacterial cell membrane. The seven major PBPs of E. coli were all present in minicell membranes. PBP 1B was altered in minicells, however, appearing as two bands on sodium dodecyl sulfate-polyacrylamide gels rather than the usual three. PBP 2, which is needed for longitudinal growth of the cell but not for septum formation, was significantly reduced in minicell membranes. This observation is consistent with the fact that minicells contain very little sidewall material and raises the possibility that the specialized function of PBP 2 may be determined or regulated by its uneven topographical distribution in the membrane. None of the PBPs appeared to be selectively enriched in minicell membranes.     

Comparative studies of penicillin-binding proteins in Pseudomonas aeruginosa and Escherichia coli.

Penicillin-binding proteins in Pseudomonas aeruginosa were compared with those of Escherichia coli. These in P. aeruginosa were found exclusively in the cytoplasmic membrane fraction (fraction soluble in sodium N-lauroyl sarcosinate). Sodium dodecyl sulfate/acrylamide gel electrophoresis of the proteins bound to [14C]penicillin G resulted in the separation of six major bands and several minor bands. The proteins in these bands are referred to as proteins 1A, 1B, 2, 3, 4 and 5 in order of increasing electrophoretical mobility. The electrophoretic mobilities and other properties of penicillin-binding proteins in P. aeruginosa and E. coli were compared and correlated. Fundamentally they seem to be very similar in the two bacteria, but proteins 1A and 1B in P. aeruginosa seem to correspond respectively to proteins 1B and 1A in E. coli, and protein 6 seems to be missing or present in only small amount in P. aeruginosa. In addition, the affinities of currently developed beta-lactam antibiotics to each protein of P. aeruginosa and E. coli were examined in relation to the morphological changes of the cells induced by these antibiotics and their antibacterial potencies. Mecillinam showed high affinity to only protein 2 in both P. aeruginosa and E. coli. At a minimal inhibitory concentration, it converted cells of both P. aeruginosa and E. coli from rods to spherical cells, although its minimal inhibitory concentration was much higher for P. aeruginosa than for E. coli.     

Approaching the physiological functions of penicillin-binding proteins in Escherichia coli

A rigid shell of peptidoglycan encases and shapes bacteria and is constructed and maintained by a diverse set of enzymes, among which are the penicillin-binding proteins (PBPs). Although a great deal has been learned about how these proteins synthesize and modify peptidoglycan, the physiological functions of the multitude of bacterial PBPs remain enigmatic. We approached this problem by combining PBP mutations in a comprehensive manner and screening for effects on biochemical processes involving the passage of proteins or nucleic acids across the cell wall. The results indicate that the PBPs or their peptidoglycan product do have significant biological functions, including roles in determination of cell shape, in phage resistance, in induction of capsule synthesis, and in regulation of autolysis.     

Multiple mechanisms of membrane anchoring of Escherichia coli penicillin-binding proteins

Abstract: The major penicillin-binding proteins (PBPs) of Escherichia coli play vital roles in cell wall biosynthesis and are located in the inner membrane. The high M _r PBPs 1A, 1B, 2 and 3 are essential bifunctional transglycosylases/transpeptidases which are thought to be type II integral inner membrane proteins with their C-terminal enzymatic domains projecting into the periplasm. The low M _r PBP4 is a DD-carboxypeptidase/endopeptidase, whereas PBPs 5 and are DD-carboxypeptidases. All three low M _r , PBPs act in the modification of peptidoglycan to allow expansion of the sacculus and are thought to be periplasmic proteins attached with varying affinities to the inner membrane via C-terminal amphiphilic α-helices. It is possible that the PBPs and other inner membrane proteins form a peptidoglycan synthesizing complex to coordinate their activities.     

The penicillin-binding proteins: structure and role in peptidoglycan biosynthesis

Penicillin-binding proteins (PBPs) have been scrutinized for over 40 years. Recent structural information on PBPs together with the ongoing long-term biochemical experimental investigations, and results from more recent techniques such as protein localization by green fluorescent protein-fusion immunofluorescence or double-hybrid assay, have brought our understanding of the last stages of the peptidoglycan biosynthesis to an outstanding level that allows a broad outlook on the properties of these enzymes. Details are emerging regarding the interaction between the peptidoglycan-synthesizing PBPs and the peptidoglycan, their mesh net-like product that surrounds and protects bacteria. This review focuses on the detailed structure of PBPs and their implication in peptidoglycan synthesis, maturation and recycling. An overview of the content in PBPs of some bacteria is provided with an emphasis on comparing the biochemical properties of homologous PBPs (orthologues) belonging to different bacteria.     

Specific location of penicillin-binding proteins within the cell envelope of Escherichia coli.

This communication deals with the location of penicillin-binding proteins in the cell envelope of Escherichia coli. For this purpose, bacterial cells have been broken by various procedures and their envelopes have been fractioned. To do so, inner (cytoplasmic) and outer membranes were separated by isopycnic centrifugation in sucrose gradients. Some separation methods (Osborn et al., J. Biol. Chem. 247:3962-3972, 1972; J. Smit, Y. Kamio, and H. Nikaido, J. Bacteriol. 124:942-958, 1975) revealed that penicillin-binding proteins are not exclusively located in the inner membrane. They are also found in the outer membrane (A. Rodríguez-Tébar, J. A. Barbas, and D. Vásquez, J. Bacteriol. 161:243-248, 1985). Under the milder conditions for cell rupture used in this work, an intermembrane fraction, sedimenting between the inner and outer membrane, can be recovered from the gradients. This fraction has a high content of both penicillin-binding proteins and phospholipase B activity and may correspond to the intermembrane adhesion sites (M. H. Bayer, G. P. Costello, and M. E. Bayer, J. Bacteriol. 149:758-769, 1982). We postulate that this intermembrane fraction is a labile structure that contains a high amount of all penicillin-binding proteins which are usually found in both the inner and outer membranes when the adhesion sites are destroyed by the cell breakage and fractionation procedures.     

Synthetic ColE1 plasmids carrying genes for penicillin-binding proteins in Escherichia coli

Clarke and Carbon's collection of 2000 Escherichia coli strains which harbor ColE1 plasmids carrying small random segments of the E. coli chromosome was screened for the correction of mutational defects in penicillin-binding proteins (PBPs): ponA (PBP-1a), ponB (PBP-1b), dacB (PBP-4), and pfv (PBP-5). We found plasmids carrying chromosomal segments containing ponA⁺-aroB⁺ (pLC29-47), ponB⁺-tonA⁺ (pLC4-43, pLC4-44, and pLC19-19), and argG⁺-dacB⁺ (pLC10-46 and pLC18-38). Characters of these plasmids were analyzed. Two other plasmids (pLC26-6 and pLC4-14) previously found to correct ftsI mutation (Y. Nishimura, Y. Takeda, A. Nishimura, H. Suzuki, M. Inouye, and Y. Hirota (1977)Plasmid1, 67–77) were also investigated further. Restriction maps of chromosomal DNAs carried by pLC29-47, pLC4-44, pLC19-19, pLC18-38, pLC26-6, and pLC4-14 were constructed. The regions of ponB-tonA on pLC4-44 and pLC19-19, and of leuA-ftsI-murE and F on pLC26-6 were located on the restriction maps. Although both pLC26-6 and pLC4-14 corrected a thermosensitive mutation, ftsI, which causes a defect in cell division due to abnormal PBP-3, only pLC26-6 led to restoration of PBP-3 production by an ftsI mutant, while pLC4-14 did not. Restriction and heteroduplex analyses of pLC26-6 and pLC4-14 have shown the absence of nucleotide sequence homology between them. The plasmids, pLC29-47 carrying ponA⁺ and pLC4-43, pLC4-44, and pLC19-19 carrying ponB⁺ led the host cell to overproduce the respective PBP.     

Effect of growth rate and cell shape on the peptidoglycan composition in Escherichia coli.

The muropeptide composition of peptidoglycan from Escherichia coli W7 cultivated at different growth rates in chemostat cultures was compared by using high-pressure liquid chromatography. At a low growth rate (D = 0.1 h-1), about 40% more covalently bound lipoprotein and at least twofold more diaminopimelyl-diaminopimelic acid cross-bridges were found than at a high growth rate (D = 0.8 h-1). The total degree of cross-linkage was only slightly increased, and the fraction of trimeric muropeptides and the average length of the glycan chains were not changed significantly. Analysis of the peptidoglycan from a morphological variant strain of W7 revealed that the altered peptidoglycan composition in slowly growing W7 cells was not correlated with the observation that these cells, due to their decreased cell length, were relatively enriched in polar material. In fact, our results suggested that peptidoglycan forming cell poles is chemically identical to that forming lateral wall.     

Properties of the penicillin-binding proteins of Escherichia coli K12,.

Benzyl[14C]penicillin binds to six proteins with molecular weights between 40000 and 91000 in the inner membrane of Escherichia coli. Two additional binding proteins with molecular weights of 29000 and 32000 were sometimes detected. All proteins were accessible to benzyl[14C]penicillin in whole cells. Proteins 5 and 6 released bound benzyl[14C]penicillin with half times of 5 and 19 min at 30 degrees C but the other binding proteins showed less than 50% release during a 60-min period at 30 degrees C. The rate of release of bound penicillin from some of the proteins was greatly stimulated by 2-mercaptoethanol and neutral hydroxylamine. Release of benzyl[14C]penicillin did not occur if the binding proteins were denatured in anionic detergent and so was probably enzymic. No additional binding proteins were detected with two [14C]cephalosporins. These beta-lactams bound to either all or some of those proteins to which benzyl[14C]penicillin bound. No binding proteins have been detected in the outer membrane of E coli with any beta-[14C]lactam. The binding of a range of unlabelled penicillins and cephalosporins were studied by measuring their competition for the binding of benzyl[14C]penicillin to the six penicillin-binding proteins. These results, together with those obtained by direct binding experiments with beta-[14C]lactams, showed that penicillins bind to all six proteins but that at least some cephalosporins fail to bind, or bind very slowly, to proteins 2, 5 and 6, although they bind to the other proteins. Since these cephalosporins inhibited cell division and caused cell lysis at concentrations where we could detect no binding to proteins 2, 5 and 6, we believe that these latter proteins are not the target at which beta-lactams bind to elicit the above physiological responses. The binding properties of proteins 1, 3, and 4 correlate reasonably well with those expected for the above killing targets.     

Effect of growth rate on the penicillin-binding proteins of Escherichia coli

Abstract In an Escherichia coli strain, the levels of penicillin-binding proteins (PBPs) 1A plus 1B, both peptidoglycan transglycosylase/transpeptidases, were found to be relatively independent of the imposed growth ratw in chemostat cultures under different nutrient limitation conditions. A considerable increase in levels of PBP 6 was observed as the growth rate was reduced, whilst, in contrast, a decrease was observed in levels of the other PBPs.     

Direct quantitation of the number of individual penicillin-binding proteins per cell in Escherichia coli.

The penicillin-binding proteins (PBPs) are a set of enzymes that participate in the terminal stages of bacterial peptidoglycan assembly. As their name implies, these proteins also covalently bind and are inhibited by beta-lactam antibiotics. Although many studies have examined the relative binding affinities of a number of beta-lactam antibiotics, a surprisingly small number of studies have addressed the absolute numbers of each of the PBPs present in the bacterial cell. In the present study, the PBP values initially reported in Escherichia coli almost 20 years ago by B. G. Spratt (Eur. J. Biochem. 72:341-352, 1977) were refined. The individual PBPs from a known number of bacteria radiolabeled with [3H]benzylpenicillin were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The radioactive bands were located, excised, and quantitatively extracted from the gel slices. The radioactivity was measured by scintillation counting, and the absolute disintegrations per minute were calculated. From the specific activity of the labeled penicillin, the absolute disintegrations per minute, and the CFU per milliliter, a determination of the number of each of the PBPs per cell was made. The measurements were performed on multiple samples to place statistical limits on the numbers obtained. The values for the individual PBPs found in E. coli deviated in several ways from the previously reported observations. Of particular significance is the higher number of molecules of PBP 2 and 3 observed, since these PBPs are known to participate in cell morphogenesis. The PBP content in both rich Luria broth medium and M9 minimal medium was determined, with the slower-growing cells in minimal medium possessing fewer of the individual PBPs per cell.     

Labeling pattern of major penicillin-binding proteins of Escherichia coli during the division cycle.

Escherichia coli cells were synchronized by the elutriation technique. The pattern of penicillin-binding proteins (PBPs) in synchronously growing cells was determined with an iodinated derivative of ampicillin in intact cells as well as in isolated membranes. This was done under nonsaturating conditions as well as under conditions in which the PBPs were saturated with [125I]ampicillin. No evidence was found for fluctuations in the PBP pattern: the PBPs seem to be present in a constant ratio throughout the division cycle. The E. coli cells exert their control on shape maintenance and cell wall growth apparently not on the level of concentration of PBPs in the cell but rather on activation of existing components.     

Staphylococcus aureus and Micrococcus luteus peptidoglycan transglycosylases that are not penicillin-binding proteins.

Major peptidoglycan transglycosylase activities, which synthesize uncross-linked peptidoglycan from lipid-linked precursors, were solubilized from the membranes of Staphylococcus aureus and Micrococcus luteus and were partially purified. The transglycosylase activities were separated from penicillin-binding proteins by solubilization and by purification steps. Therefore, we concluded that these activities were not activities of the penicillin-binding proteins, which are the presumptive peptidoglycan transpeptidases in these gram-positive cocci. Unlike Escherichia coli, in which the network structure of peptidoglycan is synthesized by multiple two-headed penicillin-binding proteins with both transpeptidase and transglycosylase activities, these gram-positive cocci have cell wall peptidoglycan which seems to be synthesized by penicillin-binding protein transpeptidases and a separate transglycosylase.     

Competitive binding of various beta-lactam compounds to penicillin-binding proteins of Escherichia coli

Competing interaction of two novel N-acyl derivatives of ampicillin i.e. N'-benzylchlorbenzimidazole (No. 48) and N-pyrazolytiazole (No. 72) derivatives and 14C-benzylpenicillin with penicillin-binding proteins (PBP) of E. coli was studied. It was shown that ampicillin and its derivative No. 48 markedly differed in their affinity to various PBPs. Derivative No. 72 did not prevent binding of the labeled benzylpenicillin to any PBP which corresponded to its low antimicrobial activity. Analogous experiments with new cephalosporin structures i.e. active and inactive N-acyl derivatives of cephalosporin showed that the active derivative No. 94 i.e. N-methyltiobenzimidazole derivative had the highest affinity to PBP-2 and PBP-5. The inactive derivative No. 68 i.e. N-chlorbenzimidazole derivative also had high affinity to PBP-1b, PBP-2 and PBP-3 essential for the cell. No activity of the latter compound against intact cells of E. coli was probably due to its low penetration through the outer membrane of the bacterial cell. Estimation of affinity of the beta-lactam structures to various PBPs not only provided data on the mechanism of their action but also made it possible to explain in some cases the peculiarities of their antimicrobial spectrum.     

Cytoplasmic steps of peptidoglycan synthesis in Escherichia coli.

The cellular pool levels of most of the cytoplasmic precursors of peptidoglycan synthesis were determined for normally growing cells of Escherichia coli K-12. In particular, a convenient method for analyzing the uridine nucleotide precursor contents was developed by associating gel filtration and reverse-phase high-pressure liquid chromatography techniques. The enzymatic parameters of the four synthetases which catalyze the stepwise addition of L-alanine, D-glutamic acid, meso-diaminopimelic acid, and D-alanyl-D-alanine to uridine diphosphate-N-acetylmuramic acid were determined. It was noteworthy that the pool levels of L-alanine, D-glutamic acid, meso-diaminopimelic acid, and D-alanyl-D-alanine were much higher than the Km values determined for these substrates, whereas the molar concentrations of the uridine nucleotide precursors were lower than or about the same order of magnitude as the corresponding Km values. Taking into consideration the data obtained, an attempt was made to compare the in vitro activities of the D-glutamic acid, meso-diaminopimelic acid, and D-alanyl-D-alanine adding enzymes with their in vivo functioning, expressed by the amounts of peptidoglycan synthesized. The results also suggested that these adding activities were not in excess in the cell under normal growth conditions, but their amounts appeared adjusted to the requirements of peptidoglycan synthesis. Under the different in vitro conditions considered, only low levels of L-alanine adding activity were observed.     

Unequal distribution of penicillin-binding proteins among inner membrane vesicles of Escherichia coli

Escherichia coli penicillin-binding proteins (PBPs) were associated only with inner membrane vesicles when separated on 30 to 65% or 19 to 49% (wt/wt) sucrose gradients. Fractionation of vesicles through the low-density gradient revealed at least two classes of PBP-inner membrane associations. The first class consisted of PBPs 1 through 4, and the second class consisted of PBPs 5 through 8. These classes were distinguished by the density of vesicles with which they were associated; class 1 PBPs migrated with vesicles of higher density than did class 2 PBPs. Such combinations suggest that PBPs are nonrandomly distributed within the inner membrane, implying potential functional relationships among the PBPs themselves and with particular membrane domains. In addition, in cell lysates and in vesicle fractions, a 60,000-dalton aztreonam-insensitive PBP or protein fragment was observed which could potentially be confused with PBP3.