Inhibition of bacterial DD-peptidases (penicillin-binding proteins) in membranes and in vivo by peptidoglycan-mimetic boronic acids

The DD-peptidases or penicillin-binding proteins (PBPs) catalyze the final steps of bacterial peptidoglycan biosynthesis and are inhibited by the β-lactam antibiotics. There is at present a question of whether the active site structure and activity of these enzymes is the same in the solubilized (truncated) DD-peptidase constructs employed in crystallographic and kinetics studies as in membrane-bound holoenzymes. Recent experiments with peptidoglycan-mimetic boronic acids have suggested that these transition state analogue-generating inhibitors may be able to induce reactive conformations of these enzymes and thus inhibit strongly. We have now, therefore, measured the dissociation constants of peptidoglycan-mimetic boronic acids from Escherichia coli and Bacillus subtilis PBPs in membrane preparations and, in the former case, in vivo, by means of competition experiments with the fluorescent penicillin Bocillin Fl. The experiments showed that the boronic acids bound measurably (K(i) < 1 mM) to the low-molecular mass PBPs but not to the high-molecular mass enzymes, both in membrane preparations and in whole cells. In two cases, E. coli PBP2 and PBP5, the dissociation constants obtained were very similar to those obtained with the pure enzymes in homogeneous solution. The boronic acids, therefore, are unable to induce tightly binding conformations of these enzymes in vivo. There is no evidence from these experiments that DD-peptidase inhibitors are more or less effective in vivo than in homogeneous solution.     

Reactivity of penicillin-binding proteins with peptidoglycan-mimetic beta-lactams: what's wrong with these enzymes?

Beta-lactams exert their antibiotic action through their inhibition of bacterial DD-peptidases (penicillin-binding proteins). Bacteria, in general, carry several such enzymes localized on the outside of their cell membrane to catalyze the final step in cell wall (peptidoglycan) synthesis. They have been classified into two major groups, one of high molecular weight, the other of low. Members of the former group act as transpeptidases in vivo, and their inhibition by beta-lactams leads to cessation of bacterial growth. The latter group consists of DD-carboxypeptidases, and their inhibition by beta-lactams is generally not fatal to bacteria. We have previously shown that representatives of the former group are ineffective at catalyzing the hydrolysis/aminolysis of peptidoglycan-mimetic peptides in vitro [Anderson et al. (2003) Biochem. J. 373, 949-955]. The theme of these experiments is expanded in the present paper where we describe the synthesis of a series of beta-lactams (penicillins and cephalosporins) containing peptidoglycan-mimetic side chains and the kinetics of their inhibition of a panel of penicillin-binding proteins spanning the major classes (Escherichia coli PBP 2 and PBP 5, Streptococcus pneumoniae PBP 1b, PBP 2x and PBP 3, the Actinomadura R39 DD-peptidase, and the Streptomyces R61 DD-peptidase). The results of these experiments mirror and expand the previous results with peptides. Neither peptides nor beta-lactams with appropriate peptidoglycan-mimetic side chains react with the solubilized constructs of membrane-bound penicillin binding proteins (the first five enzymes above) at rates exceeding those of generic analogues. Such peptides and beta-lactams do react at greatly enhanced rates with certain soluble low molecular weight enzymes (R61 and R39 DD-peptidases). The former result is unexpected and interesting. Why do the majority of penicillin-binding proteins not recognize elements of local peptidoglycan structure? Possible answers are discussed. That this question needs to be asked casts fascinating shadows on current studies of penicillin-binding proteins for new drug design.     

Crystal structures of complexes of bacterial DD-peptidases with peptidoglycan-mimetic ligands: the substrate specificity puzzle

The X-ray crystal structures of covalent complexes of the Actinomadura R39 dd-peptidase and Escherichia coli penicillin-binding protein (PBP) 5 with β-lactams bearing peptidoglycan-mimetic side chains have been determined. The structure of the hydrolysis product of an analogous peptide bound noncovalently to the former enzyme has also been obtained. The R39 dd-peptidase structures reveal the presence of a specific binding site for the d-α-aminopimelyl side chain, characteristic of the stem peptide of Actinomadura R39. This binding site features a hydrophobic cleft for the pimelyl methylene groups and strong hydrogen bonding to the polar terminus. Both of these active site elements are provided by amino acid side chains from two separate domains of the protein. In contrast, no clear electron density corresponding to the terminus of the peptidoglycan-mimetic side chains is present when these β-lactams are covalently bound to PBP5. There is, therefore, no indication of a specific side-chain binding site in this enzyme. These results are in agreement with those from kinetics studies published earlier and support the general prediction made at the time of a direct correlation between kinetics and structural evidence. The essential high-molecular-mass PBPs have demonstrated, to date, no specific reactivity with peptidoglycan-mimetic peptide substrates and β-lactam inhibitors and, thus, probably do not possess a specific substrate-binding site of the type demonstrated here with the R39 dd-peptidase. This striking deficiency may represent a sophisticated defense mechanism against low-molecular-mass substrate-analogue inhibitors/antibiotics; its discovery should focus new inhibitor design.     

Crystal structures of complexes between the R61 DD-peptidase and peptidoglycan-mimetic beta-lactams: a non-covalent complex with a "perfect penicillin"

The bacterial D-alanyl-D-alanine transpeptidases (DD-peptidases) are the killing targets of beta-lactams, the most important clinical defense against bacterial infections. However, due to the constant development of antibiotic-resistance mechanisms by bacteria, there is an ever-present need for new, more effective antimicrobial drugs. While enormous numbers of beta-lactam compounds have been tested for antibiotic activity in over 50 years of research, the success of a beta-lactam structure in terms of antibiotic activity remains unpredictable. Tipper and Strominger suggested long ago that beta-lactams inhibit DD-peptidases because they mimic the D-alanyl-D-alanine motif of the peptidoglycan substrate of these enzymes. They also predicted that beta-lactams having a peptidoglycan-mimetic side-chain might be better antibiotics than their non-specific counterparts, but decades of research have not provided any evidence for this. We have recently described two such novel beta-lactams. The first is a penicillin having the glycyl-L-alpha-amino-epsilon-pimelyl side-chain of Streptomyces strain R61 peptidoglycan, making it the "perfect penicillin" for this organism. The other is a cephalosporin with the same side-chain. Here, we describe the X-ray crystal structures of the perfect penicillin in non-covalent and covalent complexes with the Streptomyces R61 DD-peptidase. The structure of the non-covalent enzyme-inhibitor complex is the first such complex to be trapped crystallographically with a DD-peptidase. In addition, the covalent complex of the peptidyl-cephalosporin with the R61 DD-peptidase is described. Finally, two covalent complexes with the traditional beta-lactams benzylpenicillin and cephalosporin C were determined for comparison with the peptidyl beta-lactams. These structures, together with relevant kinetics data, support Tipper and Strominger's assertion that peptidoglycan-mimetic side-chains should improve beta-lactams as inhibitors of DD-peptidases.     

Substrate specificity of low-molecular mass bacterial DD-peptidases

The bacterial DD-peptidases or penicillin-binding proteins (PBPs) catalyze the formation and regulation of cross-links in peptidoglycan biosynthesis. They are classified into two groups, the high-molecular mass (HMM) and low-molecular mass (LMM) enzymes. The latter group, which is subdivided into classes A-C (LMMA, -B, and -C, respectively), is believed to catalyze DD-carboxypeptidase and endopeptidase reactions in vivo. To date, the specificity of their reactions with particular elements of peptidoglycan structure has not, in general, been defined. This paper describes the steady-state kinetics of hydrolysis of a series of specific peptidoglycan-mimetic peptides, representing various elements of stem peptide structure, catalyzed by a range of LMM PBPs (the LMMA enzymes, Escherichia coli PBP5, Neisseria gonorrhoeae PBP4, and Streptococcus pneumoniae PBP3, and the LMMC enzymes, the Actinomadura R39 dd-peptidase, Bacillus subtilis PBP4a, and N. gonorrhoeae PBP3). The R39 enzyme (LMMC), like the previously studied Streptomyces R61 DD-peptidase (LMMB), specifically and rapidly hydrolyzes stem peptide fragments with a free N-terminus. In accord with this result, the crystal structures of the R61 and R39 enzymes display a binding site specific to the stem peptide N-terminus. These are water-soluble enzymes, however, with no known specific function in vivo. On the other hand, soluble versions of the remaining enzymes of those noted above, all of which are likely to be membrane-bound and/or associated in vivo and have been assigned particular roles in cell wall biosynthesis and maintenance, show little or no specificity for peptides containing elements of peptidoglycan structure. Peptidoglycan-mimetic boronate transition-state analogues do inhibit these enzymes but display notable specificity only for the LMMC enzymes, where, unlike peptide substrates, they may be able to effectively induce a specific active site structure. The manner in which LMMA (and HMM) DD-peptidases achieve substrate specificity, both in vitro and in vivo, remains unknown.     

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.     

Contributions of PBP 5 and DD-carboxypeptidase penicillin binding proteins to maintenance of cell shape in Escherichia coli

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 DeltadacA mutants, which produce no PBP 5. Although cloned PBP 5 complemented the morphological phenotype of a DeltadacA mutant lacking a total of seven PBPs, controlled expression of PBP 4, PBP 6, or DacD did not. Also, a truncated PBP 5 protein lacking its amphipathic C-terminal membrane binding sequence did not reverse the morphological defects and was lethal at low levels of expression, implying that membrane anchoring is essential for the proper functioning of PBP 5. By examining a set of mutants from which multiple PBP genes were deleted, we found that significant morphological aberrations required the absence of at least three different PBPs. The greatest defects were observed in cells lacking, at minimum, PBPs 5 and 6 and one of the endopeptidases (either PBP 4 or PBP 7). The results further differentiate the roles of the low-molecular-weight PBPs, suggest a functional significance for the amphipathic membrane anchor of PBP 5 and, when combined with the recently determined crystal structure of PBP 5, suggest possible mechanisms by which these PBPs may contribute to maintenance of a uniform cell shape in E. coli.     

Identification and cloning of the gene encoding penicillin-binding protein 7 of Escherichia coli.

Penicillin-binding protein (PBP) 7 of Escherichia coli is a poorly characterized member of the family of enzymes that synthesize and modify the bacterial cell wall. The approximate chromosomal position of the gene encoding this protein was determined by measuring the expression of PBPs during lytic infection of E. coli by each of the 476 miniset members of the Kohara lambda phage genomic library. Phages lambda 363 and lambda 364, encompassing the region from 47.7 to 48 min of the chromosome, overproduced PBP 7. One open reading frame, yohB, was present on both these phages and directed the expression of PBPs 7 and 8. The predicted amino acid sequence of PBP 7 contains the consensus motifs associated with other PBPs and has a potential site near the carboxyl terminus where proteolysis by the OmpT protein could occur, creating an appropriately sized PBP 8. The PBP 7 gene (renamed pbpG) was interrupted by insertion of a kanamycin resistance gene cassette and was moved to the chromosome of E. coli. No obvious growth defects were observed, suggesting that PBP 7 is not essential for growth under normal laboratory conditions.     

Synthesis of mosaic peptidoglycan cross-bridges by hybrid peptidoglycan assembly pathways in gram-positive bacteria

The peptidoglycan cross-bridges of Staphylococcus aureus, Enterococcus faecalis, and Enterococcus faecium consist of the sequences Gly(5), l-Ala(2), and d-Asx, respectively. Expression of the fmhB, femA, and femB genes of S. aureus in E. faecalis led to the production of peptidoglycan precursors substituted by mosaic side chains that were efficiently used by the penicillin-binding proteins for cross-bridge formation. The Fem transferases were specific for incorporation of glycyl residues at defined positions of the side chains in the absence of any additional S. aureus factors such as tRNAs used for amino acid activation. The PBPs of E. faecalis displayed a broad substrate specificity because mosaic side chains containing from 1 to 5 residues and Gly instead of l-Ala at the N-terminal position were used for peptidoglycan cross-linking. Low affinity PBP2a of S. aureus conferred beta-lactam resistance in E. faecalis and E. faecium, thereby indicating that there was no barrier to heterospecific expression of resistance caused by variations in the structure of peptidoglycan precursors. Thus, conservation of the structure of the peptidoglycan cross-bridges in members of the same species reflects the high specificity of the enzymes for side chain synthesis, although this is not essential for the activity of the PBPs.     

Sequences near the active site in chimeric penicillin binding proteins 5 and 6 affect uniform morphology of Escherichia coli

Penicillin binding protein (PBP) 5, a DD-carboxypeptidase that removes the terminal D-alanine from peptide side chains of peptidoglycan, plays an important role in creating and maintaining the uniform cell shape of Escherichia coli. PBP 6, a highly similar homologue, cannot substitute for PBP 5 in this respect. Previously, we localized the shape-maintaining characteristics of PBP 5 to the globular domain that contains the active site (domain I), where PBPs 5 and 6 share substantial identity. To identify the specific segment of domain I responsible for shape control, we created a set of hybrids and determined which ones complemented the aberrant morphology of a misshapen PBP mutant, E. coli CS703-1. Fusion proteins were constructed in which 47, 199 and 228 amino-terminal amino acids of one PBP were fused to the corresponding carboxy-terminal amino acids of the other. The morphological phenotype was reversed only by hybrid proteins containing PBP 5 residues 200 to 228, which are located next to the KTG motif of the active site. Because residues 220 to 228 were identical in these proteins, the morphological effect was determined by alterations in amino acids 200 to 219. To confirm the importance of this segment, we constructed mosaic proteins in which these 20 amino acids were grafted from PBP 5 into PBP 6 and vice versa. The PBP 6/5/6 mosaic complemented the aberrant morphology of CS703-1, whereas PBP 5/6/5 did not. Site-directed mutagenesis demonstrated that the Asp(218) and Lys(219) residues were important for shape maintenance by these mosaic PBPs, but the same mutations in wild-type PBP 5 did not eliminate its shape-promoting activity. Homologous enzymes from five other bacteria also complemented the phenotype of CS703-1. The overall conclusion is that creation of a bacterial cell of regular diameter and uniform contour apparently depends primarily on a slight alteration of the enzymatic activity or substrate accessibility at the active site of E. coli PBP 5.     

Cloning and expression of the ponB gene, encoding penicillin-binding protein 1B of Escherichia coli, in heterologous systems.

A fragment from the ponB region of the Escherichia coli chromosome comprising the promoterless sequence encoding penicillin-binding protein 1B (PBP 1B) has been cloned in a broad-host-range expression vector under the control of the kanamycin resistance gene promoter present in the vector. The hybrid plasmid (pJP3) was used to transform appropriate strains of Salmonella typhimurium, Pseudomonas putida, and Pseudomonas aeruginosa. In all instances, the coding sequence was expressed in the heterologous hosts, yielding a product with electrophoretic mobility, protease accessibility, membrane location, and beta-lactam-binding properties identical to those of native PBP 1B in E. coli. These results indicated that PBP 1B of E. coli is compatible with the cytoplasmic membrane environment of unrelated bacterial species and support the idea that interspecific transfer of mutated alleles of genes coding for PBPs could potentially be an efficient spreading mechanism for intrinsic resistance to beta-lactams.     

Endopeptidase penicillin-binding proteins 4 and 7 play auxiliary roles in determining uniform morphology of Escherichia coli

The low-molecular-weight (LMW) penicillin-binding protein, PBP 5, plays a dominant role in determining the uniform cell shape of Escherichia coli. However, the physiological functions of six other LMW PBPs are unknown, even though the existence and enzymatic activities of four of these were established three decades ago. By applying fluorescence-activated cell sorting (FACS) to quantify the cellular dimensions of multiple PBP mutants, we found that the endopeptidases PBP 4 and PBP 7 also influence cell shape in concert with PBP 5. This is the first reported biological function for these two proteins. In addition, the combined loss of three DD-carboxypeptidases, PBPs 5 and 6 and DacD, also impaired cell shape. In contrast to previous reports based on visual inspection alone, FACS analysis revealed aberrant morphology in a mutant lacking only PBP 5, a phenotype not shared by any other strain lacking a single LMW PBP. PBP 5 removes the terminal D-alanine from pentapeptide side chains of muropeptide subunits, and pentapeptides act as donors for cross-linking adjacent side chains. As endopeptidases, PBPs 4 and 7 cleave cross-links in the cell wall. Therefore, overall cell shape may be determined by the existence or location of a specific type of peptide cross-link, with PBP 5 activity influencing how many cross-links are made and PBPs 4 and 7 acting as editing enzymes to remove inappropriate cross-links.     

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.     

Peptidoglycan synthetic activities in membranes of Escherichia coli caused by overproduction of penicillin-binding protein 2 and rodA protein

Penicillin-binding protein (PBP)-2 and the RodA protein are known to function in determining the rod shape of Escherichia coli cells. Peptidoglycan biosynthetic reactions that required these two proteins were demonstrated in the membrane fraction prepared from an E. coli strain that overproduced both of these two proteins and which lacked PBP-1B activity (the major peptidoglycan synthetase activity in the normal E. coli membranes). The cross-linked peptidoglycan was synthesized from UDP-N-acetylmuramylpentapeptide and UDP-N-acetylglucosamine in the presence of a high concentration of cefmetazole that inhibited all of PBPs except PBP-2. The peptidoglycan was synthesized via a lipid intermediate and showed up to 30% cross-linking. The cross-linking reaction was strongly inhibited by the amidinopenicillin, mecillinam, and by other beta-lactam antibiotics that have a high affinity for PBP-2, but not by beta-lactams that had very low affinity for PBP-2. The formation of peptidoglycan required the presence of high levels of both PBP-2 and the RodA protein in the membranes, but it is unclear which of the two proteins was primarily responsible for the extension of the glycan chains (transglycosylation). However, the sensitivity of the cross-linking reaction to specific beta-lactam antibiotics strongly suggested that it was catalyzed by PBP-2. The transglycosylase activity of the membranes was sensitive to enramycin and vancomycin and was unusual in being stimulated greatly by a high concentration of a chelating agent.     

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.     

Lytic response of Escherichia coli cells to inhibitors of penicillin-binding proteins 1a and 1b as a timed event related to cell division.

In growing cultures of Escherichia coli, simultaneous inhibition of penicillin-binding proteins 1a and 1b (PBPs 1) by a beta-lactam efficiently induces cell lysis. However, the lytic behavior of cultures initiating growth in the presence of beta-lactams specifically inhibiting PBPs 1 suggested that the triggering of cell lysis was a cell division-related event, at least in the first cell cycle after the resumption of growth (F. Garcia del Portillo, A. G. Pisabarro, E. J. de la Rosa, and M. A. de Pedro, J. Bacteriol. 169:2410-2416, 1987). To investigate whether this apparent correlation would hold true in actively growing cells, we studied the lytic behavior of cultures of E. coli aligned for cell division which were challenged with beta-lactams at different times after alignment. Cell division was aligned either by nutritional shift up or by chromosome replication alignment. Specific inhibition of PBPs 1 with the beta-lactam cefsulodin resulted in a delayed onset of lysis which was coincident in time with the resumption of cell division. The apparent correlation between the initiation of lysis and cell division was abolished when cefsulodin was used in combination with the PBP 2-specific inhibitor mecillinam, leading to the onset of lysis at a constant time after the addition of the beta-lactams. The results presented clearly argue in favor of the hypothesis that the triggering of cell lysis after inhibition of PBPs 1 is a cell division-correlated event dependent on the activity of PBP 2.     

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.     

Effect of aminoglycosides on the binding of C14-benzylpenicillin by penicillin-binding proteins in Escherichia coli

It was shown that preincubation of E. coli intact cells with gentamicin and streptomycin induced a marked increase in binding of 14C-benzylpenicillin to its final targets in the membrane i.e. penicillin-binding proteins (PBP). The stimulating effect of the aminoglycosides was also confirmed in experiments with a membrane fraction isolated from the cells preincubated with the aminoglycosides. The PBPs of the cells preincubated with the aminoglycosides were studied with SDS-PAAG electrophoresis. It was revealed that under the action of the aminoglycosides the quantity of the labeled substance (intensity of the bands on the fluorograms) fixed by the low molecular PBPs i.e. D-alanine carboxypeptidases increased. Moreover, the composition of the high molecular less mobile PBPs (transpeptidases) changed. The data are discussed in regard to the peculiarities of the effect of the aminoglycosides on the cells (bactericidal action, membrane tropism). The effect of the aminoglycosides can influence (along with the others) the results of their combined use with beta-lactams.     

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.