Substitution of lysine 213 with arginine in penicillin-binding protein 5 of Escherichia coli abolishes D-alanine carboxypeptidase activity without affecting penicillin binding.

All penicillin-binding proteins (PBPs) contain a conserved box of homology in the carboxyl-terminal half of their primary sequence that can be Lys-Thr-Gly, Lys-Ser-Gly, or His-Thr-Gly. Site-saturation mutagenesis was used to address the role of the lysine residue at this position (Lys213) in Escherichia coli PBP 5, a D-alanine carboxypeptidase enzyme. A soluble form of PBP 5 was used to replace Lys213 with 18 other amino acids, and the ability of these mutant proteins to bind [3H]penicillin G was assessed. Only the substitution of lysine with arginine resulted in a protein that was capable of forming a stable covalent complex with antibiotic. The affinity of [14C]penicillin G for the arginine mutant was 1.2-fold higher than for wild-type PBP 5 (4.4 versus 5.1 micrograms/ml for 20 min at 30 degrees C), and both proteins showed identical rates of hydrolysis of the [14C]penicilloyl-bound complex (t1/2 = 9.1 min). Surprisingly, the arginine-substituted protein was unable to catalyze D-alanine carboxypeptidase activity in vitro, which suggests that there is a substantial difference in the geometries of the peptide substrate and penicillin G within the active site of PBP 5.     

High-molecular-weight penicillin-binding proteins from membranes of bacilli

Mixtures of high-molecular-weight, cephalosporin-sensitive penicillin-binding proteins (PBPs) can be purified from Bacillus subtilis membranes by cephalosporin affinity chromatography (G. Kleppe and J. L. Strominger, J. Biol. Chem. 254:4856-4862, 1979). By appropriate modification of this technique, B. subtilis PBP 1 was purified to homogeneity, and a mixture of Bacillus stearothermophilus PBPs 1, 2, and 4 was isolated. [14C]penicillin-PBP complexes of high-molecular-weight PBPs purified from membranes of these two bacilli, after denaturation, were found to have chemical reactivities typical of the penicilloyl-serine derivative formed by D-alanine carboxypeptidase from B. stearothermophilus. Although enzymatic activity catalyzed by these and several other high-molecular-weight PBPs from gram-positive organisms has not been detected with cell wall-related substrates, a slow, enzymatic acylation of B. subtilis PBPs 1, 2ab, and 4 by [14C]-diacetyl-L-lysyl-D-alanyl-D-lactate was demonstrated. Further study is necessary to clarify the physiological relevance of the slow acylation by this analog of a natural cell wall biosynthetic intermediate.     

Site-directed mutants of a soluble form of penicillin-binding protein 5 from Escherichia coli and their catalytic properties

Soluble, truncated mutant and wild-type forms of penicillin-binding protein 5 (sPBP 5) from Escherichia coli were produced in large amounts by placing the dacA gene that encodes PBP 5 under the control of the trp-lac fusion promoter. The 3' end of the dacA gene used in this study contains a stop codon that results in the deletion of 15 amino acids from the carboxyl terminus and the production of a soluble protein. Using oligonucleotide-directed mutagenesis, the role of cysteine 115 in the mechanism of sPBP 5 was investigated. Alkylation of cysteine 115 with sulfhydryl reagents has previously been shown to inhibit severely the D-alanine carboxypeptidase activity of PBP 5. Alkylation also inhibits the hydrolysis of bound penicillin G, with only a slight effect on its binding. Cysteine 115 in sPBP 5 was changed to either a serine (sPBP 5C-S) or an alanine (sPBP 5C-A) residue. The wild-type and mutant sPBPs were purified in milligram amounts from induced cultures by ampicillin affinity chromatography. The mutant PBPs showed only a 2-fold increase in the half-life of the penicilloyl-PBP complex, and had a binding affinity for penicillin G identical to wild-type PBP 5. The Km for the release of D-alanine from the peptide L-Ala-D-gamma-Glu-L-Lys-D-Ala-D-Ala was 5.0, 3.5, and 7.8 mM for PBP 5, PBP 5C-S, and PBP 5C-A, respectively, while the values for Vmax were 2.5, 3.3, and 5.1 mumol/min/mg. From these data it was concluded that the cysteine residue does not directly participate in the enzymatic mechanism.     

Penicillin binding proteins of Vibrio cholerae

Eleven penicillin binding proteins (PBPs) of Vibrio cholerae have been identified using [125I] labelled p-hydroxybenzyl penicillin (PenX). These proteins are localised in the inner membrane and have molecular weights ranging from 97,000 to 22,000. Neutral hydroxylamine released the labelled PenX from the PBPs and pretreatment with cold benzyl penicillin inhibited labelling completely. The PBP 4 is the most sensitive target for cephaloridine and aztreonam. Cephaloridine also binds to three other high molecular weight PBPs, 1, 2 and 3. Aztreonam, in addition to PBP 4, has affinity for another low molecular weight PBP, PBP 7. Mecillinam has affinity for PBPs 1, 4 and 11.     

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.     

The heat shock and ethanol stress responses of yeast exhibit extensive similarity and functional overlap

Abstract We examined the penicillin-binding proteins (PBPs) of certain field strains of Streptococcus suis , as well as those from laboratory variants having different degrees of resistance to penicillin. Results indicated that (i) S. suis possesses three distinct groups of PBPs, arbitrarily named here PBP 1, PBP 2, and PBP 3, with approximate molecular weights of 97, 82, and 45 kDa respectively; (ii) PBP profiles of field strains of S. suis having different MICs (≤ 0.03 to 16.0 μg/ml) were not uniform (PBP 2 being difficult to detect in strains whose MICs exceeded 0.10 μg/ml, and PBP 3 which exhibited shifts in molecular weight of approximately 5 kDa); (iii) laboratory variant PBPs 1 and 2 showed decreased affinity for penicillin as compared to the parent strain in antibiotic competition experiments, even though the PBP profiles of both were similar. We suggest that PBP modifications (altered molecular weight and/or decreased affinity for penicillin) are involved in the mechanism of resistance to penicillin by S. suis .     

Interaction energies between beta-lactam antibiotics and E. coli penicillin-binding protein 5 by reversible thermal denaturation

Penicillin-binding proteins (PBPs) catalyze the final stages of bacterial cell wall biosynthesis. PBPs form stable covalent complexes with beta-lactam antibiotics, leading to PBP inactivation and ultimately cell death. To understand more clearly how PBPs recognize beta-lactam antibiotics, it is important to know their energies of interaction. Because beta-lactam antibiotics bind covalently to PBPs, these energies are difficult to measure through binding equilibria. However, the noncovalent interaction energies between beta-lactam antibiotics and a PBP can be determined through reversible denaturation of enzyme-antibiotic complexes. Escherichia coli PBP 5, a D-alanine carboxypeptidase, was reversibly denatured by temperature in an apparently two-state manner with a temperature of melting (T(m)) of 48.5 degrees C and a van't Hoff enthalpy of unfolding (H(VH)) of 193 kcal/mole. The binding of the beta-lactam antibiotics cefoxitin, cloxacillin, moxalactam, and imipenem all stabilized the enzyme significantly, with T(m) values as high as +4.6 degrees C (a noncovalent interaction energy of +2.7 kcal/mole). Interestingly, the noncovalent interaction energies of these ligands did not correlate with their second-order acylation rate constants (k(2)/K'). These rate constants indicate the potency of a covalent inhibitor, but they appear to have little to do with interactions within covalent complexes, which is the state of the enzyme often used for structure-based inhibitor design.     

Selective inhibition of penicillin-binding proteins and its effects on growth and architecture of Staphylococcus aureus

We found that the three high molecular weight penicillin-binding proteins (PBP) 1, 2, and 3 of Staphylococcus aureus could be blocked by the β-lactam antibiotics imipenem, cefotaxime, and mecillinam, respectively. The inhibition of any of these PBPs was not sufficient for an antibacterial effect. Even the simultaneous blocking of PBPs 2 and 3, previously supposed to be the lethal targets of β-lactam antibiotics, did not induce bacteriolysis, nor did the combined saturation of PBPs 2, 3, and 4. Instead, PBP 1 seems to play a key role, because on one hand the combined inhibition of PBP 1 with any of the other high molecular weight PBPs led to bacteriolysis, on the other hand, only inhibition of PBP 1 led to a loss of the ‘splitting system’ of the staphylococcal cross wall, similar to that observed in penicillin G-treated cells earlier.     

Growth and division of Streptococcus pneumoniae: localization of the high molecular weight penicillin-binding proteins during the cell cycle

The bacterial peptidoglycan, the main component of the cell wall, is synthesized by the penicillin-binding proteins (PBPs). We used immunofluorescence microscopy to determine the cellular localization of all the high molecular weight PBPs of the human pathogen Streptococcus pneumoniae, for a wild type and for several PBP-deficient strains. Progression through the cell cycle was investigated by the simultaneous labelling of DNA and the FtsZ protein. Our main findings are: (i) the temporal dissociation of cell wall synthesis, inferred by the localization of PBP2x and PBP1a, from the constriction of the FtsZ-ring; (ii) the localization of PBP2b and PBP2a at duplicated equatorial sites indicating the existence of peripheral peptidoglycan synthesis, which implies a similarity between the mechanism of cell division in bacilli and streptococci; (iii) the abnormal localization of some class A PBPs in PBP-defective mutants which may explain the apparent redundancy of these proteins in S. pneumoniae.     

Structural and functional difference of pheromone binding proteins in discriminating chemicals in the gypsy moth, Lymantria dispar

Pheromone-binding proteins (PBPs) of the gypsy moth, Lymantria dispar L., play an important role in olfaction. Here structures of PBPs were first built by Homology Modeling, and each model of PBPs had seven α-helices and a large hydrophobic cavity including 25 residues for PBP1 and 30 residues for PBP2. Three potential semiochemicals were first screened by CDOCKER program based on the PBP models and chemical database. These chemicals were Palmitic acid n-butyl ester (Pal), Bis(3,4-epoxycyclohexylmethyl) adipate (Bis), L-trans-epoxysuccinyl-isoleucyl-proline methyl ester propylamide (CA-074). The analysis of chemicals docking the proteins showed one hydrogen bond was established between the residues Lys94 and (+)-Disparlure ((+)-D), and л-л interactions were present between Phe36 of PBP1 and (+)-D. The Lys94 of PBP1 formed two and three hydrogen bonds with Bis and CA-074, respectively. There was no residue of PBP2 interacting with these four chemicals except Bis forming one hydrogen bond with Lys121. After simulating the conformational changes of LdisPBPs at pH7.3 and 5.5 by constant pH molecular dynamics simulation in implicit solvent, the N-terminal sequences of PBPs was unfolded, only having five α-helices, and PBP2 had larger binding pocket at 7.3 than PBP1. To investigate the changes of α-helices at different pH, far-UV and near-UV circular dichroism showed PBPs consist of α-helices, and the tertiary structures of PBP1 and PBP2 were influenced at pH7.3 and 5.5. The fluorescence binding assay indicated that PBP1 and PBP2 have similarly binding affinity to (+)-D at pH 5.5 and 7.3, respectively. At pH 5.5, the dissociation constant of the complex between PBP1 and 2-decyl-1-oxaspiro [2.2] pentane (OXP1) was 0.68 ± 0.01 μM, for (+)-D was 5.32 ± 0.11 μM, while PBP2 with OXP1 and (+)-D were 1.88 ± 0.02 μM and 5.54 ± 0.04 μM, respectively. Three chemicals screened had higher affinity to PBP1 than (+)-D except Pal at pH5.5, and had lower affinity than (+)-D at pH7.3. To PBP2, these chemicals had lower affinity than the sex pheromone except Bis at pH 5.5 and pH 7.3. Only PBP1 had higher affinity with Sal than the sex pheromone at pH 5.5. Therefore, the structures of PBP1 and PBP2 had different changes at pH5.5 and 7.3, showing different affinity to chemicals. This study helps understanding the role of PBPs as well as in developing more efficient chemicals for pest control.     

One or two low affinity penicillin-binding proteins may be responsible for the range of susceptibility of Enterococcus faecium to benzylpenicillin

Three benzylpenicillin-resistant, clinical isolates of Enterococcus faecium (MIC values 16-64 micrograms ml-1) contained six penicillin-binding proteins (PBPs), of which PBP5 was the most abundant and had the lowest affinity for the antibiotic. Four benzylpenicillin-susceptible strains (MIC values 0.031-0.5 microgram ml-1) were obtained as spontaneous derivatives from these above organisms. There were significant decreases in the amounts of PBP5 in each of the derivatives, with the concomitant appearance of a new, higher affinity PBP (5*) in three strains. Increased amounts of PBP5, with no changes in PBP5*, were found in several mutants with intermediate-level benzylpenicillin-resistance (MIC values 1-8 micrograms ml-1) selected from two of the susceptible strains. Examination of 18 other clinical isolates, with a wide range of susceptibilities to benzylpenicillin (MIC values 0.062-128 micrograms ml-1), showed that PBP5* was present in 13 strains, and PBP5 in all of them, but in differing amounts. The results concerning the relative amounts and relative affinities of PBPs 5* and 5 allowed the categorization of the various strains into six groups, within which organisms had somewhat similar susceptibilities to benzylpenicillin.     

Identification of two penicillin-binding multienzyme complexes in Haemophilus influenzae.

Dansyl-labeled penicillin, reversed-phase chromatography, and peptide mapping have been used to detect, separate, and study penicillin-binding proteins (PBPs) and PBP multienzyme complexes of H. influenzae. The cross-linking of proteins in the multienzyme complex was accomplished with the aid of cyanogen, a salt-bridge specific cross-linking agent. The chromatographic profile of the PBPs clearly showed a dramatic change in the number and identity of peaks after treatment of the bacterial cells with cyanogen. The disappearance of all seven peaks corresponding to the PBPs was accompanied by the emergence of two new peaks with molecular weights between 400 kDa and 600 kDa. The results hint at the existence of two penicillin-binding multienzyme complexes, each containing subunits that interact via salt-bridges. Chromatographic active site peptide mapping of PBPs and PBP complexes was used to determine the identity of PBPs involved in each complex. It is postulated that one multienzyme complex containing PBP 2 may be involved in cell elongation while the other complex containing PBP 3 may be responsible for cell division.     

Three pheromone-binding proteins in olfactory sensilla of the two silkmoth species Antheraea polyphemus and Antheraea pernyi.

Females of the sibling silkmoth species Antheraea polyphemus and A. pernyi use the same three sex pheromone components in different ratios to attract conspecific males. Accordingly, the sensory hairs on the antennae of males contain three receptor cells sensitive to each of the pheromone components. In agreement with the number of pheromones used, three different pheromone-binding proteins (PBPs) could be identified in pheromone-sensitive hairs of both species by combining biochemical and molecular cloning techniques. MALDI-TOF MS of sensillum lymph droplets from pheromone-sensitive sensilla trichodea of male A. polyphemus revealed the presence of three major peaks with m/z of 15702, 15752 and 15780 and two minor peaks of m/z 15963 and 15983. In Western blots with four antisera raised against different silkmoth odorant-binding proteins, immunoreactivity was found only with an anti-(Apol PBP) serum. Free-flow IEF, ion-exchange chromatography and Western blot analyses revealed at least three anti-(Apol PBP) immunoreactive proteins with pI values between 4.4 and 4.7. N-Terminal sequencing of these three proteins revealed two proteins (Apol PBP1a and Apol PBP1b) identical in the first 49 amino acids to the already known PBP (Apol PBP1) [Raming, K. , Krieger, J. & Breer, H. (1989) FEBS Lett. 256, 2215-2218] and a new PBP having only 57% identity with this amino-acid region. Screening of antennal cDNA libraries with an oligonucleotide probe corresponding to the N-terminal end of the new A. polyphemus PBP, led to the discovery of full length clones encoding this protein in A. polyphemus (Apol PBP3) and in A. pernyi (Aper PBP3). By screening the antennal cDNA library of A. polyphemus with a digoxigenin-labelled A. pernyi PBP2 cDNA [Krieger, J., Raming, K. & Breer, H. (1991) Biochim. Biophys. Acta 1088, 277-284] a homologous PBP (Apol PBP2) was cloned. Binding studies with the two main pheromone components of A. polyphemus and A. pernyi, the (E,Z)-6, 11-hexadecadienyl acetate (AC1) and the (E,Z)-6,11-hexadecadienal (ALD), revealed that in A. polyphemus both Apol PBP1a and the new Apol PBP3 bound the 3H-labelled acetate, whereas no binding of the 3H-labelled aldehyde was found. In A. pernyi two PBPs from sensory hair homogenates showed binding affinity for the AC1 (Aper PBP1) and the ALD (Aper PBP2), respectively.     

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.     

Discrete and overlapping functions of peptidoglycan synthases in growth,cell division and virulence of Listeria monocytogenes

Upon ingestion of contaminated food, Listeria monocytogenes can cause serious infections in humans that are normally treated with β‐lactam antibiotics. These target Listeria's five high molecular weight penicillin‐binding proteins (HMW PBPs), which are required for peptidoglycan biosynthesis. The two bi‐functional class A HMW PBPs PBP A1 and PBP A2 have transglycosylase and transpeptidase domains catalyzing glycan chain polymerization and peptide cross‐linking, respectively, whereas the three class B HMW PBPs B1, B2 and B3 are monofunctional transpeptidases. The precise roles of these PBPs in the cell cycle are unknown. Here we show that green fluorescent protein (GFP)‐PBP fusions localized either at the septum, the lateral wall or both, suggesting distinct and overlapping functions. Genetic data confirmed this view: PBP A1 and PBP A2 could not be inactivated simultaneously, and a conditional double mutant strain is largely inducer dependent. PBP B1 is required for rod‐shape and PBP B2 for cross‐wall biosynthesis and viability, whereas PBP B3 is dispensable for growth and cell division. PBP B1 depletion dramatically increased β‐lactam susceptibilities and stimulated spontaneous autolysis but had no effect on peptidoglycan cross‐linkage. Our in vitro virulence assays indicated that the complete set of all HMW PBPs is required for maximal virulence.     

Studies on the mechanism of intrinsic resistance to beta-lactam antibiotics in group D streptococci

Six penicillin-binding proteins (PBPs) were detected in clinical isolates of each one of three group D streptococci: Streptococcus bovis, S. faecalis and S. faecium. When examined in whole organisms, the PBPs of S. faecium, the most penicillin-resistant species of group D streptococci, generally had lower affinities for the antibiotic than those of S. faecalis (intermediate penicillin resistance), which in turn were of lower affinity than those of S. bovis (penicillin-sensitive). On the other hand, no quantitative correlation could be established between the binding of penicillin to any one PBP or group of PBPs, and the penicillin MIC value for the corresponding micro-organism. Examination of the amounts of antibiotic bound and the rates of binding to PBPs of equal numbers of protoplasts and whole bacteria of S. faecalis and S. faecium, indicated that there was no permeability barrier to benzylpenicillin in the cell walls of these species. The lower antibacterial effectiveness of cephalothin compared with ampicillin in group D streptococci was paralleled by the higher concentrations of cephalothin needed in competition assays to inhibit the lower molecular size PBPs of these bacteria.     

Mutational analysis of the Streptococcus pneumoniae bimodular class A penicillin-binding proteins.

One group of penicillin target enzymes, the class A high-molecular-weight penicillin-binding proteins (PBPs), are bimodular enzymes. In addition to a central penicillin-binding-transpeptidase domain, they contain an N-terminal putative glycosyltransferase domain. Mutations in the genes for each of the three Streptococcus pneumoniae class A PBPs, PBP1a, PBP1b, and PBP2a, were isolated by insertion duplication mutagenesis within the glycosyltransferase domain, documenting that their function is not essential for cellular growth in the laboratory. PBP1b PBP2a and PBP1a PBP1b double mutants could also be isolated, and both showed defects in positioning of the septum. Attempts to obtain a PBP2a PBP1a double mutant failed. All mutants with a disrupted pbp2a gene showed higher sensitivity to moenomycin, an antibiotic known to inhibit PBP-associated glycosyltransferase activity, indicating that PBP2a is the primary target for glycosyltransferase inhibitors in S. pneumoniae.     

Penicillin-binding protein 2b of Streptococcus pneumoniae in piperacillin-resistant laboratory mutants.

In Streptococcus pneumoniae, alterations in penicillin-binding protein 2b (PBP 2b) that reduce the affinity for penicillin binding are observed during development of beta-lactam resistance. The development of resistance was now studied in three independently obtained piperacillin-resistant laboratory mutants isolated after several selection steps on increasing concentrations of the antibiotic. The mutants differed from the clinical isolates in major aspects: first-level resistance could not be correlated with alterations in the known PBP genes, and the first PBP altered was PBP 2b. The point mutations occurring in the PBP 2b genes were characterized. Each mutant contained one single point mutation in the PBP 2b gene. In one mutant, this resulted in a mutation of Gly-617 to Ala within one of the homology boxes common to all PBPs, and in the other two cases, the same Gly-to-Asp substitution at the end of the penicillin-binding domain had occurred. The sites affected were homologous to those determined previously in the S. pneumoniae PBP 2x of mutants resistant to cefotaxime, indicating that, in both PBPs, similar sites are important for interaction with the respective beta-lactams.     

Penicillin-binding proteins of Thiobacillus versutus

The cytoplasmic membrane of Thiobacillus versutus was found to contain at least nine penicillin-binding proteins (PBPs) with apparent molecular weights as judged by sodium dodecyl sulphate polyacrylamide slab gel electrophoresis of 87000 (PBP1), 81000 (PBP2), 68000 (PBP3), 63000 (PBP4), 57000 (PBP5), 40000 (PBP6), 37000 (PBP70, 33000 (PBP8) and 31000 (PBP9). The PBP pattern of T. versutus was thus quite different from that of the Enterobacteria and the Pseudomonads. Also the properties of the PBPs of T. versutus such as affinity for various beta-lactam antibiotics, heat stability and release of bound penicillin were different from similar properties of Escherichia coli, Pseudomonas aeruginosa and other gram-negative bacteria.     

Resistance to β-Lactam Antibiotics Conferred by Point Mutations in Penicillin-Binding Proteins PBP3, PBP4 and PBP6 in Salmonella enterica

Penicillin-binding proteins (PBPs) are enzymes responsible for the polymerization of the glycan strand and the cross-linking between glycan chains as well as the target proteins for β-lactam antibiotics. Mutational alterations in PBPs can confer resistance either by reducing binding of the antibiotic to the active site or by evolving a β-lactamase activity that degrades the antibiotic. As no systematic studies have been performed to examine the potential of all PBPs present in one bacterial species to evolve increased resistance against β-lactam antibiotics, we explored the ability of fifteen different defined or putative PBPs in Salmonella enterica to acquire increased resistance against penicillin G. We could after mutagenesis and selection in presence of penicillin G isolate mutants with amino-acid substitutions in the PBPs, FtsI, DacB and DacC (corresponding to PBP3, PBP4 and PBP6) with increased resistance against β-lactam antibiotics. Our results suggest that: (i) most evolved PBPs became ‘generalists” with increased resistance against several different classes of β-lactam antibiotics, (ii) synergistic interactions between mutations conferring antibiotic resistance are common and (iii) the mechanism of resistance of these mutants could be to make the active site more accessible for water allowing hydrolysis or less binding to β-lactam antibiotics.