Crystal structure of PBP2x from a highly penicillin-resistant Streptococcus pneumoniae clinical isolate: a mosaic framework containing 83 mutations.

Penicillin-binding proteins (PBPs) are the main targets for beta-lactam antibiotics, such as penicillins and cephalosporins, in a wide range of bacterial species. In some Gram-positive strains, the surge of resistance to treatment with beta-lactams is primarily the result of the proliferation of mosaic PBP-encoding genes, which encode novel proteins by recombination. PBP2x is a primary resistance determinant in Streptococcus pneumoniae, and its modification is an essential step in the development of high level beta-lactam resistance. To understand such a resistance mechanism at an atomic level, we have solved the x-ray crystal structure of PBP2x from a highly penicillin-resistant clinical isolate of S. pneumoniae, Sp328, which harbors 83 mutations in the soluble region. In the proximity of the Sp328 PBP2x* active site, the Thr(338) --> Ala mutation weakens the local hydrogen bonding network, thus abrogating the stabilization of a crucial buried water molecule. In addition, the Ser(389) --> Leu and Asn(514) --> His mutations produce a destabilizing effect that generates an "open" active site. It has been suggested that peptidoglycan substrates for beta-lactam-resistant PBPs contain a large amount of abnormal, branched peptides, whereas sensitive strains tend to catalyze cross-linking of linear forms. Thus, in vivo, an "open" active site could facilitate the recognition of distinct, branched physiological substrates.     

Pneumococcal beta-lactam resistance due to a conformational change in penicillin-binding protein 2x

Streptococcus pneumoniae is a life-threatening human pathogen that is increasingly resistant to a wide array of drugs. Resistance to beta-lactams, the most widely used antibiotics, is correlated with tens of amino acid substitutions in their targets; that is, the penicillin-binding proteins (PBPs), resulting from multiple events of recombination. To discriminate relevant substitutions from those that are incidental to the recombination process, we report the exhaustive characterization of all the mutations in the transpeptidase domain of PBP2x from the highly resistant strain 5204. A semi-automated method combining biochemical and microbiological approaches singled out 6 mutations of 41 (15%) that are essential for high level resistance. The hitherto uncharacterized I371T, R384G, M400T, and N605T together with the previously studied T338M and M339F account for nearly all the loss of affinity of PBP2x for beta-lactams. Most interestingly, I371T and R384G cause the conformational change of a loop that borders the entrance of the active site cavity, hampering antibiotic binding. For the first time all the mutations of a PBP relevant to beta-lactam resistance have been identified, providing new mechanistic insights. Most notable is the relationship between the decreased susceptibility to beta-lactams and the dynamic behavior of a loop.     

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.     

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.     

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.     

Five independent combinations of mutations can result in low-affinity penicillin-binding protein 2x of Streptococcus pneumoniae.

Penicillin-binding protein 2x (PBP 2x) of Streptococcus pneumoniae is one of the high-molecular-weight PBPs involved in the development of intrinsic beta-lactam resistance. Point mutations in the PBP 2x genes (pbpX) have now been characterized in five independent spontaneous laboratory mutants in order to identify protein regions which are important for interaction with beta-lactam antibiotics. All mutant genes contained two to four mutations resulting in amino acid substitutions within the penicillin-binding domain of PBP 2x, and none of the mutants carried an identical set of mutations. For one particular mutant, C606, carrying four mutations in pbpX, the mutations at positions 601 and 597 conferred first- and second-level resistance when introduced into the susceptible parent strain S. pneumoniae R6. However, the other two mutations, at amino acid positions 289 and 422, which were originally selected at the fifth and sixth isolation steps, did not contribute at all to resistance in similar experiments. This suggests that they are phenotypically expressed only in combination with mutations in other genes. Three PBP 2x regions were mutated in from two to all four mutants carrying a low-affinity PBP 2x. However, in a fifth mutant containing a PBP 2x with apparent zero affinity for beta-lactams, the three mutations in pbpX mapped at entirely different positions. This demonstrates that different mutational pathways exist for remodeling this PBP during resistance development.     

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.     

The structural modifications induced by the M339F substitution in PBP2x from Streptococcus pneumoniae further decreases the susceptibility to beta-lactams of resistant strains

PBP2x is a primary determinant of beta-lactams resistance in Streptococcus pneumoniae. Altered PBP2x with multiple mutations have a reduced "affinity" for the antibiotics. An important polymorphism is found in PBP2x sequences from clinical resistant strains. To understand the mechanism of resistance, it is necessary to identify and characterize the relevant substitutions. Many similar PBP2x sequences from resistant isolates have the previously studied T338A mutation, adjacent to the active site Ser337. We report here the structural and functional analysis of the M339F substitution that is found in a subset of these sequences, originating from highly resistant strains. The M339F mutation causes a 4-10-fold reduction of the reaction rate with beta-lactams, depending on the molecular context. In addition, release of the inactivated antibiotic from the active site is up to 3-fold faster as a result from the M339F mutation. These effects measured in vitro are correlated with the level of beta-lactam resistance in vivo conferred by several PBP2x variants. Thus, a single amino acid difference between similar PBP2x from clinical isolates can strongly modulate the degree of beta-lactam resistance. The crystal structure of the double mutant T338A/M339F solved to a resolution of 2.4 A shows a distortion of the active site and a reorientation of the hydroxyl group of the active site Ser337, which can explain the kinetic effects of the mutations.     

The crystal structure of the penicillin-binding protein 2x from Streptococcus pneumoniae and its acyl-enzyme form: implication in drug resistance

Penicillin-binding proteins (PBPs), the primary targets for beta-lactam antibiotics, are periplasmic membrane-attached proteins responsible for the construction and maintenance of the bacterial cell wall. Bacteria have developed several mechanisms of resistance, one of which is the mutation of the target enzymes to reduce their affinity for beta-lactam antibiotics. Here, we describe the structure of PBP2x from Streptococcus pneumoniae determined to 2.4 A. In addition, we also describe the PBP2x structure in complex with cefuroxime, a therapeutically relevant antibiotic, at 2.8 A. Surprisingly, two antibiotic molecules are observed: one as a covalent complex with the active-site serine residue, and a second one between the C-terminal and the transpeptidase domains. The structure of PBP2x reveals an active site similar to those of the class A beta-lactamases, albeit with an absence of unambiguous deacylation machinery. The structure highlights a few amino acid residues, namely Thr338, Thr550 and Gln552, which are directly related to the resistance phenomenon.     

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.     

Interspecies recombinational events during the evolution of altered PBP 2x genes in penicillin-resistant clinical isolates of Streptococcus pneumoniae

Penicillin resistance in pneumococci is due to the appearance of high molecular-weight penicillin-binding proteins (PBPs) that have reduced affinity for the antibiotic. We have compared the PBX 2x genes (pbpX) of one penicillin-susceptible and five penicillin-resistant clinical isolates of Streptococcus pneumoniae isolated from various parts of the world. All of the resistant isolates contained a low-affinity form of PBP 2x. The 2 kb region of the two penicillin-susceptible isolates differed at only eight nucleotide sites (0.4%) and resulted in one single amino acid difference in PBP 2x. In contrast, the sequences of the PBP 2x genes from the resistant isolates differed overall from those of the susceptible isolates at between 7 and 18% of nucleotide sites and resulted in between 27 and 86 amino acid substitutions in PBP 2x. The altered PBP 2x genes consisted of regions that were similar to those of susceptible strains (less than 3% diverged), alternating with regions that were very different (18-23% diverged). The presence of highly diverged regions within the PBP 2x genes of the resistant isolates contrasts with the uniformity of the sequences of the amylomaltase genes from the same isolates, and with the uniformity of the PBP 2x genes in the two susceptible isolates. It suggests that the altered PBP 2x genes have arisen by localized interspecies recombinational events involving the PBP 2x genes of closely related streptococci, as has been suggested to occur for altered PBP 2b genes (Dowson et al., 1989b). The PBP 2x genes from the resistant isolates could transform the susceptible strain R6 to increased levels of resistance to beta-lactam antibiotics, indicating that the altered forms of PBP 2x in the resistant isolates contribute to their resistance to penicillin.     

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.     

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.     

A mass-spectrometric analysis of genetic markers of S. pneumoniae resistance to beta-lactam antibiotics

Beta-lactam antibiotics remain the drugs of choice for treatment of S. pneumoniae infections in spite of growing level of resistance. The formation of S. pneumoniae resistance to these drugs is mediated by modifications of the penicillin-binding proteins (PBPs), the targets of the antibiotic action. A new approach to detection of mutations in PBP1A, 2B and 2X genes based on minisequencing reaction followed by MALDI-ToF (Matrix-Assisted Laser Desorption/Ionization Time of Flight) mass spectrometry was developed in this study. The evaluation of these mutations prevalence in clinical S. pneumoniae isolates (n = 194) with different susceptibility level to beta-lactam antibiotics was performed. Twenty-four different combinations of mutations in PBPs (genotypes) were detected. All isolates susceptible to penicillin (n = 49, MIC > or = 0.06 > or = gamma/ml) carried no mutations in all analyzed loci. For 145 S. pneumoniae isolates with reduced susceptibility to penicillin (MIC > 0.06 > or = gamma/ml) the mutations in PBPs were detected in 133 (91.7 %) cases that testify to high diagnostic sensitivity of such approach. The isolates with MIC > or = 4 > or = gamma/ml (n = 20) carried multiple mutations in all analyzed genes that confirms cumulative effects of penicillin resistance formation. However, it was not possible to associate observed mutations in PBPs genes with decrease of susceptibility to cefotaxime that allows suggesting the entire difference in molecular mechanisms of formation of resistance to penicillins and cephalosporins. The offered method of S. pneumoniae genotyping is suitable for susceptibility testing to penicillin of individual isolates and for molecular monitoring of the resistance determinants in population.     

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 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.     

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.     

Relatedness between Streptococcus pneumoniae and viridans streptococci: transfer of penicillin resistance determinants and immunological similarities of penicillin-binding proteins

The occurrence of highly variable penicillin-binding proteins (PBPs) in penicillin-resistant Streptococcus pneumoniae suggested that transfer of homologous genes from related species may be involved in resistance development. Antiserum and monoclonal antibodies raised against PBPs 1a and 2b from the susceptible S. pneumoniae R6 strain were used to identify related PBPs in 41 S. mitis, S. sanguis I and S. sanguis II strains mostly isolated in South Africa with MIC values ranging from less than 0.15 to 16 mg/ml. Furthermore, the possibility of genetic exchange was examined with 30 penicillin-resistant strains of this collection (MIC greater than 0.06 mg/ml) as donors using S. pneumoniae R6 as recipient in transformation experiments. The majority of S. mitis and S. sanguis II strains but none of the S. sanguis I strains could transform penicillin resistance genes into S. pneumoniae R6. All positive donor strains and all susceptible isolates of S. mitis and S. sanguis II strains contained PBPs which cross-reacted with the anti-PBP 1a and/or anti-PBP 2b antibodies. On the other hand, only five of the 14 S. sanguis I strains contained a PBP that reacted with one of the antibodies. This strongly suggested the presence of genes homologous to the pneumococcal PBP 1a and 2b genes in viridans streptococci, and documents that penicillin resistance determinants can be transformed from viridans streptococci into the pneumococcus.     

Nucleotide sequences of genes encoding penicillin-binding proteins from Streptococcus pneumoniae and Streptococcus oralis with high homology to Escherichia coli penicillin-binding proteins 1a and 1b.

The nucleotide sequence of a 3,378-bp DNA fragment of Streptococcus pneumoniae that included the structural gene for penicillin-binding protein (PBP) 1a (ponA), which encodes 719 amino acids, was determined. Homologous DNA fragments from an S. oralis strain were amplified with ponA-specific oligonucleotides. The 2,524-bp S. oralis sequence contained the coding region for the first 636 amino acids of a PBP. The coding sequence differed by 437 nucleotides (27%) and one additional triplet, resulting in 87 amino acid substitutions (14%), from S. pneumoniae PBP 1a. Both PBPs are highly homologous to bifunctional high-M(r) Escherichia coli PBPs 1a and 1b.     

Mutations in the active site of penicillin-binding protein PBP2x from Streptococcus pneumoniae. Role in the specificity for beta-lactam antibiotics.

Penicillin-binding protein 2x (PBP2x) isolated from clinical beta-lactam-resistant strains of Streptococcus pneumoniae (R-PBP2x) have a reduced affinity for beta-lactam antibiotics. Their transpeptidase domain carries numerous substitutions compared with homologous sequences from beta-lactam-sensitive streptococci (S-PBP2x). Comparison of R-PBP2x sequences suggested that the mutation Gln552 --> Glu is important for resistance development. Mutants selected in the laboratory with cephalosporins frequently contain a mutation Thr550 --> Ala. The high resolution structure of a complex between S-PBP2x* and cefuroxime revealed that Gln552 and Thr550, which belong to strand beta3, are in direct contact with the cephalosporin. We have studied the effect of alterations at positions 552 and 550 in soluble S-PBP2x (S-PBP2x*) expressed in Escherichia coli. Mutation Q552E lowered the acylation efficiency for both penicillin G and cefotaxime when compared with S-PBP2x*. We propose that the introduction of a negative charge in strand beta3 conflicts with the negative charge of the beta-lactam. Mutation T550A lowered the acylation efficiency of the protein for cefotaxime but not for penicillin G. The in vitro data presented here are in agreement with the distinct resistance profiles mediated by these mutations in vivo and underline their role as powerful resistance determinants.