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Vancomycin-resistant Staphylococcus aureus (S. aureus) (VRSA) uses depsipeptide-containing modified cell-wall precursors for the biosynthesis of peptidoglycan. Transglycosylase is responsible for the polymerization of the peptidoglycan, and the penicillin-binding protein 2 (PBP2) plays a major role in the polymerization among several transglycosylases of wild-type S. aureus. However, it is unclear whether VRSA processes the depsipeptide-containing peptidoglycan precursor by using PBP2. Here, we describe the total synthesis of depsi-lipid I, a cell-wall precursor of VRSA. By using this chemistry, we prepared a depsi-lipid II analogue as substrate for a cell-free transglycosylation system. The reconstituted system revealed that the PBP2 of S. aureus is able to process a depsi-lipid II intermediate as efficiently as its normal substrate. Moreover, the system was successfully used to demonstrate the difference in the mode of action of the two antibiotics moenomycin and vancomycin.

To make progress in understanding peptidoglycan metabolism, we will reconstitute in vitro the assembly process and the molecular machineries that carry out this formidable task. We review here the reports of isolation of complexes comprising penicillin-binding proteins (PBPs), the enzymes that synthesize the peptidoglycan from its lipid-linked precursor.

The enzymatic synthesis of dipeptide units of the D-D-configuration in aqueous media, catalysed by muramoyl-pentapeptide carboxypeptidase (E.C.3.4.17.8), is described. Ac-L-Lys(Ac)-D-Ala-D-Lac-OH and Ac-D-Ala-OMe were used as acyl-components. Neutral, basic, and hydrophobic amino acids acting as nucleophiles were incorporated. The enzyme is stereospecific in that only the D-enantiomers of amino acids or amino acid derivatives were incorporated. As nucleophiles, the unmodified amino acids resulted in higher product yields compared with using the corresponding amino acid derivatives. Product yields ranged from 40 to 87%.

The sequence homology found by Waxman & Strominger between penicillin-sensitive D-alanine-carboxypeptidases and penicillin-inactivating beta-lactamases is shown to extend to the level of secondary structure as predicted by the method of Chou & Fasman or by the informational method of Garnier et al. Thermodynamic similarity of homologous segments of these proteins is demonstrated by means of a sequence-independent parameter, the hydration potential of Wolfenden at al. Although the 40- to 70-residue amino-terminal sequences examined contain a common serine reactive with penicillins and (in the case of the carboxypeptidases) an R-D-alanyl-D-alanine substrate analog, no homology in secondary structure or hydration potential could be found with a serine protease such as alpha-chymotrypsin.

Resistance to third-generation cephalosporins in a clinical isolate of Streptococcus pneumoniae was shown to be due to the production of altered forms of penicillin-binding proteins (PBPs) 2X and 1A. The cloned PBP2X gene from the resistant strain was able to transform a susceptible strain to an intermediate level of resistance. The resulting transformant could be transformed to the full level of resistance of the clinical isolate using the cloned PBP1A gene from the latter strain. Chromosomal DNA from the resistant strain (and from other resistant strains) could readily transform a susceptible strain to the full level of resistance to third-generation cephalosporins (greater than 250-fold for cefotaxime; greater than 100-fold for ceftriaxone) in a single step (transformation frequency of about 10(-5)). The resistant transformants obtained with chromosomal DNA were shown by gene fingerprinting to have gained both the PBP1A and PBP2X genes from the DNA donor.

The monobactams are a novel family of monocyclic beta-lactam antibiotics characterized by the 2-oxoazetidine-1-sulfonic acid moiety. A series of monobactams bind covalently to the Streptomyces R61 DD-carboxypeptidase in a manner similar to that for bicyclic beta-lactams, especially cephalosporins. The similarity of interaction was established by the following criteria: inhibition of binding by diisopropylfluorophosphate and alpha-dicarbonyls; stoichiometry of binding; similarity of partial proteolysis products of radiolabelled enzyme; rates of release of bound beta-lactams; nature of hydrolysis and hydroxylaminolysis products.

The 22076-Mr Zn2+-containing D-alanyl-D-alanine-cleaving carboxypeptidase of Streptomyces abuls G effectively catalyses the transfer of the N alpha, N epsilon-diacetyl-L-lysyl-D-alanyl electrophilic group of the standard tripeptide substrate N alpha, N epsilon-diacetyl-L-lysyl-D-alanyl-D-alanine to water. It also performs a weak beta-lactamase activity, hydrolysing penicillin into penicilloate at a very low rate. This protein consists of 212 amino acid residues in a single polypeptide chain. The N terminus is partially blocked as a result of the cyclization of the dipeptide Asn-Gly into anhydroaspartylglycine imide. The protein has been fragmented by cyanogen bromide into five fragments whose sequences have been determined via appropriate subcleavages with various proteases. The ordering of the cyanogen bromide peptide fragments has been carried out (a) by submitting the S-carboxymethylated protein to complete tryptic digestion and labelling the methionine-containing peptides thus obtained with iodo[14C]-acetamide, and (b) by submitting to limited tryptic digestion the S-[2-(4'-pyridyl)ethyl]-cysteine protein whose amino groups have been blocked by reaction with exo-cis-3,6-endoxo-delta 4-tetrahydrophthalic anhydride prior to digestion. The protein contains six cysteine residues in the form of three disulfide bridges. No homology is found by comparing this peptidase with other Zn2+-containing enzymes (carboxypeptidase A, thermolysin, carbonic anhydrase B and alcohol dehydrogenase) and several completely or partially sequenced, serine-containing D-alanyl-D-alanine-cleaving peptidases and Zn2+/serine-containing beta-lactamases.

The cell-wall composition and degree of cross-linking of peptidoglycan in a strain of Staphylococcus aureus (strain MR-1) which is highly resistant to methicillin were similar to those of other strains of S. aureus. When the organism was grown in the presence of very low concentrations of methicillin (equivalent to 3 x 10(-4) x minimum growth-inhibitory concentration [MGIC] there was a large decrease in the degree of cross-linking of the peptidoglycan. Increasing concentrations of methicillin (up to 1.25 x 10(-2) x MGIC) caused a further decrease in cross-linkage but thereafter a minimum value was reached. This remained unchanged even after growth of the organisms in much higher concentrations of the antibiotic up to 0.3 x minimum growth-inhibitory concentration. S. aureus MR-1 was able to grow normally for many generations under these conditions and reduction in cross-linkage of peptidoglycan was the only change detected in wall chemistry. Growth in the presence of methicillin (up to 0.3 x MGIC) (or other beta-lactam antibiotics) did not lead to an imbalance in the biosynthesis of peptidoglycan since no soluble polymers were secreted into the growth medium and nucleotide-linked precursors did not accumulate intracellularly. High concentrations of beta-lactam antibiotics (5 x MGIC) were bacteriostatic not bactericidal and this may be related to an apparent deficiency in the endogenous autolytic enzymes of strain MR-1. Studies of the penicillin-binding proteins after growth in the presence of methicillin suggest that one of these proteins remains resistant to very high concentrations of the antibiotic. We propose that this protein acts as the primary transpeptidase responsible for the incorporation of newly synthesised peptidoglycan into the growing wall.

Streptomyces K15 possesses a set of exocellular and cell-bound D-alanyl-D-alanine carboxypeptidases. Four of them have been isolated to the stage where each enzyme preparation contains on single penicillin-binding protein. The exocellular 54000-Mr enzyme is extremely sensitive to benzylpenicillin and performs low transpeptidase activity on the carbonyl-donor/amino-acceptor tetrapeptide ACLLys(Gly)-DAla-DAla. The exocellular 40 000-Mr enzyme and the two lysozyme-releasable 40 000-Mr and 38 000-Mr enzymes are moderately sensitive to benzylpenicillin and have a high propensity to catalyse dimer formation from the aforementioned tetrapeptide monomer.

The mechanisms by which bacteria adopt and maintain individual shapes remain enigmatic. Outstanding questions include why cells are a certain size, length, and width; why they are uniform or irregular; and why some branch while others do not. Previously, we showed that Escherichia coli mutants lacking multiple penicillin binding proteins (PBPs) display extensive morphological diversity. Because defective sites in these cells exhibit the structural and functional characteristics of improperly localized poles, we investigated the connection between cell division and shape. Here we show that under semipermissive conditions the temperature-sensitive FtsZ84 protein produces branched and aberrant cells at a high frequency in mutants lacking PBP 5, and this phenotype is exacerbated by the loss of additional peptidoglycan endopeptidases. Surprisingly, certain ftsZ84 strains lyse at the nonpermissive temperature instead of filamenting, and inhibition of wild-type FtsZ forces some mutants into tightly wound spirillum-like morphologies. The results demonstrate that significant aspects of bacterial shape are dictated by a previously unrecognized relationship between the septation machinery and ostensibly minor peptidoglycan-modifying enzymes and that under certain circumstances improper FtsZ function can destroy the structural integrity of the cell.

Penicillin-binding protein 2a (PBP2a) of Staphylococcus aureus is refractory to inhibition by available beta-lactam antibiotics, resulting in resistance to these antibiotics. The strains of S. aureus that have acquired the mecA gene for PBP2a are designated as methicillin-resistant S. aureus (MRSA). The mecA gene was cloned and expressed in Escherichia coli, and PBP2a was purified to homogeneity. The kinetic parameters for interactions of several beta-lactam antibiotics (penicillins, cephalosporins, and a carbapenem) and PBP2a were evaluated. The enzyme manifests resistance to covalent modification by beta-lactam antibiotics at the active site serine residue in two ways. First, the microscopic rate constant for acylation (k2) is attenuated by 3 to 4 orders of magnitude over the corresponding determinations for penicillin-sensitive penicillin-binding proteins. Second, the enzyme shows elevated dissociation constants (Kd) for the non-covalent pre-acylation complexes with the antibiotics, the formation of which ultimately would lead to enzyme acylation. The two factors working in concert effectively prevent enzyme acylation by the antibiotics in vivo, giving rise to drug resistance. Given the opportunity to form the acyl enzyme species in in vitro experiments, circular dichroism measurements revealed that the enzyme undergoes substantial conformational changes in the course of the process that would lead to enzyme acylation. The observed conformational changes are likely to be a hallmark for how this enzyme carries out its catalytic function in cross-linking the bacterial cell wall.

The enzymes involved in the biosynthesis of peptidoglycan are targets for the development of new antibiotics. The bifunctional high molecular weight (HMW) penicillin-binding proteins (PBPs), which contain both glycosyltransferase (GTase) and transpeptidase (TPase) activities, are particularly attractive targets because of their extracellular location. However, there is limited mechanistic or structural information about the GTase modules of these enzymes. In this paper, we describe the overexpression and characterization of the GTase module of Escherichia coli PBP1b, a paradigm of the HMW PBPs. We define the C-terminal boundary of the GTase module and show that the isolated module can be overexpressed at significantly higher levels than the full-length protein. The catalytic efficiency and other characteristics of the isolated module are comparable in most respects to the full-length enzyme. This work lays the groundwork for mechanistic and structural analysis of GTase modules.

Beta-lactamases and penicillin-binding proteins are bacterial enzymes involved in antibiotic resistance to beta-lactam antibiotics and biosynthetic assembly of cell wall, respectively. Members of these large families of enzymes all experience acylation by their respective substrates at an active site serine as the first step in their catalytic activities. A Ser-X-X-Lys sequence motif is seen in all these proteins, and crystal structures demonstrate that the side-chain functions of the serine and lysine are in contact with one another. Three independent methods were used in this report to address the question of the protonation state of this important lysine (Lys-73) in the TEM-1 beta-lactamase from Escherichia coli. These techniques included perturbation of the pK(a) of Lys-73 by the study of the gamma-thialysine-73 variant and the attendant kinetic analyses, investigation of the protonation state by titration of specifically labeled proteins by nuclear magnetic resonance, and by computational treatment using the thermodynamic integration method. All three methods indicated that the pK(a) of Lys-73 of this enzyme is attenuated to 8.0-8.5. It is argued herein that the unique ground-state ion pair of Glu-166 and Lys-73 of class A beta-lactamases has actually raised the pK(a) of the active site lysine to 8.0-8.5 from that of the parental penicillin-binding protein. Whereas we cannot rule out that Glu-166 might activate the active site water, which in turn promotes Ser-70 for the acylation event, such as proposed earlier, we would like to propose as a plausible alternative for the acylation step the possibility that the ion pair would reconfigure to the protonated Glu-166 and unprotonated Lys-73. As such, unprotonated Lys-73 could promote serine for acylation, a process that should be shared among all active-site serine beta-lactamases and penicillin-binding proteins.

ME1036, formerly CP5609, is a novel parenteral carbapenem with a 7-acylated imidazo[5,1- b ]thiazole-2-yl group directly attached to the carbapenem moiety of the C-2 position. The present study evaluated the in vitro activities of ME1036 against clinical isolates of gram-positive and gram-negative bacteria. ME1036 displayed broad activity against aerobic gram-positive and gram-negative bacteria. Unlike other marketed β-lactam antibiotics, ME1036 maintained excellent activity against multiple-drug-resistant gram-positive bacteria, such as methicillin-resistant staphylococci and penicillin-resistant Streptococcus pneumoniae (PRSP). The MICs of this compound at which 90% of isolates were inhibited were 2 μg/ml for methicillin-resistant Staphylococcus aureus (MRSA), 2 μg/ml for methicillin-resistant coagulase-negative staphylococci, and 0.031 μg/ml for PRSP. In time-kill studies with six strains of MRSA, ME1036 at four times the MIC caused a time-dependent decrease in the numbers of viable MRSA cells. The activity of ME1036 against MRSA is related to its high affinity for penicillin-binding protein 2a, for which the 50% inhibitory concentration of ME1036 was approximately 300-fold lower than that of imipenem. In conclusion, ME1036 demonstrated a broad antibacterial spectrum and high levels of activity in vitro against staphylococci, including β-lactam-resistant strains.

Objectives: To determine the epidemiological relationship between non-invasive penicillin nonsusceptible Streptococcus pneumoniae isolates collected in the Christchurch community between 1997 and 2001. Methods: One hundred and ninety-seven pneumococcal isolates were examined by macrorestriction profile analysis of SmaI-digested genomic DNA separated by PFGE and restriction fragment length polymorphism analysis of penicillin binding protein genes. Results: Four major clonal lineages were identified, the largest and most homogenous containing 95 (48.2%) of the isolates, the bulk of which (93.7%), had identical macrorestriction patterns. Members of this clonal group were multidrug-resistant and exhibited high resistance to third-generation cephalosporins, with MICs >8.0 mg/L not uncommon (23.1%). Two of the clonal groups, each containing 24 (12.2%) isolates, appeared indistinguishable from the globally widespread Spain 23F -1 and France 9V -3 strains, respectively. The fourth (12.7% of isolates) multidrug-resistant clone possessed intermediate penicillin susceptibility (MIC 0.12 mg/L). Conclusions: This study shows that several distinct penicillin-resistant pneumococcal clones are present in the Christchurch community, most of which appear to have been imported into New Zealand.

The origin of the substantial difference in deacylation rates for acyl-enzyme intermediates in penicillin-binding proteins (PBPs) and beta-lactamases has remained an unsolved puzzle whose solution is of great importance to understanding bacterial antibiotic resistance. In this work, accurate, large-scale mixed ab initio quantum mechanical/molecular mechanical (QM/MM) calculations have been used to study the hydrolysis of acyl-enzyme intermediates formed between cephalothin and the dd-peptidase of Streptomyces sp. R61, a PBP, and the Enterobacter cloacae P99 cephalosporinase, a class C beta-lactamase. Qualitative and, in the case of P99, quantitative agreement was achieved with experimental kinetics. The faster rate of deacylation in the beta-lactamase is attributed to a more favorable electrostatic environment around Tyr150 in P99 (as compared to that for Tyr159 in R61) which facilitates this residue's function as the general base. This is found to be in large part accomplished by the ability of P99 to covalently bind the ligand without concurrent elimination of hydrogen bonds to Tyr150, which proves not to be the case with Tyr159 in R61. This work provides an essential foundation for further work in this area, such as selecting mutations capable of converting the PBP into a beta-lactamase.

Extensive use of β-lactam antibiotics has led to the selection of pathogenic streptococci resistant to β-lactams due to modifications of the penicillin-binding proteins (PBPs). PBP2b from Streptococcus pneumoniae is a monofunctional (class B) high-molecular-weight PBP catalyzing the transpeptidation between adjacent stem peptides of peptidoglycan. The transpeptidase domain of PBP2b isolated from seven clinical resistant (CR) strains contains 7 to 44 amino acid changes over the sequence of PBP2b from the R6 β-lactam-sensitive strain. We show that the extracellular soluble domains of recombinant PBP2b proteins (PBP2b*) originating from these CR strains have an in vitro affinity for penicillin G that is reduced by up to 99% from that of the R6 strain. The Thr446Ala mutation is always observed in CR strains and is close to the key conserved motif (S 443 SN). The Thr446Ala mutation in R6 PBP2b* displays a 60% reduction in penicillin G affinity in vitro compared to that for the wild-type protein. A recombinant R6 strain expressing the R6 PBP2b Thr446Ala mutation is twofold less sensitive to piperacillin than the parental S. pneumoniae strain. Analysis of the Thr446Ala mutation in the context of the PBP2b CR sequences revealed that its influence depends upon the presence of other unidentified mutations.

Previous studies with beta-lactamase-negative, ampicillin-resistant (BLNAR) Haemophilus influenzae from Japan, France, and North America indicate that mutations in ftsI encoding PBP3 confer ampicillin MICs of 1 to 4 μg/ml. Several BLNAR strains with ampicillin MICs of 4 to 16 μg/ml recently isolated from North America were studied. Pulsed-field gel electrophoresis identified 12 unique BLNAR strains; sequencing of their ftsI transpeptidase domains identified 1 group I and 11 group II mutants, as designated previously (K. Ubukata, Y. Shibasaki, K. Yamamoto, N. Chiba, K. Hasegawa, Y. Takeuchi, K. Sunakawa, M. Inoue, and M. Konno, Antimicrob. Agents Chemother. 45: 1693-1699, 2001). Geometric mean ampicillin MICs for several clinical isolates were 8 to 10.56 μg/ml. Replacement of the ftsI gene in H. influenzae Rd with the intact ftsI from several clinical isolates resulted in integrants with typical BLNAR geometric mean ampicillin MICs of 1.7 to 2.2 μg/ml. Cloning and purification of His-tagged PBP3 from three clinical BLNAR strains showed significantly reduced Bocillin binding compared to that of PBP3 from strain Rd. Based on these data, changes in PBP3 alone could not account for the high ampicillin MICs observed for these BLNAR isolates. In an effort to determine the presence of additional mechanism(s) of ampicillin resistance, sequencing of the transpeptidase regions of pbp1a , - 1b , and - 2 was performed. While numerous changes were observed compared to the sequences from Rd, no consistent pattern correlating with high-level ampicillin resistance was apparent. Additional analysis of the resistant BLNAR strains revealed frame shift insertions in acrR for all four high-level, ampicillin-resistant isolates. acrR was intact for all eight low-level ampicillin-resistant and four ampicillin-susceptible strains tested. A knockout of acrB made in one clinical isolate (initial mean ampicillin MIC of 10.3 μg/ml) lowered the ampicillin MIC to 3.67 μg/ml, typical for BLNAR strains. These studies illustrate that BLNAR strains with high ampicillin MICs exist that have combined resistance mechanisms in PBP3 and in the AcrAB efflux pump.

The antibiotic susceptibilities, genotypes of penicillin (PEN)-binding protein genes ( pbp ), and serotype distributions of Streptococcus pneumoniae isolates from meningitis patients were investigated by a nationwide surveillance group in Japan between 1999 and 2002. We analyzed 146 isolates from children (≤17 years old) and 73 from adults (≥18 years old). Isolates with or without abnormal pbp1a , pbp2x , or pbp2b genes identified by PCR were classified into six genotype patterns and 90% MIC (MIC 90 ) values for PEN: (i) strains with three normal genes (17.2% of isolates; MIC 90 , 0.031 μg/ml); (ii) strains with abnormal pbp2x (22.1%, 0.063 μg/ml); (iii) strains with abnormal pbp2b (1.0%, 0.125 μg/ml); (iv) strains with abnormal pbp2x and pbp2b (7.4%, 0.25 μg/ml); (v) strains with abnormal pbp1a and pbp2x (12.7%, 0.25 μg/ml); and (vi) strains with three abnormal PBP genes (39.7%, 4 μg/ml), which are termed genotypic PEN-resistant S. pneumoniae (gPRSP). Panipenem, a carbapenem, showed an excellent MIC 90 (0.125 μg/ml) against gPRSP, followed by meropenem and vancomycin (0.5 μg/ml), cefotaxime and ceftriaxone (1 μg/ml), and ampicillin (4 μg/ml). Strains of gPRSP were significantly more prevalent in children (45.2%) than in adults (27.4%). The most frequent serotypes were 6B, 19F, 23F, 6A, and 14 in children and 23F, 22, 3, 10, 6B, and 19F in adults. Serotypes 6B, 6A, 19F, 23F, and 14 predominated among gPRSP. In children, 7- and 11-valent pneumococcal conjugate vaccines would cover 76.2 and 81.3% of isolates, respectively, although coverage would be lower in adults (43.9 and 56.0%, respectively). These findings suggest the need for early introduction of pneumococcal conjugate vaccines and continuous bacteriological surveillance for meningitis.

The aim of this study was to establish an identification method for the anginosus group within the genus Streptococcus by polymerase chain reaction (PCR). Using a primer pair based on the group-specific sequences of penicillin-binding protein 2B (pbp2b) gene, a 275-bp fragment was amplified from each species in the group but no size-matched products were obtained in other streptococci. Further identification in the species or subspecies level was possible by a multiplex PCR with primers for the 16S ribosomal RNA gene of Streptococcus anginosus, the hyaluronate lyase genes both of Streptococcus intermedius and Streptococcus constellatus subsp. constellatus, and the intermedilysin (ily) gene of S. intermedius. In the case ofStreptococcus constellatus subsp. pharyngis, the amplified fragment from the S. intermedius-type hyaluronate lyase gene was obtained, while that from the ily gene was not. These results also indicate that two different hyaluronate lyase genes are distributed among the anginosus group.

The importance of amino acid sequence differences in the C-terminal part and levels of mRNA expression of penicillin-binding protein 5 (PBP5) for ampicillin resistance in Enterococcus faecium was investigated. Seventeen isolates from Norwegian hospitalized patients (ampicillin MIC 0.064->256 mg/L) with different C-terminal pbp5 DNA sequences encoding 11 different amino acid sequences were analyzed with a 14C-radiolabeled penicillin- binding assay to PBP5 and with real-time PCR quantification of pbp5 mRNA expression. Using multiple logistic regression analysis the amino acid substitution Met 485 was linked to ampicillin MIC and levels of 14C-radiolabeled penicillin bound to PBP5; however, there were isolates with identical PBP5 alleles and different ampicillin MICs. There was no relation between the quantity of pbp5 mRNA transcripts and ampicillin resistance. The results cannot explain ampicillin resistance in Norwegian clinical strains of E. faecium and indicate that other factors besides the properties of the C-terminal part of PBP5 are most likely involved.

Several non-β-lactam compounds were active against various gram-positive and gram-negative bacterial strains. The MICs of arylalkylidene rhodanines and arylalkylidene iminothiazolidin-4-ones were lower than those of ampicillin and cefotaxime for methicillin-resistant Staphylococcus aureus MI339 and vancomycin-resistant Enterococcus faecium EF12. Several compounds were found to inhibit the cell wall synthesis of S. aureus and the last two steps of peptidoglycan biosynthesis catalyzed by ether-treated cells of Escherichia coli or cell wall membrane preparations of Bacillus megaterium . The effects of the arylalkylidene rhodanines and arylalkylidene iminothiazolidin-4-one derivatives on E. coli PBP 3 and PBP 5, Streptococcus pneumoniae PBP 2xS (PBP 2x from a penicillin-sensitive strain) and PBP 2xR (PBP 2x from a penicillin-resistant strain), low-affinity PBP 2a of S. aureus , and the Actinomadura sp. strain R39 and Streptomyces sp. strain R61 dd -peptidases were studied. Some of the compounds exhibited inhibitory activities in the 10 to 100 μM concentration range. The inhibition of PBP 2xS by several of them appeared to be noncompetitive. The dissociation constant for the best inhibitor ( Ki = 10 μM) was not influenced by the presence of the substrate.

Peptidoglycan polymerization complexes contain multimodular penicillin-binding proteins (PBP) of classes A and B that associate a conserved C-terminal transpeptidase module to an N-terminal glycosyltransferase or morphogenesis module, respectively. In Enterococcus faecalis , class B PBP5 mediates intrinsic resistance to the cephalosporin class of β-lactam antibiotics, such as ceftriaxone. To identify the glycosyltransferase partner(s) of PBP5, combinations of deletions were introduced in all three class A PBP genes of E. faecalis JH2-2 ( ponA , pbpF , and pbpZ ). Among mutants with single or double deletions, only JH2-2 Δ ponA Δ pbpF was susceptible to ceftriaxone. Ceftriaxone resistance was restored by heterologous expression of pbpF from Enterococcus faecium but not by mgt encoding the monofunctional glycosyltransferase of Staphylococcus aureus . Thus, PBP5 partners essential for peptidoglycan polymerization in the presence of β-lactams formed a subset of the class A PBPs of E. faecalis , and heterospecific complementation was observed with an ortholog from E. faecium . Site-directed mutagenesis of pbpF confirmed that the catalytic serine residue of the transpeptidase module was not required for resistance. None of the three class A PBP genes was essential for viability, although deletion of the three genes led to an increase in the generation time and to a decrease in peptidoglycan cross-linking. As the E. faecalis chromosome does not contain any additional glycosyltransferase-related genes, these observations indicate that glycan chain polymerization in the triple mutant is performed by a novel type of glycosyltransferase. The latter enzyme was not inhibited by moenomycin, since deletion of the three class A PBP genes led to high-level resistance to this glycosyltransferase inhibitor.

This study examines the role of the penicillin-binding proteins (PBPs) of Bacteroides fragilis in the mechanism of resistance to different beta-lactam antibiotics. Six of the eight strains used were beta-lactamase-positive by the nitrocefin assay. These strains displayed reduced susceptibility to imipenem (MIC, 2-16 mg l(-1)) and some of them were resistant to the actions of ampicillin, cefuroxime, cephalexin, cefoxitin and piperacillin. When studying specific enzymic activity, the capacity to degrade cefuroxime was only detected in strains AK-4, R212 and 0423 and the capacity to degrade cephalexin was only detected in strains R212 and 2013E; no specific activity was detected on imipenem. Metallo-beta-lactamase activity was only detected in strains AK-2 and 119, despite the fact that the cfiA gene was identified in four strains (AK-2, 2013E, 119 and 7160). The cepA gene was detected in six of the eight strains studied. Three high-molecular-mass PBPs were detected in all strains; however, in some cases, PBP2Bfr and/or PBP3Bfr appeared as a faint band. PBP4Bfr and PBP5Bfr were detected in six strains. PBP6Bfr only was detected in B. fragilis strains AK-2, 0423, 119 and 7160. By analysis of the sequence of B. fragilis chromosomal DNA and comparison with genes that are known to encode PBPs in Escherichia coli, six genes that encode PBP-like proteins were detected in the former organism. The gene that encodes the PBP2 orthologue of E. coli (pbpABfr, PBP3Bfr) was sequenced in six of the eight strains and its implications for resistance were examined. Differences in the PBP3Bfr amino acid sequences of strains AK-2 and 119 and their production of beta-lactamases indicate that these differences are not involved in the mechanism of resistance to imipenem and/or cephalexin.

Bacillus subtilis contains seven extracytoplasmic-function σ factors that activate partially overlapping regulons. We here identify four additional members of the σ X regulon, pbpX (penicillin-binding protein), ywnJ , the dlt operon ( d -alanylation of teichoic acids), and the pss ybfM psd operon (phosphatidylethanolamine biosynthesis). Modification of teichoic acids by esterification with d -alanine and incorporation of phosphatidylethanolamine into the cell membrane have a common consequence: in both cases positively charged amino groups are introduced into the cell envelope. The resulting reduction in the net negative charge of the cell envelope has been previously implicated as a resistance mechanism specific for cationic antimicrobial peptides. Consistent with this notion, we find that both sigX and dltA mutants are more sensitive to nisin than wild-type cells. We conclude that activation of the σ X regulon serves to alter cell surface properties to provide protection against antimicrobial peptides.

The ubiquitous occurrence of β-lactamases in bacteria and their association with clinical resistance to β-lactams have allowed strong interest in these enzymes to be sustained (3, 26). β-Lactamases are periplasmic in gram-negative bacteria, while they are exocellular in gram-positive bacteria. They are conveniently divided into four classes (classes A, B, C, and D) on the basis of amino acid sequence homologies (Ambler classification) (1, 15). Of these classes, classes A and C are of the greatest clinical significance. β-Lactamase inhibitors, such as clavulanic acid and the penicillanic acid sulfones, mostly target class A enzymes; and their use in combination with older compounds has restored the broad-spectrum activities of older compounds, such as amoxicillin (18, 22, 25, 26). Inhibitors that target class C and class B β-lactamases have yet to reach clinical development. Exploiting common β-lactamases to generate novel antibacterials is a strategy originally used by O'Callaghan et al. (24) and subsequently by Mobashery and Johnston (20) and other investigators (6, 10, 16). It has been part of NewBiotics' general enzyme-catalyzed therapeutic activation (ECTA) prodrug approach that harnesses unique enzymes in bacteria to achieve selective release of cytotoxic agents from substrate-like molecules (16; M. V. Sergeeva, G. H. Khambatta, B. E. Cathers, R. S. Castillo, V. R. Doppalapudi, H. H. Bendall, A. R. Bueno, J. Y. Lee, Q. Li, and N. H. Georgopapadakou, Abstr. 102nd Gen. Meet. Am. Soc. Microbiol., abstr. A-113, 2002). These antibacterials developed by the ECTA approach are designed so that neither they nor their products inactivate the enzymes that activate them. We have synthesized a variety of cephalosporins with different C-7 side chains that affect enzymatic hydrolysis, cephalosporins with different C-3 linkers that affect chemical stability, and different toxophores that are released upon opening of the β-lactam ring. Two of these compounds, NB2001 and NB2030, contain cephalothin (thienyl) and cefazolin (tetrazole) side chains at position C-7, respectively and the enoyl reductase inhibitor triclosan at the C-3 position of the cephem nucleus (Fig. ​(Fig.1).1). Triclosan is designed to act as a toxophore that is released upon β-lactam hydrolysis in β-lactamase-positive bacteria. Here we describe studies aimed at elucidating the mechanisms of action of NB2001 and NB2030 in Escherichia coli and Staphylococcus aureus. In E. coli, the overall mechanisms of action of these compounds were deduced from the cell morphologies observed after treatment of the cells with the compounds. Their levels of transport into bacterial cells, their levels of penicillin-binding protein (PBP) binding, their activities as β-lactamase substrates, and the activity of the bound triclosan moiety relative to that of free triclosan against the enoyl reductase target were also determined. FIG. 1. Chemical structures of NB2001 and NB2030.

Resistance genes were determinded for 81 strains of Streptococcus pneumoniae isolated from Ehime University hospital, during 2002 and 2003 by various clinical material. In penicillin-binding proteins of mutation, there were 74 strains; pbp2x mutation 23 strains (28.4%), pbp2b mutation one strain (1.2%), pbp1a + pbp2x mutations 5 strains (6.2%), pbp2x + pbp2b mutations 18 strains (22.2%) and all mutations 27 strains (33.3%). As for the result of macrolide resistance genes, there were 67 strains; mefA gene 20 strains (24.7%), ermB gene 46 strains (56.8%) and both gene one strain (1.2%). In the analysis of gyrA gene and parC gene, 3 strains (3.7%) had both gene mutations, and 26 strains (32.1%) had only parC gene mutation. There was more of an increase than before in isolates, two or more mutation strains with PBPs gene, ermB gene holding strains and the levofloxacin resistance strain. These results suggest that the gyrA gene or parC gene mutation strains hold PBPs gene mutation and macrolide resistance genes in a high rate, and there will be more drug resistance in the future.

Corilagin and tellimagrandin I are polyphenols isolated from the extract of Arctostaphylos uvaursi and Rosa canina L. (rose red), respectively. We have reported that corilagin and tellimagrandin I remarkably reduced the minimum inhibitory concentration (MIC) of beta-lactams in methicillin-resistant Staphylococcus aureus(MRSA). In this study, we investigated the effect of corilagin and tellimagrandin I on the penicillin binding protein 2 '(2a) (PBP2 '(PBP2a)) which mainly confers the resistance to beta-lactam antibiotics in MRSA. These compounds when added to the culture medium were found to decrease production of the PBP2 '(PBP2a) slightly. Using BOCILLIN FL, a fluorescent-labeled benzyl penicillin, we found that PBP2 '(PBP2a) in MRSA cells that were grown in medium containing corilagin or tellimagrandin I almost completely lost the ability to bind BOCILLIN FL. The binding activity of PBP2 and PBP3 were also reduced to some extent by these compounds. These results indicate that inactivation of PBPs, especially of PBP2 '(PBP2a), by corilagin or tellimagrandin I is the major reason for the remarkable reduction in the resistance level of beta-lactams in MRSA. Corilagin or tellimagrandin I suppressed the activity of beta-lactamase to some extent.

Production of low-affinity forms of penicillin-binding proteins (PBPs), although essential, is not sufficient to protect pneumococci against the inhibitory action of penicillin. Resistance also requires the newly identified protein MurM which, together with MurN, is involved with the synthesis of short peptide branches in the pneumococcal cell wall. Cells in which murM was inactivated produced cell walls without branches and also completely lost penicillin resistance. To understand these surprising observations a 3D-model of MurM was constructed, which helped to put into structural context several of the biochemical and genetic observations made about this protein.

Penicillin-binding proteins (PBPs) are responsible for the final stages of bacterial cell wall assembly. These enzymes are targets of beta-lactam antibiotics. Two of the PBP activities include dd-transpeptidase and DD-carboxypeptidase activities, which carry out the cross-linking of the cell wall and trimming of the peptidoglycan, the major constituent of the cell wall, by an amino acid, respectively. The activity of the latter enzyme moderates the degree of cross-linking of the cell wall, which is carried out by the former. Both these enzymes go through an acyl-enzyme species in the course of their catalytic events. Compound 6, a cephalosporin derivative incorporated with structural features of the peptidoglycan was conceived as an inhibitor specific for DD-transpeptidases. On acylation of the active sites of dd-transpeptidases, the molecule would organize itself in the two active site subsites such that it mimics the two sequestered strands of the bacterial peptidoglycan en route to their cross-linking. Hence, compound 6 is the first inhibitor conceived and designed specifically for inhibition of DD-transpeptidases. The compound was synthesized in 13 steps and was tested with recombinant PBP1b and PBP5 of Escherichia coli, a dd-transpeptidase and a dd-carboxypeptidase, respectively. Compound 6 was a time-dependent and irreversible inhibitor of PBP1b. On the other hand, compound 6 did not interact with PBP5, neither as an inhibitor (reversible or irreversible) nor as a substrate.

Summary We have developed several new fluorescent staining procedures that enabled us to study the synthesis of cell wall material in the spherical Gram-positive bacterium Staphylococcus aureus. The results obtained support previous proposals that these cells synthesize new wall material specifically at cell division sites, in the form of a flat circular plate that is subsequently cleaved and remodelled to produce the new hemispherical poles of the daughter cells. We have shown that formation of the septal peptidoglycan is dependent on the key cell division protein FtsZ, which recruits penicillin-binding protein (PBP) 2. Unexpectedly, in FtsZ-depleted cells, the cell wall synthetic machinery becomes dispersed and new wall material is made in dispersed patches over the entire surface of the cells, which increase in volume by up to eightfold before lysing. The results have implications for understanding the nature of S. aureus morphogenesis and for inhibitors of cell division proteins as drug targets.

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.

Objective: To determine the prevalence of penicillin resistance and molecular characteristics of pneumococcal isolates at the University of Malaya Medical Center. Methods: From March 1999 to July 2000, 100 clinical isolates of Streptococcus pneumoniae were obtained from 93 patients of various ages and from various body sites. The minimum inhibitory concentrations (MICs) for penicillin and ceftriaxone were determined by E test, and results were interpreted according to guidelines recommended by the National Committee for Clinical Laboratory Standards (NCCLS). Fifty isolates were further serotyped, and analyzed by pulsed-field gel electrophoresis (PFGE) and polymerase chain reaction/restriction fragment length polymorphism (PCR-RFLP) of the penicillin-binding protein ( pbp ) 2b and 2x genes. Results: The majority of the isolates were from respiratory sites. Thirty-one isolates showed decreased susceptibility to penicillin (PRSP), and many of these also showed decreased susceptibility to ceftriaxone. Twelve serogroup/types (SGTs) were present, with 19F being the most common. PFGE analysis identified two dominant profiles, consisting mainly of PRSPs that had common serotypes (19F) and pbp gene patterns within their respective groups, although PCR-RFLP analysis showed different patterns of pbp genes among the PRSPs as compared to penicillin-susceptible strains, which had a uniform pattern. Conclusion: PRSPs were genetically related as shown by PFGE and serotype. The consistency of pbp gene patterns, observed among many of the PRSPs within their respective PFGE profiles, supported their relatedness as established by PFGE.

The peptidoglycan cell wall determines the shape and structural integrity of a bacterial cell. Class B penicillin-binding proteins (PBPs) carry a transpeptidase activity that cross-links peptidoglycan strands via their peptide side chains, and some of these proteins are directly involved in cell shape determination. No Bacillus subtilis PBP with a clear role in rod shape maintenance has been identified. However, previous studies showed that during outgrowth of pbpA mutant spores, the cells grew in an ovoid shape for several hours before they recovered and took on a normal rod shape. It was postulated that another PBP, expressed later during outgrowth, was able to compensate for the lack of the pbpA product, PBP2a, and to guide the formation of a rod shape. The B. subtilis pbpH ( ykuA ) gene product is predicted to be a class B PBP with greatest sequence similarity to PBP2a. We found that a pbpH-lacZ fusion was expressed at very low levels in early log phase and increased in late log phase. A pbpH null mutant was indistinguishable from the wild-type, but a pbpA pbpH double mutant was nonviable. When pbpH was placed under the control of an inducible promoter in a pbpA mutant, viability was dependent on pbpH expression. Growth of this strain in the absence of inducer resulted in conversion of the cells from rods to ovoid/round shapes and lysis. We conclude that PBP2a and PbpH play redundant roles in formation of a rod-shaped peptidoglycan cell wall.

author, Penicillin binding protein (pbp) gene alterations of 328 clinical isolates of Streptococcus pneumoniae were examined for a correlation with their antibiotic-resistance. The frequency of penicillin G (PEN-G) resistance was determined to clarify susceptibility to several antibiotics, namely PEN-G, ampicillin, sulbactam/ampicillin, cefozopram, panipenem (PAPM), clarithromycin (CLR), azithromycin (AZM) and levofloxacin (LVX). Oligonucleotide primers for three pbp genes (pbp1a, pbp2x and pbp2b) were used to detect mutations in pbp. Of the strains, 25.9% were classified as Pen-G^s, 68.0% as Pen-G^ir and 6.1% as Pen-G^r. The polymerase chain reaction product for wild-type pbp1a was found in 185 isolates, that for wild-type pbp2x was found in 66 isolates and that for wild-type pbp2b was found in 213 isolates. None of these three genes was detectable in 100 isolates while all of them were detected in 64 isolates (1a^w/2x^w/2b^w). Of those 64 isolates with 1a^w/2x^w/2b^w, the minimum inhibitory concentration (MIC) of PEN-G was ≤0.06 mg/l for 54 isolates and 0.12 mg/l for 10 isolates. Of the 272 strains for which the MIC of PAPM was ≤0.03 mg/l, there were 85 Pen-G^s, 184 Pen-G^ir and three Pen-G^r isolates. Three strains for which the MIC of LVX was ≥4.0 mg/l included one Pen-G^s and two Pen-G^ir isolates. The MICs of CLR correlated significantly with those of AZM. The MIC of CLR was ≥1 mg/l for 216 isolates, and the MIC of AZM was ≥1 mg/l for 244 of them. These data suggested that PAPM may be effective against S. pneumoniae infection, although acquisition of resistance should be considered. LVX also seemed to be effective against S. pneumoniae.

The bacterial peptidoglycan consists of glycan chains of repeating β-1,4-linked N -acetylglucosaminyl- N -acetylmuramyl units cross-linked through short peptide chains. The polymerization of the glycans, or glycosyltransfer (GT), and transpeptidation (TP) are catalyzed by bifunctional penicillin-binding proteins (PBPs). The β-lactam antibiotics inhibit the TP reaction, but their widespread use led to the development of drug resistance in pathogenic bacteria. In this context, the GT catalytic domain represents a potential target in the antibacterial fight. In this work, the in vitro polymerization of glycan chains by the extracellular region of recombinant Streptococcus pneumoniae PBP2a, namely, PBP2a* (the asterisk indicates the soluble form of the protein) is presented. Dansylated lipid II was used as the substrate, and the kinetic parameters K m and k cat / K m were measured at 40.6 μM (± 15.5) and 1 × 10 −3 M −1 s −1 , respectively. The GT reaction catalyzed by PBP2a* was inhibited by moenomycin and vancomycin. Furthermore, the sequence between Lys 78 and Ser 156 is required for enzymatic activity, whereas it is dispensable for lipid II binding. In addition, we confirmed that this region of the protein is also involved in membrane interaction, independently of the transmembrane anchor. The characterization of the catalytically active GT domain of S . pneumoniae PBP2a may contribute to the development of new inhibitors, which are urgently needed to renew the antibiotic arsenal.

Multidrug-resistant Acinetobacter baumannii has emerged as a serious nosocomial pathogen in certain areas. In Brooklyn, New York, citywide surveillance revealed that approximately 2 of every 3 isolates were resistant to carbapenem antibiotics. Genetic fingerprinting revealed that 2 strains accounted for 82% of these resistant isolates. Compared with carbapenem-susceptible isolates, carbapenem-resistant isolates had reduced expression of 47-, 44-, and 37-kDa outer-membrane proteins. No specific carbapenemase was found; however, carbapenem-resistant isolates expressed greater levels of a class C cephalosporinase. Although expression of penicillin-binding proteins varied among strains, no consistent pattern appeared to account for carbapenem resistance. An efflux pump, present in several strains, did not appear to contribute to carbapenem resistance. Clonal spread of carbapenem-resistant A. baumannii has occurred in hospitals in Brooklyn. The preliminary findings for a small number of strains suggest that diminished production of outer-membrane porins, together with increased expression of a class C cephalosporinase, appear to be important factors leading to carbapenem resistance in this region.

Streptomyces cacaoi beta-lactamase genes are controlled by two regulators named blaA and blaB. Whereas BlaA has been identified as a LysR-type activator, the function of BlaB is still unknown. Its primary structure is similar to that of the serine penicillin-recognizing enzymes (PREs). Indeed, the SXXK and KTG motifs are perfectly conserved in BlaB, whereas the common SXN element found in PREs is replaced by a SDG motif. Site-directed mutations were introduced in these motifs and they all disturb beta-lactamase regulation. A water-soluble form of BlaB was also overexpressed in the Streptomyces lividans TK24 cytoplasm and purified. To elucidate the activity of BlaB, several compounds recognized by PREs were tested. BlaB could be acylated by some of them, and it can therefore be considered as a penicillin-binding protein. BlaB is devoid of beta-lactamase, D-aminopeptidase, DD-carboxypeptidase or thiolesterase activity.

The interaction of a moenomycin derivative with the enzyme penicillin binding protein 1b (PBP 1b) has been studied by means of STD NMR. The results obtained initiated the synthesis of a number of moenomycin derivatives modified in unit A including a moenomycin-ampicillin conjugate and determination of their antibiotic activities. A protocol is described that allows studying the interaction of moenomycin analogues with PBP 1b by fluorescence correlation spectroscopy.

Context.—A rapid, real-time, duplex, fluorescent molecular beacon probe–based polymerase chain reaction (PCR) assay was recently developed for the detection of methicillin-resistant Staphylococcus aureus. Objective.—To describe the development and validation of this unique assay. Design.—Prospective laboratory analysis. Setting.—Urban health region/centralized diagnostic microbiology laboratory. Bacterial Strains.—One hundred eighty-one previously characterized clinical and American Type Culture Collection isolates, including 50 strains each of methicillin-resistant and methicillin-sensitive S aureus, plus 50 strains of coagulase-negative staphylococci and 31 nonstaphylococcal isolates to ensure assay specificity. Intervention.—Assays were performed on purified genomic DNA extracted from growing bacterial colonies. Two sets of oligonucleotide primers were used to specifically amplify the mecA and nuc genes, followed by detection of amplicons using fluorophore-labeled molecular beacon probes. Assays were performed on the Mx4000 Multiplex Quantitative PCR System (Stratagene Inc, La Jolla, Calif). Main Outcome Measures.—(1) Assay sensitivity and specificity, and (2) analytical sensitivity. Results.—The assay demonstrated 100% sensitivity and 100% specificity, and accurately characterized isolates as methicillin-resistant S aureus, methicillin-sensitive S aureus, or methicillin-resistant coagulase-negative staphylococci, with test results available in 2.5 hours. The analytical sensitivity of the assay was determined to be between 6 and 60 genomic equivalents. Conclusions.—This assay is rapid, accurate, easy to perform, and is compatible with other real-time PCR instruments, making it a suitable alternative to conventional PCR methodologies.

The genetic relationship of 81 ampicillin-resistant and 21 ampicillin-susceptible Enterococcus faecium isolates from clinical infections and rectal screening in hospitalized patients in Norway was studied by pulsed-field gel electrophoresis (PFGE) and amplified fragment length polymorphism (AFLP). PFGE showed 55 different banding patterns, and 65 of the isolates could be grouped into one large group. With AFLP, 46 patterns were discerned, and 74 isolates clustered in one group. In general, the isolates had a higher degree of similarity than with PFGE. The purK gene, which is one of the targets of the E. faecium multilocus sequence typing scheme, was sequenced. Eleven different purK alleles could be discerned, with the majority of isolates ( n = 80) harboring allele 1. With only two exceptions, all strains carrying purK-1 clustered in the same PFGE and AFLP groups, indicating a good correlation between PFGE type, AFLP type, and purK allele. Genetic polymorphism of a 571-bp PCR fragment of the C-terminal domain of the penicillin-binding protein 5 gene ( pbp5 ) was determined, and sequence differences were associated with the level of ampicillin resistance. This study indicates that the majority of ampicillin-resistant E. faecium strains in Norway belong to a distinct genetic lineage of closely related genotypes. Rectal and clinical isolates were generally indistinguishable, and differences in clonal distribution and allele polymorphism were found mainly between ampicillin-resistant and -susceptible isolates.

Crystalline 1 (TAK-599) is a novel N-phosphono prodrug of anti-methicillin-resistant Staphylococcus aureus (MRSA) cephalosporin 2a (T-91825) that has high affinity for penicillin-binding protein (PBP) 2' (IC(50); 0.90 microg/mL) and shows potent in vitro anti-MRSA activity (MIC against MRSA N133; 1.56 microg/mL), comparable to that of vancomycin (1.56 microg/mL). Although 2a had insufficient water solubility (2.3 mg/mL) for parenteral administration, 1 showed excellent water solubility (>100 mg/mL, pH 7) as well as good chemical stability in the solid state and solution. In pharmacokinetic studies, when 1 was administered intravenously to rats and monkeys, it was rapidly converted into 2a in the blood. These results show that 1 (TAK-599) is a highly promising parenteral cephalosporin targeted for MRSA infection.

Staphylococci, a leading cause of infections worldwide, have devised two mechanisms for resistance to beta-lactam antibiotics. One is production of beta-lactamases, hydrolytic resistance enzymes, and the other is the expression of penicillin-binding protein 2a (PBP 2a), which is not susceptible to inhibition by beta-lactam antibiotics. The beta-lactam sensor-transducer (BlaR), an integral membrane protein, binds beta-lactam antibiotics on the cell surface and transduces the information to the cytoplasm, where gene expression is derepressed for both beta-lactamase and penicillin-binding protein 2a. The gene for the sensor domain of the sensor-transducer protein (BlaR(S)) of Staphylococcus aureus was cloned, and the protein was purified to homogeneity. It is shown that beta-lactam antibiotics covalently modify the BlaR(S) protein. The protein was shown to contain the unusual carboxylated lysine that activates the active site serine residue for acylation by the beta-lactam antibiotics. The details of the kinetics of interactions of the BlaR(S) protein with a series of beta-lactam antibiotics were investigated. The protein undergoes acylation by beta-lactam antibiotics with microscopic rate constants (k(2)) of 1-26 s(-1), yet the deacylation process was essentially irreversible within one cell cycle. The protein undergoes a significant conformational change on binding with beta-lactam antibiotics, a process that commences at the preacylation complex and reaches its full effect after protein acylation has been accomplished. These conformational changes are likely to be central to the signal transduction events when the organism is exposed to the beta-lactam antibiotic.

Constantly expressed genes are used as internal controls in relative quantification studies. Suitable internal controls for such studies have not yet been defined for Pseudomonas aeruginosa. In this study, the genes ampC, fabD, proC, pbp-2, rpoD and rpoS of P. aeruginosa were compared in terms of expression stability by real-time quantitative RT-PCR. A total of 23 strains with diverse resistance phenotypes were studied. Stability of expression among the housekeeping genes was assessed on the basis of correlation coefficients, with the best-correlated pair accepted as being the most stable one. Eventually, proC and rpoD formed the most stable pair (r = 0.958; P < 0.001). Next, in four ciprofloxacin-selected nfxC-like mutants, levels of oprD, oprM and oprN mRNA were compared with those of their wild-type counterparts. The comparison was made after correcting the raw values by the geometric mean of the internal control genes proC and rpoD. The level of oprN mRNA was significantly up-regulated, while the oprD gene was down-regulated (although this difference was statistically insignificant), in the mutants. This expression pattern was consistent with that of the expected expression profile of nfxC-type mutants; this experiment therefore lends further support to the use of proC and rpoD genes simultaneously as internal controls for such studies.

Bacterial transglycosylases are enzymes that couple the disaccharide subunits of peptidoglycan to form long carbohydrate chains. These enzymes are the target of the pentasaccharide antibiotic moenomycin as well as the proposed target of certain glycopeptides that overcome vancomycin resistance. Because bacterial transglycosylases are difficult enzymes to study, it has not previously been possible to evaluate how moenomycin inhibits them or to determine whether glycopeptide analogues directly target them. We have identified transglycosylase assay conditions that enable kinetic analysis of inhibitors and have examined the inhibition of Escherichia coli penicillin-binding protein 1b (PBP1b) by moenomycin as well as by various glycopeptides. We report that chlorobiphenyl vancomycin analogues that are incapable of binding substrates nevertheless inhibit E. coli PBP1b, which shows that these compounds interact directly with the enzyme. These findings support the hypothesis that chlorobiphenyl vancomycin derivatives overcome vanA resistance by targeting bacterial transglycosylases. We have also found that moenomycin is not competitive with respect to the lipid II substrate of PBP1b, as has long been believed. With the development of suitable methods to evaluate bacterial transglycosylases, it is now possible to probe the mechanism of action of some potentially very important antibiotics.

This review summarizes some of the published attempts to incorporate protein and NMR structures in the design of new antibiotics that specifically target Cell Wall biosynthesis. Most of the steps involved in peptidglycan synthesis have been investigated as potential strategies against cell wall inhibition. Structural information has been most useful in the design of molecules in the Mur enzyme pathway, penicillin binding proteins and lactamases, as well as proteins that are part of the final steps of transglycosylation - in particular, d-Ala-d-Ala ligase. Several unique issues exist in the design of effective antibacterials, such as the significant differences in protein structure between organisms, such as the case of MurB in which a large amino acid loop that occupies the active site of the E. Coli is gone in the Staph aureus enzyme. Additionally, bacterial resistance is an important issue, and in some cases, structural information can be used to understand the source of this resistance. For example, mutations within the d-Ala-d-Ala ligases lead to the inability of Vancomycin antibiotics to bind.

Methicillin-resistant Staphylococcus aureus (MRSA) is still one of major problems of drug-resistant microorganisms and healthcare-acquired infections. Methicillin-resistant Staphylococcus aureus is highly prevalent in patients in neonatal intensive care units (NICU) in Japan. The most predominant MRSA in NICU is multidrug resistant and produces superantigenic exotoxin, toxic shock syndrome toxin−1 (TSST−1) and staphylococcal enterotoxin C (SEC). These predominant MRSA strains belong to coagulase type II, SCCmec type II, mecA-Tn554 polymorph type I-A and show closely related pulse field gel electrophoresis types. The dissemination of MRSA is wide, and there is a pandemic distribution of a single MRSA clone in the NICU of Japan. Since 1992, the nationwide spread of this clone has also led to the development of a new neonatal disease known as neonatal toxic shock-like exanthematous disease (NTED), which is caused by overactivation of vβ2+ T cells induced by TSST−1. The spread of MRSA in NICU in Japan has been attributed to overcrowding, high rates of extremely low birthweight babies, understaffing, low control measures of infection and overuse of antibiotics. The environment of NICU and infection control intervention should be improved and a new strategy for control like vaccination or probiotics is required.

The transpeptidase encoding region of the penA gene was sequenced in 44 meningococcal strains (41 serogroup C [23 characterized as serotype 2b and 18 as serotype 2a] and 3 serogroup B [B:2b:P1.2,5]). All strains were characterized by multilocus sequence typing and were determined to be susceptible or intermediate resistant to penicillin (Pen(s) or Pen(i), respectively). A high degree of homology was found among the penA alleles identified in the Pen(s) strains. All the Pen(i) C:2b strains, which belonged to 2 different clonal complexes, showed the same penA gene allele. This fact suggests that 1 of the clonal complexes acquired that allele, spreading it to the other by horizontal transfer. The same allele also was found in the B:2b strains studied, indicating that 1 of the Pen(i) C:2b strains underwent a capsular switching event. A different mosaic penA allele was identified in the Pen(i) C:2a strains, which belonged to the ET37 cluster.

SUMMARY Work on two diverse rod-shaped bacteria, Escherichia coli and Bacillus subtilis, has defined a set of about 10 conserved proteins that are important for cell division in a wide range of eubacteria. These proteins are directed to the division site by the combination of two negative regulatory systems. Nucleoid occlusion is a poorly understood mechanism whereby the nucleoid prevents division in the cylindrical part of the cell, until chromosome segregation has occurred near midcell. The Min proteins prevent division in the nucleoid-free spaces near the cell poles in a manner that is beginning to be understood in cytological and biochemical terms. The hierarchy whereby the essential division proteins assemble at the midcell division site has been worked out for both E. coli and B. subtilis. They can be divided into essentially three classes depending on their position in the hierarchy and, to a certain extent, their subcellular localization. FtsZ is a cytosolic tubulin-like protein that polymerizes into an oligomeric structure that forms the initial ring at midcell. FtsA is another cytosolic protein that is related to actin, but its precise function is unclear. The cytoplasmic proteins are linked to the membrane by putative membrane anchor proteins, such as ZipA of E. coli and possibly EzrA of B. subtilis, which have a single membrane span but a cytoplasmic C-terminal domain. The remaining proteins are either integral membrane proteins or transmembrane proteins with their major domains outside the cell. The functions of most of these proteins are unclear with the exception of at least one penicillin-binding protein, which catalyzes a key step in cell wall synthesis in the division septum.

The chemical diversity of peptide and protein libraries generated from biological display systems is typically confined to the 20 naturally occurring amino acids. Here, we have developed a general strategy to introduce non-natural side chains into mRNA-display libraries via specific chemical derivatization. We constructed a mRNA-display library containing 3 × 1012 different peptides bearing a pendant penicillin moiety in a fixed position. In vitro selection using this hybrid peptide-drug library resulted in novel inhibitors of the Staphylococcus aureus penicillin binding protein 2a (PBP2a). This strategy resulted in a penicillin-peptide conjugate that has at least 100-fold higher activity than the parent penicillin itself. Our approach provides a convenient way to enhance the efficacy of known drugs and facilitates the discovery of powerful new hybrid ligands with functionalities beyond those provided by the 20 naturally occurring residues.