The glycosyltransferase domain of penicillin-binding protein 2a from Streptococcus pneumoniae catalyzes the polymerization of murein glycan chains.

The bacterial peptidoglycan consists of glycan chains of repeating beta-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 beta-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 micro M (+/- 15.5) and 1 x 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.

Distribution of mecA among methicillin-resistant clinical staphylococcal strains isolated at hospitals in Naples, Italy.

Two hundred and twenty strains of Staphylococcus isolated in Naples, Italy, were surveyed for the distribution of the mecA, the structural gene for penicillin-binding protein 2a, which is the genetic determinant for methicillin-resistance in staphylococci. Screening by a cloned mecA, revealed that of 220 strains, 43 were methicillin-resistant (19.5%) and 177 were methicillin-susceptible (80.5%). Among the 43 resistant strains 23 (53.5%) carried mecA in their genome and 20 (46.5%) did not carry mecA, in spite of their resistance to methicillin. Every group was submitted to the AP-PCR profiling. A quantitative analysis of the patterns divided strains into four different clusters for methicillin-resistant mecA-negative and two different clusters for methicillin-resistant mecA-positive with primer 1, while no clusters were noted with primer 7. We conclude that these clinical isolates from our area, were not found to belong to a single clone, although the predominance of four methicillin-resistant mecA-negative genotypes were noted.

Purification and characterization of VanXY(C), a D,D-dipeptidase/D,D-carboxypeptidase in vancomycin-resistant Enterococcus gallinarum BM4174.

VanXY(C), a bifunctional enzyme from VanC-phenotype Enterococcus gallinarum BM4174 that catalyses D,D-peptidase and D,D-carboxypeptidase activities, was purified as the native protein, as a maltose-binding protein fusion and with an N-terminal tag containing six histidine residues. The kinetic parameters of His(6)-VanXY(C) were measured for a variety of precursors of peptidoglycan synthesis involved in resistance: for D-Ala-D-Ala, the K(m) was 3.6 mm and k(cat), 2.5 s(-1); for UDP-MurNAc-L-Ala-D-Glu-L-Lys-DAla-D-Ala (UDP-MurNAc-pentapeptide[Ala]), K(m) was 18.8 mm and k(cat) 6.2 s(-1); for D-Ala-D-Ser, K(m) was 15.5 mm and k(cat) 0.35 s(-1). His(6)-VanXYC was inactive against the peptidoglycan precursor UDP-MurNAc-L-Ala-D-Glu-L-Lys-D-Ala-D-Ser (UDP-MurNAc-pentapeptide[Ser]). The rate of hydrolysis of the terminal D-Ala of UDP-MurNAc-pentapeptide[Ala] was inhibited 30% by 2 mm D-Ala-D-Ser or UDP-MurNAc-pentapeptide[Ser]. Therefore preferential hydrolysis of substrates terminating in D-Ala would occur during peptidoglycan synthesis in E. gallinarum BM4174, leaving precursors ending in D-Ser with a lower affinity for glycopeptides to be incorporated into peptidoglycan. Mutation of an aspartate residue (Asp59) of His-tagged VanXY(C) corresponding to Asp68 in VanX to Ser or Ala, resulted in a 50% increase and 73% decrease, respectively, of the specificity constant (k(cat)/K(m)) for D-Ala-D-Ala. This situation is in contrast to VanX in which mutation of Asp68-->Ala produced a greater than 200,000-fold decrease in the substrate specificity constant. This suggests that Asp59, unlike Asp68 in VanX, does not have a pivotal role in catalysis.

Genome and virulence determinants of high virulence community-acquired MRSA.

BACKGROUND: A new type of meticillin-resistant Staphylococcus aureus (MRSA), designated community-acquired MRSA, is becoming increasingly noticeable in the community, some strains of which cause fatal infections in otherwise healthy individuals. By contrast with hospital-acquired MRSA, community-acquired MRSA is more susceptible to non b-lactam antibiotics. We investigated the high virulence potential of certain strains of this bacterium. METHODS: We ascertained the whole genome sequence of MW2, a strain of community-acquired MRSA, by shotgun cloning and sequencing. MW2 caused fatal septicaemia and septic arthritis in a 16-month-old girl in North Dakota, USA, in 1998. The genome of this strain was compared with those of hospital-acquired MRSA strains, including N315 and Mu50. FINDINGS: Meticillin resistance gene (mecA) in MW2 was carried by a novel allelic form (type IVa) of staphylococcal cassette chromosome mec (SCCmec), by contrast with type II in N315 and Mu50. Type IVa SCCmec did not carry any of the multiple antibiotic resistance genes reported in type II SCCmec. By contrast, 19 additional virulence genes were recorded in the MW2 genome. All but two of these virulence genes were noted in four of the seven genomic islands of MW2. INTERPRETATION: MW2 carried a range of virulence and resistance genes that was distinct from those displayed on the chromosomes of extant S aureus strains. Most genes were carried by specific allelic forms of genomic islands in the MW2 chromosome. The combination of allelic forms of genomic islands is the genetic basis that determines the pathogenicity of medically important phenotypes of S aureus, including those of community-acquired MRSA strains.

Mutations in ponA, the gene encoding penicillin-binding protein 1, and a novel locus, penC, are required for high-level chromosomally mediated penicillin resistance in Neisseria gonorrhoeae.

Chromosomally mediated penicillin resistance in Neisseria gonorrhoeae occurs in part through alterations in penicillin-binding proteins (PBPs) and a decrease in outer membrane permeability. However, the genetic and molecular mechanisms of transformation of a penicillin-susceptible strain of N. gonorrhoeae to high-level penicillin resistance have not been clearly elucidated. Previous studies suggested that alterations in PBP 1 were involved in high-level penicillin resistance. In this study, we identified a single amino acid mutation in PBP 1 located 40 amino acids N terminal to the active-site serine residue that was present in all chromosomally mediated resistant N. gonorrhoeae (CMRNG) strains for which MICs of penicillin were > or = 1 microg/ml. PBP 1 harboring this point mutation (PBP 1*) had a three- to fourfold lower rate of acylation (k2/K') than wild-type PBP 1 with a variety of beta-lactam antibiotics. Consistent with its involvement in high-level penicillin resistance, replacement of the altered ponA gene (ponA1) in several CMRNG strains with the wild-type ponA gene resulted in a twofold decrease in the MICs of penicillin. Surprisingly, transformation of an intermediate-level penicillin-resistant strain (PR100; FA19 penA4 mtr penB5) with the ponA1 gene did not increase the MIC of penicillin for this strain. However, we identified an additional resistance locus, termed penC, which was required along with ponA1 to increase penicillin resistance of PR100 to a high level (MIC = 4 microg/ml). The penC locus by itself, when present in PR100, increases the MICs of penicillin and tetracycline twofold each. These data indicate that an additional locus, penC, is required along with ponA1 to achieve high-level penicillin resistance.

Cloning and overexpression of Bacillus cereus penicillin-binding protein 3 gene in Escherichia coli.

The pbp3 gene encoding PBP3 of Bacillus cereus was cloned and sequenced. For this purpose, PBP3 was first purified from B. cereus ts-4, and N-terminal amino acid sequences of the peptides obtained from the protease digests of the protein were analyzed. The B. cereus ts-4 pbp3 gene consisted of an open reading frame of 1,986 bp encoding 662 amino acid residues with a calculated molecular mass of 73,044 Da. The active site-motifs SXXK, SXN, and KTG are present at the positions 393, 452, and 590, respectively, in the deduced amino acid sequence. The pbp3 structural gene was ligated into the pET17 x b expression vector and pET-pbp3 was constructed. A protein was produced by the cells of E. coli carrying pET-pbp3. The produced protein migrated at about 75 kDa in SDS-polyacrylamide gel and strongly reacted with biotinylated ampicillin.

Overexpression, purification and biochemical characterization of a class A high-molecular-mass penicillin-binding protein (PBP), PBP1* and its soluble derivative from Mycobacterium tuberculosis.

The product of the gene ponA present in cosmid MTCY21D4, one of the collection of clones representing the genome of Mycobacterium tuberculosis, has been named penicillin-binding protein 1* (PBP1*), by analogy to the previously characterized PBP1* of M. leprae. This gene has been overexpressed in Escherichia coli. His(6)-tagged PBP1* localizes to the membranes of induced E. coli cells. Its susceptibility to degradation upon proteinase K digestion of spheroplasts from E. coli expressing the protein supports the view that the majority of the protein translocates to the periplasmic side of the membrane. Recombinant PBP1* binds benzylpenicillin and several other beta-lactams, notably cefotaxime, with high affinity. Truncation of the N-terminal 64 amino acid residues results in an expressed protein present exclusively in inclusion bodies and unable to associate with the membrane. The C-terminal module encompassing amino acids 272-663 can be extracted from inclusion bodies under denaturing conditions using guanidine/HCl and refolded to give a protein fully competent in penicillin-binding. Deletion of Gly(95)-Gln(143) results in the expression of a protein, which is localized in the cytosol. The soluble derivative of PBP1* binds benzylpenicillin with the same efficiency as the full-length protein. This is the first report of a soluble derivative of a class A high-molecular-mass PBP.

Prediction of mecA-positive coagulase-negative staphylococci: assessment of different phenotypic methods, breakpoints, culture media and culture conditions.

Sensitivity for the detection of mecA-positive coagulase-negative staphylococci (CNS) was evaluated for different breakpoints of resistance for oxacillin using three different susceptibility tests, either on Mueller-Hinton agar supplemented with 2% NaCl (MH-NaCl agar) or on paper disc method agar supplemented with 5% defibrinated blood (PDM-blood agar). The Etest, multipoint inoculation test and disc diffusion test showed comparable sensitivity (0.96, 0.96 and 0.95, respectively) using an oxacillin breakpoint of > or = 0.5 mg/L or < or = 17 mm for the disc test, after incubation at 35 degrees C for 24 h on MH-NaCl agar. The sensitivity decreased for breakpoints > or = 1 mg/L and when PDM-blood agar was used instead of MH-NaCl agar.

Isolation and molecular characterization of methicillin-resistant coagulase-negative staphylococci from nasal flora of healthy humans at three community institutions in Rio de Janeiro City.

We describe the isolation and molecular characterization of methicillin-resistant coagulase-negative staphylococci (MRCNS) from the nasal flora of healthy humans from three institutions located in Rio de Janeiro City. Swabs were obtained from the nares of students attending a non-residential public school and adults from two military quarters. Isolates of staphylococci were tested for the presence of the mecA gene by hybridization with a specific probe. S. epidermidis was the most frequent MRCNS (38 of the total 45 CNS isolated). Twenty-five percent of nasal staphylococcal carriers studied were colonized with MRCNS. Pulsed-field gel electrophoresis (PFGE) of SmaI-digested genomic DNA was carried out to study the clonality of the methicillin-resistant S. epidermidis (MRSE) isolates. In addition to cross-colonization among individuals belonging to the same institution, familial cross-colonization appeared to contribute to the spread of the methicillin-resistant isolates among two inter-communicable institutions. Indeed, the wide genomic diversity among the MRSE flora suggests that the spread of the mecA gene among these isolates might also have occurred via horizontal transmission. Despite the limited number of institutions analysed, it is reasonable to conclude that our data do not represent a situation unique to the three organizations but may reflect other communities in Rio with respect to transmission of MRCNS.

Fluorescent Bocillins: synthesis and application in the detection of penicillin-binding proteins.

Novel fluorescent analogs of penicillin V were synthesized and evaluated for efficacy in the detection of penicillin binding proteins (PBPs). These molecules include the full structure of penicillin V, with the potent Bodipy fluorophore attached to the para-position of the penicillin V phenyl group. The green fluorescent Bocillin FL and the near-infrared (IR) fluorescent Bocillin 650/665 probes were shown to bind to PBPs, both purified and from membrane preparations, with high affinity and specificity. These reagents allow for facile detection of 2-4 ng of purified PBP with the aid of a fluorescent scanner.

Purification and characterization of PBP4a, a new low-molecular-weight penicillin-binding protein from Bacillus subtilis.

Penicillin-binding protein 4a (PBP4a) from Bacillus subtilis was overproduced and purified to homogeneity. It clearly exhibits DD-carboxypeptidase and thiolesterase activities in vitro. Although highly isologous to the Actinomadura sp. strain R39 DD-peptidase (B. Granier, C. Duez, S. Lepage, S. Englebert, J. Dusart, O. Dideberg, J. van Beeumen, J. M. Frère, and J. M. Ghuysen, Biochem. J. 282:781-788, 1992), which is rapidly inactivated by many beta-lactams, PBP4a is only moderately sensitive to these compounds. The second-order rate constant (k(2)/K) for the acylation of the essential serine by benzylpenicillin is 300,000 M(-1) s(-1) for the Actinomadura sp. strain R39 peptidase, 1,400 M(-1) s(-1) for B. subtilis PBP4a, and 7,000 M(-1) s(-1) for Escherichia coli PBP4, the third member of this class of PBPs. Cephaloridine, however, efficiently inactivates PBP4a (k(2)/K = 46,000 M(-1) s(-1)). PBP4a is also much more thermostable than the R39 enzyme.

Rapid detection of mecA-positive and mecA-negative coagulase-negative staphylococci by an anti-penicillin binding protein 2a slide latex agglutination test.

A rapid slide latex agglutination (LA) test, MRSA-Screen (Denka Seiken Co., Niigata, Japan), which detects PBP 2a, was tested for its ability to differentiate between mecA-positive and -negative coagulase-negative staphylococci. A total of 463 isolates from 13 species were included in the study. The mecA gene was detected by PCR, and the oxacillin MIC was determined by the agar dilution method according to the guidelines of the National Committee for Clinical Laboratory Standards (NCCLS). The LA test was performed with oxacillin-induced isolates. The true-positive and true-negative results were defined on the basis of the presence or the absence of the mecA gene. By PCR, 251 isolates were mecA positive and 212 were mecA negative. The sensitivities, specificities, and positive and negative predictive values for the LA test compared to the NCCLS breakpoint for oxacillin resistance (>/=0.5 mg/liter) were as follows: for the LA test, 100, 99.5, 99.6, and 100%, respectively; for the NCCLS breakpoint, 100, 60.8, 75.1, and 100%, respectively. One hundred twenty-five mecA-positive isolates were also tested by the LA test without induction of PBP 2a; only 72 (57.6%) gave a positive result and required 3 to 15 min for reaction. With induction, all 251 isolates were positive within 3 min. The LA test was reliable in classifying mecA-negative isolates, but it classified isolates for which the oxacillin MIC was >/=0.5 mg/liter as oxacillin susceptible. For the reliable detection of oxacillin resistance by the MRSA-Screen in coagulase-negative staphylococci, induction of the mecA gene appears to be necessary.

Evaluation of three rapid methods for detection of methicillin resistance in Staphylococcus aureus.

The probe-based Velogene Rapid MRSA Identification Assay (ID Biomedical Corp., Vancouver, British Columbia, Canada) and the latex agglutination MRSA-Screen (Denka Seiken Co., Tokyo, Japan) were evaluated for their ability to identify methicillin-resistant Staphylococcus aureus (MRSA) and to distinguish strains of MRSA from borderline oxacillin-resistant S. aureus (BORSA; mecA-negative, oxacillin MICs of 2 to 8 microgram/ml). The Velogene is a 90-min assay using a chimeric probe to detect the mecA gene. MRSA-Screen is a 15-min latex agglutination test with penicillin-binding protein 2a antibody-sensitized latex particles. We compared these assays with the BBL Crystal MRSA ID System (Becton Dickinson, Cockeysville, Md.) and with PCR for mecA gene detection. A total of 397 clinical isolates of S. aureus were tested, consisting of 164 methicillin-susceptible strains, 197 MRSA strains, and 37 BORSA strains. All assays performed well for the identification of MRSA with sensitivities and specificities for Velogene, MRSA-Screen, and BBL Crystal MRSA ID of 98.5 and 100%, 98.5 and 100%, and 98.5 and 98%, respectively. Three MRSA strains were not correctly identified by each of the Velogene and MRSA-Screen assays, but repeat testing with a larger inoculum resolved the discrepancies. The BBL Crystal MRSA ID test misclassified four BORSA strains as MRSA. Both the Velogene and the MRSA-Screen assays are easy to perform, can accurately differentiate BORSA isolates from MRSA isolates, and provide a rapid alternative for the detection of methicillin resistance in S. aureus in clinical laboratories, especially when mecA PCR gene detection is unavailable.

Use of digoxigenin-labelled ampicillin in the identification of penicillin-binding proteins in Helicobacter pylori.

Amoxycillin is used in current therapeutic regimens to treat the infection caused by the human gastric pathogen, Helicobacter pylori. The penicillin-binding proteins (PBPs) are the primary targets for the beta-lactam antibiotics, such as amoxycillin, and are involved in the terminal stages of peptidoglycan synthesis. They also play active roles in the determination and maintenance of cellular morphology. It was believed that an organism with a complex morphology, such as H. pylori, would have more than the three PBPs previously suggested. Using digoxigenin-labelled ampicillin (DIG-ampicillin), we report the identification of eight PBPs in H. pylori with masses of 72, 62, 54, 50, 44, 33.5, 30.5 and 28 kDa. A smaller (21 kDa) ninth band was also detected, which may represent another PBP. However, the relatively small size of this apparent PBP raises questions as to whether this is a true PBP. In an attempt to identify the PBPs to which amoxycillin preferentially binds, amoxycillin was used in competition assays with DIG-ampicillin. It appeared that amoxycillin inhibited the binding of DIG-ampicillin to only the 72 kDa PBP. The experimental data were also compared with the seven putative PBPs identified in the two published H. pylori genomes, most of which correlate with the experimental data. To investigate further the properties of these PBPs, the seven putative PBP genes identified in the H. pylori genomes were examined. The derived amino acid sequences of the putative PBPs were examined for the three characteristic motifs found in all conventional PBPs, SXXK, SXN and KTG. We were able to determine that all of the putative PBPs had at least one of these motifs, but none possessed all three motifs with the characteristics of conventional PBPs. These findings suggest that the PBPs of H. pylori are unique.

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

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

Identification of a novel penicillin-binding protein from Helicobacter pylori.

The Helicobacter pylori genome encodes four penicillin-binding proteins (PBPs). PBPs 1, 2, and 3 exhibit similarities to known PBPs. The sequence of PBP 4 is unique in that it displays a novel combination of two highly conserved PBP motifs and an absence of a third motif. Expression of PBP 4, but not PBP 1, 2, or 3, is significantly increased during mid- to late-log-phase growth.

The crystal structure of a penicilloyl-serine transferase of intermediate penicillin sensitivity. The DD-transpeptidase of streptomyces K15.

The serine DD-transpeptidase/penicillin-binding protein of Streptomyces K15 catalyzes peptide bond formation in a way that mimics the penicillin-sensitive peptide cross-linking reaction involved in bacterial cell wall peptidoglycan assembly. The Streptomyces K15 enzyme is peculiar in that it can be considered as an intermediate between classical penicillin-binding proteins, for which benzylpenicillin is a very efficient inactivator, and the resistant penicillin-binding proteins that have a low penicillin affinity. With its moderate penicillin sensitivity, the Streptomyces K15 DD-transpeptidase would be helpful in the understanding of the structure-activity relationship of this penicillin-recognizing protein superfamily. The structure of the Streptomyces K15 enzyme has been determined by x-ray crystallography at 2.0-A resolution and refined to an R-factor of 18.6%. The fold adopted by this 262-amino acid polypeptide generates a two-domain structure that is close to those of class A beta-lactamases. However, the Streptomyces K15 enzyme has two particular structural features. It lacks the amino-terminal alpha-helix found in the other penicilloyl-serine transferases, and it exhibits, at its surface, an additional four-stranded beta-sheet. These two characteristics might serve to anchor the enzyme in the plasma membrane. The overall topology of the catalytic pocket of the Streptomyces K15 enzyme is also comparable to that of the class A beta-lactamases, except that the Omega-loop, which bears the essential catalytic Glu(166) residue in the class A beta-lactamases, is entirely modified. This loop adopts a conformation similar to those found in the Streptomyces R61 DD-carboxypeptidase and class C beta-lactamases, with no equivalent acidic residue.

Penicillin-binding protein 2a of Streptococcus pneumoniae: expression in Escherichia coli and purification and refolding of inclusion bodies into a soluble and enzymatically active enzyme.

Penicillin-binding proteins (PBPs), targets of beta-lactam antibiotics, are membrane-bound enzymes essential for the biosynthesis of the bacterial cell wall. PBPs possess transpeptidase and transglycosylase activities responsible for the final steps of the bacterial cell wall cross-linking and polymerization, respectively. To facilitate our structural studies of PBPs, we constructed a 5'-truncated version (lacking bp from 1 to 231 encoding the N-terminal part of the protein including the transmembrane domain) of the pbp2a gene of Streptococcus pneumoniae and expressed the truncated gene product as a GST fusion protein in Escherichia coli. This GST fusion form of PBP2a, designated GST-PBP2a*, was expressed almost exclusively as inclusion bodies. Using a combination of high- and low-speed centrifugation, large amounts of purified inclusion bodies were obtained. These purified inclusion bodies were refolded into a soluble and enzymatically active enzyme using a single-step refolding method consisting of solubilization of the inclusion bodies with urea and direct dialysis of the solubilized preparations. Using these purification and refolding methods, approximately 37 mg of soluble GST-PBP2a* protein was obtained from 1 liter of culture. The identity of this refolded PBP2a* protein was confirmed by N-terminal sequencing. The refolded PBP2a*, with or without the GST-tag, was found to bind to BOCILLIN FL, a beta-lactam, and to hydrolyze S2d, an analog of the bacterial cell wall stem peptides. The S2d hydrolysis activity of PBP2a* was inhibited by penicillin G. In conclusion, using this expression system, and the purification and refolding methods, large amounts of the soluble GST-PBP2a* protein were obtained and shown to be enzymatically active.

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.

Penicillin-binding proteins of pathogenic Escherichia coli strains.

The penicillin-binding proteins of 11 pathogenic Escherichia coli strains, including enteropathogenic, enterotoxigenic, enteroinvasive, enteroaggregative, and enterohemorrhagic E. coli, were detected in gels following the labeling of isolated cell envelopes with [3H]benzylpenicillin. The electrophoretic profiles, sensitivities to and morphological changes induced by beta-lactam antibiotics showed that the penicillin-binding proteins of most pathogenic E. coli possess structural and physiological functions similar to those of E. coli K12.