RESUMEN
Methicillin resistant Staphylococcus is an important worldwide problem. Resistance is verified in strains harboring the mecA gene and laboratory methods used to detect resistance are object of constant investigation. In the present study, 99 clinical isolates of staphylococci (41 S. aureus, 33 S. epidermidis, 12 S. saprophyticus and 13 members of other species) were submitted to different phenotypic methods and conditions. Detection of the mecA gene by PCR was used as the reference method and detected 14/41, 10/33, and 10/25 isolates of S. aureus, S. epidermidis and other species, respectively. Results showed that, for S. aureus and S. epidermidis, agar diffusion, agar dilution, and the E test incubated during 24h at 35 degrees C correctly discriminated mecA positive from mecA negative isolates. For other species, all methods and conditions presented low specificity (ranging from 20% to 66.7%) and, particularly S. saprophyticus, may need molecular methods to correctly assess methicillin resistance.
Asunto(s)
Proteínas Bacterianas , Proteínas Portadoras/análisis , Hexosiltransferasas , Inmunodifusión/métodos , Resistencia a la Meticilina , Pruebas de Sensibilidad Microbiana/métodos , Muramoilpentapéptido Carboxipeptidasa/análisis , Peptidil Transferasas , Staphylococcus/fisiología , Agar , Técnicas Bacteriológicas/métodos , Secuencia de Bases , Proteínas Portadoras/genética , Proteínas Portadoras/inmunología , Difusión , Genes Bacterianos/genética , Resistencia a la Meticilina/genética , Muramoilpentapéptido Carboxipeptidasa/genética , Muramoilpentapéptido Carboxipeptidasa/inmunología , Proteínas de Unión a las Penicilinas , Reacción en Cadena de la Polimerasa , Staphylococcus/enzimología , Staphylococcus/genética , Staphylococcus/inmunologíaRESUMEN
More than 90% of methicillin-resistant Staphylococcus aureus (MRSA) isolates produce a penicillin-binding protein PBP2' (or PBP2a) with low affinity for beta-lactam antibiotics. PBP2' is encoded by the mecA gene, a foreign gene integrated into the chromosome of methicillin-susceptible S. aureus (MSSA). DNA vaccination by injection of transgene-expressing plasmids has been demonstrated to elicit an immune response against transgene-encoded protein. We hypothesized that the application of DNA vaccination with the mecA sequence would elicit protective immunity against MRSA. This immunity was evoked by injection of a mecA-expressing plasmid into BALB/c mice. Anti-PBP2' antibody was detected in the sera obtained from the DNA-vaccinated mice. These sera produced a five-fold increase in phagocytosis of MRSA compared with sera from mice treated with control plasmid. However, there was no difference in phagocytosis of MSSA among these groups. In addition, the in-vivo antibacterial effect of DNA vaccination was demonstrated in mice infected with MRSA. Eight days after iv inoculation of 10(8) cfu of MRSA into mice, the number of bacteria in the kidneys obtained from mice vaccinated with mecA-expressing plasmid (1.48 +/- 0.27 x 10(5) cfu/mg kidney; n = 18) was significantly lower than that from mice vaccinated with negative control plasmid (3.59 +/- 0.57 x 10(5) cfu/mg kidney; n = 17) (P < 0.02) or that from sham-treated mice (3. 43 +/- 0.66 x 10(5) cfu/mg kidney; n = 9) (P < 0.02). Interestingly, PBP2' was found in both the bacterial membrane fraction and the supernatant, thus being accessible to serum antibodies. Together these observations indicate that PBP2' or the mecA sequence may be eligible as a candidate molecule for vaccination against MRSA.
Asunto(s)
Proteínas Bacterianas , Proteínas Portadoras/genética , Proteínas Portadoras/inmunología , Hexosiltransferasas , Resistencia a la Meticilina , Muramoilpentapéptido Carboxipeptidasa/genética , Muramoilpentapéptido Carboxipeptidasa/inmunología , Peptidil Transferasas , Infecciones Estafilocócicas/inmunología , Staphylococcus aureus/inmunología , Vacunas de ADN/inmunología , Animales , Anticuerpos Antibacterianos/sangre , Bacteriemia/inmunología , Bacteriemia/microbiología , Proteínas Portadoras/metabolismo , Humanos , Resistencia a la Meticilina/genética , Ratones , Ratones Endogámicos BALB C , Muramoilpentapéptido Carboxipeptidasa/metabolismo , Proteínas de Unión a las Penicilinas , Fagocitosis , Plásmidos/genética , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/efectos de los fármacos , VacunaciónRESUMEN
A previously unrecognized penicillin binding protein (PBP) gene, pbpF, was identified in Staphylococcus aureus. This gene encodes a protein of 691 amino acid residues with an estimated molecular mass of 78 kDa. The molecular mass is very close to that of S. aureus PBP2 (81 kDa), and the protein is tentatively named PBP2B. PBP2B has three motifs, SSVK, SSN, and KTG, that can be found in PBPs and beta-lactamases. Recombinant PBP2B (rPBP2B), which lacks a putative signal peptide at the N terminus and has a histidine tag at the C terminus, was expressed in Escherichia coli. The purified rPBP2B was shown to have penicillin binding activity. A protein band was detected from S. aureus membrane fraction by immunoblotting with anti-rPBP2B serum. Also, penicillin binding activity of the protein immunoprecipitated with anti-rPBP2B serum was detected. These results suggest the presence of PBP2B in S. aureus cell membrane that covalently binds penicillin. The internal region of pbpF and PBP2B protein were found in all 12 S. aureus strains tested by PCR and immunoblotting.
Asunto(s)
Proteínas Bacterianas , Proteínas Portadoras/genética , Genes Bacterianos , Hexosiltransferasas , Muramoilpentapéptido Carboxipeptidasa/genética , Peptidil Transferasas , Staphylococcus aureus/genética , Secuencia de Aminoácidos , Secuencia de Bases , Proteínas Portadoras/química , Proteínas Portadoras/inmunología , Clonación Molecular , Datos de Secuencia Molecular , Muramoilpentapéptido Carboxipeptidasa/química , Muramoilpentapéptido Carboxipeptidasa/inmunología , Penicilina G/metabolismo , Proteínas de Unión a las Penicilinas , Pruebas de PrecipitinaRESUMEN
The MRSA-Screen test (Denka Seiken Co., Ltd., Tokyo, Japan), consisting of a slide latex agglutination kit that detects PBP 2a with a monoclonal antibody, was blindly compared to the oxacillin disk diffusion test, the oxacillin-salt agar screen, and PCR of the mecA gene for the detection of methicillin resistance in Staphylococcus aureus. A total of 120 methicillin-susceptible S. aureus (MSSA) and 80 methicillin-resistant S. aureus (MRSA) isolates, defined by the absence or presence of the mecA gene, respectively, were tested. The MRSA-Screen test, the oxacillin disk diffusion test, and the oxacillin-salt agar screening test showed sensitivities of 100, 61.3, and 82.5% and specificities of 99.2, 96.7, and 98.3%, respectively. We conclude that the MRSA-Screen is a very accurate, reliable, and fast test (15 min) for differentiation of MRSA from MSSA colonies on agar plates.
Asunto(s)
Proteínas Portadoras/análisis , Hexosiltransferasas , Resistencia a la Meticilina , Muramoilpentapéptido Carboxipeptidasa/análisis , Peptidil Transferasas , Juego de Reactivos para Diagnóstico , Staphylococcus aureus/efectos de los fármacos , Anticuerpos Monoclonales/inmunología , Proteínas Bacterianas/genética , Proteínas Portadoras/inmunología , Humanos , Pruebas de Fijación de Látex , Muramoilpentapéptido Carboxipeptidasa/inmunología , Oxacilina/farmacología , Proteínas de Unión a las Penicilinas , Sensibilidad y EspecificidadRESUMEN
Screening of a large transposon library constructed in the background of a highly and homogeneously methicillin-resistant Staphylococcus aureus (MRSA) strain (methicillin MIC 1,600 micrograms/ml) for Tn551 mutants with reduced resistance, identified mutant RUSA130 with a methicillin MIC of 12 micrograms/ml. Cloning in E. coli followed by sequencing located the Tn551 insert omega 703 near the C-terminal of the PBP2 gene. Penicillin-binding assays with mutant RUSA130 showed the presence of normal amounts of penicillin-binding protein 2A (PBP2A) but the absence of PBP2. These observations suggest that the mecA gene product PBP2A is not the sole catalyst of peptidoglycan synthesis in MRSA growing in the presence of beta-lactam antibiotics, since an intact PBP2 is also essential for the optimal expression of methicillin resistance in MRSA.
Asunto(s)
Proteínas Bacterianas , Proteínas Portadoras/inmunología , Elementos Transponibles de ADN/efectos de los fármacos , Hexosiltransferasas , Resistencia a la Meticilina/genética , Muramoilpentapéptido Carboxipeptidasa/inmunología , Peptidil Transferasas , Staphylococcus aureus/efectos de los fármacos , Bacteriófagos/genética , Medios de Cultivo , Elementos Transponibles de ADN/genética , ADN Bacteriano/genética , ADN Bacteriano/inmunología , Mutación , Proteínas de Unión a las Penicilinas , Staphylococcus aureus/genética , Transducción Genética/genéticaRESUMEN
This paper reports the first attempt to characterize the penicillin-binding proteins (PBPs) of Shigella dysenteriae, an important human pathogen. The PBP pattern of the membranes of S. dysenteriae closely resembles that of Escherichia coli membranes. A 38 kDa PBP which is an important target for the penem SCH34343, the cephamycin cefoxitin and the oxacephem moxalactam, has been purified. This PBP is immunologically related to a PBP of similar molecular mass in E. coli and is present at high levels in stationary-phase cells of S. dysenteriae.
Asunto(s)
Antibacterianos/metabolismo , Proteínas Bacterianas , Proteínas Portadoras/aislamiento & purificación , Dipeptidasas , Hexosiltransferasas , Muramoilpentapéptido Carboxipeptidasa/aislamiento & purificación , Peptidil Transferasas , Shigella dysenteriae/química , Secuencia de Aminoácidos , Antibacterianos/farmacología , Proteínas Portadoras/inmunología , Reacciones Cruzadas , Endopeptidasas/análisis , Escherichia coli/química , Escherichia coli/inmunología , Lactamas , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Muramoilpentapéptido Carboxipeptidasa/inmunología , Proteínas de Unión a las Penicilinas , Análisis de SecuenciaRESUMEN
The recent model of Treponema pallidum molecular architecture proposes that the vast majority of the bacterium's integral membrane proteins are lipoprotein immunogens anchored in the cytoplasmic membrane while the outer membrane contains only a limited number of surface-exposed transmembrane proteins. This unique model explains, in part, the organism's remarkable ability to evade host immune defenses and establish persistent infection. Our strategy for refining this model involves demonstrating that the physiological functions of treponemal membrane proteins are consistent with their proposed cellular locations. In this study, we used an ampicillin-digoxigenin conjugate to demonstrate by chemiluminescence that the 47-kDa lipoprotein immunogen of T. pallidum (Tpp47) is a penicillin-binding protein. Reexamination of the Tpp47 primary sequence revealed the three amino acid motifs characteristic of penicillin-binding proteins. A recombinant, nonlipidated, soluble form of Tpp47 was used to demonstrate that Tpp47 is a zinc-dependent carboxypeptidase. Escherichia coli expressing Tpp47 was characterized by cell wall abnormalities consistent with altered peptidoglycan biosynthesis. Though the inability to cultivate T. pallidum in vitro and the lack of genetic exchange systems continue to impede treponemal research, this study advances strategies for utilizing E. coli molecular genetics as a means of elucidating the complex relationships between syphilis pathogenesis and T. pallidum membrane biology.
Asunto(s)
Antígenos Bacterianos/química , Proteínas Bacterianas , Carboxipeptidasas/inmunología , Proteínas Portadoras/inmunología , Hexosiltransferasas , Lipoproteínas/inmunología , Muramoilpentapéptido Carboxipeptidasa/inmunología , Peptidil Transferasas , Treponema pallidum/inmunología , Secuencia de Aminoácidos , Antígenos Bacterianos/metabolismo , Secuencia de Bases , Carboxipeptidasas/química , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Cationes Bivalentes/metabolismo , Cartilla de ADN/química , Lipoproteínas/metabolismo , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Peso Molecular , Muramoilpentapéptido Carboxipeptidasa/química , Proteínas de Unión a las Penicilinas , Treponema pallidum/enzimologíaRESUMEN
For Staphylococcus aureus, it is hypothesized that two genes located upstream of the beta-lactamase gene, blaZ, are required for the inducible expression of beta-lactamase. blaR1 is predicted to encode a signal-transducing membrane protein, and blaI is predicted to encode a repressor protein. These same two genes may also regulate the production of penicillin-binding protein 2a (PBP 2a), a protein essential for expression of methicillin resistance. To confirm that these two genes encode products that can control both beta-lactamase and PBP 2a production, blaI, blaR1, and blaZ with a 150-nucleotide deletion at the 3' end were subcloned from a 30-kb staphylococcal beta-lactamase plasmid and three beta-lactamase-negative strains of methicillin-resistant S. aureus were transformed with the recombinant plasmid containing that insert. The production of PBP 2a and a nonfunctional beta-lactamase was detected by fluorography and by immunoblots with polyclonal antisera directed against each of the proteins. Whereas the parent strains did not produce beta-lactamase and constitutively produced PBP 2a, PBP 2a and a truncated beta-lactamase were now inducible in the transformants. Therefore, two plasmid-derived genes regulate the production of both PBP 2a and beta-lactamase.
Asunto(s)
Proteínas Bacterianas/biosíntesis , Proteínas Portadoras/biosíntesis , Hexosiltransferasas , Metaloendopeptidasas/biosíntesis , Resistencia a la Meticilina , Muramoilpentapéptido Carboxipeptidasa/biosíntesis , Peptidil Transferasas , Staphylococcus aureus/efectos de los fármacos , beta-Lactamasas/biosíntesis , Proteínas Bacterianas/genética , Secuencia de Bases , Southern Blotting , Proteínas Portadoras/genética , Proteínas Portadoras/inmunología , Clonación Molecular , Inducción Enzimática , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Sueros Inmunes , Metaloendopeptidasas/genética , Datos de Secuencia Molecular , Muramoilpentapéptido Carboxipeptidasa/genética , Muramoilpentapéptido Carboxipeptidasa/inmunología , Hibridación de Ácido Nucleico , Proteínas de Unión a las Penicilinas , Plásmidos , Staphylococcus aureus/genética , Transformación Bacteriana , beta-Lactamasas/genética , beta-Lactamasas/inmunologíaRESUMEN
The occurrence of highly variable penicillin-binding proteins (PBPs) in penicillin-resistant Streptococcus pneumoniae suggested that transfer of homologous genes from related species may be involved in resistance development. Antiserum and monoclonal antibodies raised against PBPs 1a and 2b from the susceptible S. pneumoniae R6 strain were used to identify related PBPs in 41 S. mitis, S. sanguis I and S. sanguis II strains mostly isolated in South Africa with MIC values ranging from less than 0.15 to 16 mg/ml. Furthermore, the possibility of genetic exchange was examined with 30 penicillin-resistant strains of this collection (MIC greater than 0.06 mg/ml) as donors using S. pneumoniae R6 as recipient in transformation experiments. The majority of S. mitis and S. sanguis II strains but none of the S. sanguis I strains could transform penicillin resistance genes into S. pneumoniae R6. All positive donor strains and all susceptible isolates of S. mitis and S. sanguis II strains contained PBPs which cross-reacted with the anti-PBP 1a and/or anti-PBP 2b antibodies. On the other hand, only five of the 14 S. sanguis I strains contained a PBP that reacted with one of the antibodies. This strongly suggested the presence of genes homologous to the pneumococcal PBP 1a and 2b genes in viridans streptococci, and documents that penicillin resistance determinants can be transformed from viridans streptococci into the pneumococcus.
Asunto(s)
Proteínas Bacterianas , Proteínas Portadoras/inmunología , Hexosiltransferasas , Muramoilpentapéptido Carboxipeptidasa/inmunología , Resistencia a las Penicilinas/genética , Peptidil Transferasas , Streptococcus pneumoniae/genética , Streptococcus/genética , Transformación Genética , Western Blotting , Proteínas Portadoras/análisis , Reacciones Cruzadas , Genes Bacterianos , Pruebas de Sensibilidad Microbiana , Muramoilpentapéptido Carboxipeptidasa/análisis , Resistencia a las Penicilinas/inmunología , Proteínas de Unión a las Penicilinas , Especificidad de la Especie , Streptococcus/inmunología , Streptococcus pneumoniae/inmunologíaRESUMEN
Penicillin-binding protein (PBP) 5 of Enterococcus hirae ATCC 9790 belongs to the class of the high-molecular mass, low-affinity PBPs which have been correlated with penicillin resistance in most Enterococcus species. Polyclonal antibodies were raised against PBP 5 and used to detect immunologically related membrane proteins in E. faecium and E. faecalis strains. Several strains of both species were found to have a membrane protein of similar molecular mass to E. hirae PBP 5 which reacted with the antibodies. Some E. faecium strains did not react with antibodies but their derivatives with increased penicillin minimal inhibitory concentrations did. In some E. faecalis strains the lack of a PBP 5-related protein was associated with failure to select stable penicillin-resistant derivatives.
Asunto(s)
Proteínas Bacterianas , Proteínas Portadoras/análisis , Enterococcus faecalis/química , Enterococcus faecium/química , Enterococcus/química , Hexosiltransferasas , Muramoilpentapéptido Carboxipeptidasa/análisis , Penicilinas , Peptidil Transferasas , Western Blotting , Proteínas Portadoras/inmunología , Enterococcus/inmunología , Enterococcus faecalis/inmunología , Enterococcus faecium/inmunología , Sueros Inmunes , Proteínas de la Membrana/análisis , Proteínas de la Membrana/inmunología , Muramoilpentapéptido Carboxipeptidasa/inmunología , Proteínas de Unión a las PenicilinasRESUMEN
Penicillin-binding proteins (PBPs) from penicillin-susceptible strains of Streptococcus pneumoniae are believed to be fairly similar in contrast to PBPs occurring in resistant isolates. The antigenic variation of PBPs 1a and 2b in 65 penicillin-susceptible strains from different geographic areas and a wide variety of isolation sites was analyzed using a set of specific antisera and monoclonal antibodies. Three strains contained different antigenic variants of PBP 1a, and 50 strains contained one of three antigenic variants found in PBP 2b. Seven patterns of antibody reactivity could be defined; all but one were distinct from those found recently in resistant strains. In addition, electrophoretic mobilities of all six PBPs, compared after conventional SDS-PAGE and fluorography, revealed an unexpected variation of PBP-profiles even for strains of one sero-group. Few strains appeared identical to each other or to the laboratory reference strain R6.
Asunto(s)
Proteínas Bacterianas , Proteínas Portadoras/análisis , Hexosiltransferasas , Muramoilpentapéptido Carboxipeptidasa/análisis , Resistencia a las Penicilinas , Penicilinas/farmacología , Peptidil Transferasas , Streptococcus pneumoniae/efectos de los fármacos , Anticuerpos Antibacterianos/inmunología , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Variación Antigénica , Antígenos Bacterianos/análisis , Western Blotting , Proteínas Portadoras/inmunología , Electroforesis en Gel de Poliacrilamida , Humanos , Sueros Inmunes/inmunología , Muramoilpentapéptido Carboxipeptidasa/inmunología , Proteínas de Unión a las Penicilinas , Streptococcus pneumoniae/clasificación , Streptococcus pneumoniae/inmunologíaRESUMEN
Penicillin-resistant strains of Streptococcus pneumoniae that are isolated with increasing frequency worldwide contain low-affinity penicillin-binding proteins (PBPs). The relatedness of PBPs from 55 resistant strains isolated on three continents was investigated by testing the reactivity of antibodies specific for PBP 1a or 2b and by comparing the PBP patterns. Seventeen patterns of antibody reactivity could be distinguished, 12 of which were specific to one isolate. Most strains, including all German and South African strains, had a unique PBP profile. A few groups of Spanish and Finnish isolates were identified where the strains within each group shared the same PBP profile, the same antigenic variants of PBPs 1a and 2b, and the same serogroup, suggesting that they represent different clones of S. pneumoniae. The results demonstrated highly variable pathways of resistance development and confirmed that resistant strains have emerged independently in different locations.
Asunto(s)
Antígenos Bacterianos/análisis , Proteínas Bacterianas , Proteínas Portadoras/inmunología , Hexosiltransferasas , Muramoilpentapéptido Carboxipeptidasa/inmunología , Resistencia a las Penicilinas , Peptidil Transferasas , Streptococcus pneumoniae/inmunología , Anticuerpos Antibacterianos/inmunología , Variación Antigénica , Western Blotting , Proteínas Portadoras/análisis , Electroforesis en Gel de Poliacrilamida , Finlandia , Alemania , Humanos , Sueros Inmunes/inmunología , Muramoilpentapéptido Carboxipeptidasa/análisis , Proteínas de Unión a las Penicilinas , Sudáfrica , España , Streptococcus pneumoniae/efectos de los fármacosRESUMEN
A previous study in our laboratory identified a surface-exposed peptidoglycan-associated protein of Neisseria gonorrhoeae which had an apparent molecular mass of 44,000 daltons (44kDa) (Hill and Judd, 1989). This paper reports results which confirm that the 44kDa protein is surface-exposed, and that the protein is expressed in, and is structurally invariant among, 14 strains of N. gonorrhoeae. The fact that the 44kDa outer-membrane protein is found in a conserved form in all gonococci examined strongly suggests that it is crucial to the bacterium's survival. Moreover, it appears that this protein is a penicillin-binding protein (PBP3) (Shafer and Judd, 1991). This invariant, surface-exposed, peptidoglycan-associated outer-membrane protein deserves further investigation to elucidate its role in the immunobiology of N. gonorrhoeae, and its possible use as an immunoprophylactic reagent.
Asunto(s)
Antígenos de Superficie/aislamiento & purificación , Proteínas de la Membrana Bacteriana Externa/aislamiento & purificación , Proteínas Bacterianas , Proteínas Portadoras/aislamiento & purificación , Hexosiltransferasas , Muramoilpentapéptido Carboxipeptidasa/aislamiento & purificación , Neisseria gonorrhoeae/química , Peptidil Transferasas , Antígenos de Superficie/inmunología , Antígenos de Superficie/metabolismo , Proteínas de la Membrana Bacteriana Externa/inmunología , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Portadoras/inmunología , Proteínas Portadoras/metabolismo , Peso Molecular , Muramoilpentapéptido Carboxipeptidasa/inmunología , Muramoilpentapéptido Carboxipeptidasa/metabolismo , Neisseria gonorrhoeae/inmunología , Neisseria gonorrhoeae/metabolismo , Proteínas de Unión a las Penicilinas , Penicilinas/metabolismoRESUMEN
We have analyzed the location of the epitope areas of the four monoclonal antibody groups against penicillin-binding protein 1B (PBP 1B; T. den Blaauwen, F. B. Wientjes, A. H. J. Kolk, B. G. Spratt, and N. Nanninga, J. Bacteriol. 171:1393-1401). They could be specified by studying monoclonal antibody binding patterns to amino- and carboxy-terminal truncated PBP 1B molecules. Monoclonal antibodies against conformational epitopes, with the exception of one epitope area, did not recognize PBP 1B molecules that had not been translocated across the membrane. Apparently, translocation is required for PBP 1B to fully obtain its native conformation.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Proteínas Bacterianas , Proteínas Portadoras/inmunología , Escherichia coli/inmunología , Hexosiltransferasas , Muramoilpentapéptido Carboxipeptidasa/inmunología , Peptidil Transferasas , Western Blotting , Proteínas Portadoras/genética , Clonación Molecular , Análisis Mutacional de ADN , Epítopos , Escherichia coli/genética , Muramoilpentapéptido Carboxipeptidasa/genética , Proteínas de Unión a las Penicilinas , Conformación Proteica , Proteínas Recombinantes de Fusión/inmunología , Relación Estructura-ActividadRESUMEN
We prepared monoclonal antibodies against penicillin-binding protein 1B (PBP 1B) of Escherichia coli to study the membrane topology, spatial organization, and enzyme activities of this protein. The majority of the antibodies derived with PBP 1B as the immunogen reacted against the carboxy terminus. To obtain monoclonal antibodies recognizing other epitopes, we used PBP 1B lacking the immunodominant carboxy-terminal 65 amino acids as the immunogen. Eighteen monoclonal antibodies directed against membrane-bound PBP 1B were isolated and characterized. The epitopes recognized by those monoclonal antibodies were located with various truncated forms of PBP 1B. We could distinguish four different epitope areas located on different parts of the molecule. Interestingly, we could not isolate monoclonal antibodies against the amino terminus, although they were specifically selected for. This is attributed to its predicted extreme hydrophilicity and flexibility, which could make the amino terminus very sensitive to proteolytic degradation. All antibodies reacted against native PBP 1B in a dot-blot immunobinding assay. One monoclonal antibody also recognized PBP 1B in a completely sodium dodecyl sulfate-denatured form. This suggests that all the other monoclonal antibodies recognize conformational epitopes. These properties make the monoclonal antibodies suitable tools for further studies.