RESUMEN
beta-lactams have a long history in the treatment of infectious diseases, though their use has been and continues to be confounded by the development of resistance in target organisms. beta-lactamases, particularly in Gram-negative pathogens, are a major determinant of this resistance, although alterations in the beta-lactam targets, the penicillin-binding proteins (PBPs), are also important, especially in Gram-positive pathogens. Mechanisms for the efflux and/or exclusion of these agents also contribute, though often in conjunction these other two. Approaches for overcoming these resistance mechanisms include the development of novel beta-lactamase-stable beta-lactams, beta-lactamase inhibitors to be employed with existing beta-lactams, beta-lactam compounds that bind strongly to low-affinity PBPs and agents that potentiate the activity of existing beta-lactams against low-affinity PBP-producing organisms.
Asunto(s)
Resistencia betalactámica , Antibacterianos/metabolismo , Proteínas Bacterianas/fisiología , Proteínas Portadoras/fisiología , Resistencia a Múltiples Medicamentos , Hexosiltransferasas/fisiología , Muramoilpentapéptido Carboxipeptidasa/fisiología , Proteínas de Unión a las Penicilinas , Peptidil Transferasas/fisiología , Permeabilidad , Plásmidos , beta-Lactamasas/metabolismoRESUMEN
We tested the impact of individual PBP 5 mutations on expression of ampicillin resistance in Enterococcus faecium using a shuttle plasmid designed to facilitate expression of cloned pbp5 in ampicillin-susceptible E. faecium D344SRF. Substitutions that had been implicated in contributing to the resistance of clinical strains conferred only modest levels of resistance when they were present as single point mutations. The levels of resistance were amplified when some mutations were present in combination. In particular, a methionine-to-alanine change at position 485 (in close proximity to the active site) combined with the insertion of a serine at position 466 (located in a loop that forms the outer edge of the active site) was associated with the highest levels of resistance to all beta-lactams. Affinity for penicillin generally correlated with beta-lactam MICs for the mutants, but these associations were not strictly proportional.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/fisiología , Proteínas Portadoras/genética , Proteínas Portadoras/fisiología , Enterococcus faecium/efectos de los fármacos , Enterococcus faecium/genética , Hexosiltransferasas/genética , Hexosiltransferasas/fisiología , Muramoilpentapéptido Carboxipeptidasa/genética , Muramoilpentapéptido Carboxipeptidasa/fisiología , Mutación/genética , Mutación/fisiología , Peptidil Transferasas/genética , Peptidil Transferasas/fisiología , Resistencia betalactámica/genética , Resistencia a la Ampicilina/genética , Cristalografía por Rayos X , Vectores Genéticos/genética , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Proteínas de Unión a las Penicilinas , Penicilinas/metabolismo , Plásmidos/genética , Unión ProteicaRESUMEN
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.
Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/química , Bacteroides fragilis/efectos de los fármacos , Proteínas Portadoras/química , Hexosiltransferasas/química , Muramoilpentapéptido Carboxipeptidasa/química , Peptidil Transferasas/química , beta-Lactamasas/química , beta-Lactamas/farmacología , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/fisiología , Bacteroides fragilis/química , Bacteroides fragilis/enzimología , Bacteroides fragilis/genética , Secuencia de Bases , Proteínas Portadoras/genética , Proteínas Portadoras/fisiología , Dermatoglifia del ADN , ADN Bacteriano/química , Farmacorresistencia Bacteriana , Hexosiltransferasas/genética , Hexosiltransferasas/fisiología , Humanos , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Peso Molecular , Muramoilpentapéptido Carboxipeptidasa/genética , Muramoilpentapéptido Carboxipeptidasa/fisiología , Proteínas de Unión a las Penicilinas , Peptidil Transferasas/genética , Peptidil Transferasas/fisiología , Reacción en Cadena de la Polimerasa , beta-Lactamasas/genética , beta-Lactamasas/fisiologíaRESUMEN
The penicillin-binding proteins (PBPs) polymerize and modify peptidoglycan, the stress-bearing component of the bacterial cell wall. As part of this process, the PBPs help to create the morphology of the peptidoglycan exoskeleton together with cytoskeleton proteins that regulate septum formation and cell shape. Genetic and microscopic studies reveal clear morphological responsibilities for class A and class B PBPs and suggest that the mechanism of shape determination involves differential protein localization and interactions with specific cell components. In addition, the low molecular weight PBPs, by varying the substrates on which other PBPs act, alter peptidoglycan synthesis or turnover, with profound effects on morphology.
Asunto(s)
Proteínas Bacterianas/fisiología , Proteínas Portadoras/fisiología , Bacterias Gramnegativas/crecimiento & desarrollo , Bacterias Gramnegativas/fisiología , Hexosiltransferasas/fisiología , Muramoilpentapéptido Carboxipeptidasa/fisiología , Peptidil Transferasas/fisiología , Proteínas de Unión a las PenicilinasRESUMEN
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.
Asunto(s)
Bacterias/metabolismo , Resistencia a las Penicilinas , Péptido Sintasas/química , Péptido Sintasas/fisiología , Peptidoglicano/biosíntesis , Bacterias/efectos de los fármacos , Bacterias/ultraestructura , Proteínas Bacterianas/química , Proteínas Bacterianas/fisiología , Proteínas Portadoras/fisiología , Pared Celular/química , Pared Celular/metabolismo , Pared Celular/ultraestructura , Hexosiltransferasas/fisiología , Modelos Moleculares , Muramoilpentapéptido Carboxipeptidasa/fisiología , Proteínas de Unión a las Penicilinas , Peptidil Transferasas/fisiología , Streptococcus pneumoniae/efectos de los fármacos , Streptococcus pneumoniae/metabolismo , Streptococcus pneumoniae/ultraestructuraRESUMEN
In free-living eubacteria an external shell of peptidoglycan opposes internal hydrostatic pressure and prevents membrane rupture and death. At the same time, this wall imposes on each cell a shape. Because shape is both stable and heritable, as is the ability of many organisms to execute defined morphological transformations, cells must actively choose from among a large repertoire of available shapes. How they do so has been debated for decades, but recently experiment has begun to catch up with theory. Two discoveries are particularly informative. First, specific protein assemblies, nucleated by FtsZ, MreB or Mbl, appear to act as internal scaffolds that influence cell shape, perhaps by correctly localizing synthetic enzymes. Second, defects in cell shape are correlated with the presence of inappropriately placed, metabolically inert patches of peptidoglycan. When combined with what we know about mutants affecting cellular morphology, these observations suggest that bacteria may fabricate specific shapes by directing the synthesis of two kinds of cell wall: a long-lived, rigid framework that defines overall topology, and a metabolically plastic peptidoglycan whose shape is directed by internal scaffolds.
Asunto(s)
Bacterias/citología , Pared Celular/ultraestructura , Proteínas de Escherichia coli/fisiología , Peptidoglicano/metabolismo , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/fisiología , Bacterias/efectos de los fármacos , Bacterias/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/fisiología , Proteínas Portadoras/genética , Proteínas Portadoras/fisiología , Proteínas de Ciclo Celular , División Celular , Pared Celular/efectos de los fármacos , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/fisiología , Replicación del ADN , ADN Bacteriano/biosíntesis , Proteínas de Escherichia coli/genética , Hexosiltransferasas/genética , Hexosiltransferasas/fisiología , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Modelos Biológicos , Morfogénesis , Muramoilpentapéptido Carboxipeptidasa/genética , Muramoilpentapéptido Carboxipeptidasa/fisiología , Presión Osmótica , Proteínas de Unión a las Penicilinas , Penicilinas/farmacología , Peptidoglicano/ultraestructura , Peptidil Transferasas/genética , Peptidil Transferasas/fisiología , Especificidad de la EspecieRESUMEN
Streptococcus pneumoniae and Neisseria meningitidis have very similar mechanisms of resistance to penicillin G. Although penicillin resistance is now common in S. pneumoniae, it is still rare in N. meningitidis. Using a mathematical model, we studied determinants of this difference and attempted to anticipate trends in meningococcal resistance to penicillin G. The model predicted that pneumococcal resistance in a population similar to that of France might emerge after 20 years of widespread use of beta-lactam antibiotics; this period may vary from 10 to 30 years. The distribution of resistance levels became bimodal with time, a pattern that has been observed worldwide. The model suggests that simple differences in the natural history of colonization, interhuman contact, and exposure to beta-lactam antibiotics explain major differences in the epidemiology of resistance of S. pneumoniae and N. meningitidis.
Asunto(s)
Proteínas Bacterianas , Hexosiltransferasas , Neisseria meningitidis/efectos de los fármacos , Penicilina G/farmacología , Peptidil Transferasas , Streptococcus pneumoniae/efectos de los fármacos , Proteínas Portadoras/fisiología , Modelos Logísticos , Pruebas de Sensibilidad Microbiana , Muramoilpentapéptido Carboxipeptidasa/fisiología , Proteínas de Unión a las PenicilinasRESUMEN
Insertional mutagenesis was used to isolate clones from Streptococcus thermophilus CNRZ368 that were modified in their abilities to tolerate oxidative stress. During this process, two menadione-sensitive clones (6G4 and 18C3) were found to display abnormal cell morphologies and distorted chain topologies and were further studied. Molecular characterization of both 6G4 and 18C3 mutants indicated that they were disrupted in open reading frames homologous to rodA and pbp2b, respectively. Both genes encoded proteins in Escherichia coli that were described as being implicated in peptidoglycan synthesis during the process of cell elongation and to function in determining the rod shape of the cell. This work reports a possible connection between peptidoglycan biosynthesis and oxidative stress defense in S. thermophilus CNRZ368.
Asunto(s)
Aminoaciltransferasas , Proteínas Bacterianas/fisiología , Proteínas Portadoras/fisiología , Proteínas de Escherichia coli , Hexosiltransferasas , Proteínas de la Membrana , Muramoilpentapéptido Carboxipeptidasa/fisiología , Estrés Oxidativo , Peptidil Transferasas , Streptococcus/metabolismo , Secuencia de Bases , Genoma Bacteriano , Peróxido de Hidrógeno/toxicidad , Datos de Secuencia Molecular , Mutación , Proteínas de Unión a las Penicilinas , Peptidoglicano/biosíntesis , Streptococcus/genética , Streptococcus/ultraestructura , Superóxidos/metabolismo , Transcripción GenéticaRESUMEN
It has been reported that penicillin-binding protein 4 (PBP4) activity decreases when a vancomycin-susceptible Staphylococcus aureus isolate is passaged in vitro to vancomycin resistance. We analyzed the PBP profiles of four vancomycin intermediately susceptible S. aureus (VISA) clinical isolates and found that PBP4 was undetectable in three isolates (HIP 5827, HIP 5836, and HIP 6297) and markedly reduced in a fourth (Mu50). PBP4 was readily visible in five vancomycin-susceptible, oxacillin-resistant S. aureus (ORSA) isolates. The nucleotide sequences of the pbp4 structural gene and flanking sequences did not different between the VISA and vancomycin-susceptible isolates. Overproduction of PBP4 on a high-copy-number plasmid in the VISA isolates produced a two- to threefold decrease in vancomycin MICs. Inactivation of pbp4 by allelic replacement mutagenesis in three vancomycin-susceptible ORSA strains (COL, RN450M, and N315) led to a decrease in vancomycin susceptibility, an increase in highly vancomycin-resistant subpopulations, and decreased cell wall cross-linking by high-performance liquid chromatography analysis. Complementation of the COL mutant with plasmid-encoded pbp4 restored the vancomycin MIC and increased cell wall cross-linking. These data suggest that alterations in PBP4 expression are at least partially responsible for the VISA phenotype.
Asunto(s)
Proteínas Bacterianas , Proteínas Portadoras/fisiología , Hexosiltransferasas , Muramoilpentapéptido Carboxipeptidasa/fisiología , Oxacilina/farmacología , Resistencia a las Penicilinas/genética , Peptidil Transferasas , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/genética , Resistencia a la Vancomicina/genética , Medios de Cultivo , ADN Bacteriano/genética , Humanos , Pruebas de Sensibilidad Microbiana , Mutación/genética , Proteínas de Unión a las Penicilinas , Peptidoglicano/metabolismo , Fenotipo , Plásmidos/genética , Eliminación de Secuencia/genética , Staphylococcus aureus/metabolismoRESUMEN
The contribution of penicillin-binding protein 5 (PBP 5) to intrinsic and acquired beta-lactam resistance was investigated by constructing isogenic strains of Enterococcus faecium producing different PBP 5. The pbp5 genes from three E. faecium clinical isolates (BM4107, D344, and H80721) were cloned into the shuttle vector pAT392 and introduced into E. faecium D344S, a spontaneous derivative of E. faecium D344 highly susceptible to ampicillin due to deletion of pbp5 (MIC, 0.03 microg/ml). Immunodetection of PBP5 indicated that cloning of the pbp5 genes into pAT392 resulted in moderate overproduction of PBP 5 in comparison to wild-type strains. This difference may be attributed to a difference in gene copy number. Expression of the pbp5 genes from BM4107 (MIC, 2 microg/ml), D344 (MIC, 24 microg/ml), and H80721 (MIC, 512 microg/ml) in D344S conferred relatively low levels of resistance to ampicillin (MICs, 6, 12, and 20 microg/ml, respectively). A methionine-to-alanine substitution was introduced at position 485 of the BM4107 PBP 5 by site-directed mutagenesis. In contrast to previous hypotheses based on comparison of nonisogenic strains, this substitution resulted in only a 2.5-fold increase in the ampicillin MIC. The reversed-phase high-performance liquid chromatography muropeptide profiles of D344 and D344S were similar, indicating that deletion of pbp5 was not associated with a detectable defect in cell wall synthesis. These results indicate that pbp5 is a nonessential gene responsible for intrinsic resistance to moderate levels of ampicillin and by itself cannot confer high-level resistance.
Asunto(s)
Resistencia a la Ampicilina/fisiología , Proteínas Bacterianas , Proteínas Portadoras/fisiología , Enterococcus faecium/fisiología , Hexosiltransferasas , Muramoilpentapéptido Carboxipeptidasa/fisiología , Peptidoglicano/química , Peptidil Transferasas , Proteínas Portadoras/química , Proteínas Portadoras/genética , Enterococcus faecium/química , Enterococcus faecium/genética , Eliminación de Gen , Humanos , Muramoilpentapéptido Carboxipeptidasa/química , Muramoilpentapéptido Carboxipeptidasa/genética , Mutagénesis Sitio-Dirigida , Proteínas de Unión a las Penicilinas , Análisis de Secuencia de ProteínaRESUMEN
BACKGROUND: Protein phosphatase 2A (PP2A) is a serine/threonine phosphatase distributed in eukaryotes from yeast to human, and plays pivotal roles in diverse cellular functions such as metabolism, cell cycle progression, gene expression and development. PP2A holoenzyme is a heterodimer of a catalytic subunit C and a regulatory subunit A, or a heterotrimer of C, A and a variable regulatory subunit consisting of three families; B, B', and PR72. Specific functions for each variable subunit are not well understood. RESULTS: Two fission yeast genes pbp1+ and pbp2+ homologous to the regulatory subunit B' were isolated. Physical in vivo interaction of the gene products with the catalytic subunit was demonstrated. A double disruption haploid mutant (Deltapbp1Deltapbp2) showed growth defect, cell shape and size abnormality, multiseptation and anucleated cell formation due to abnormality in septum positioning. These phenotypes were suppressed by human B' cDNA, indicating the striking conservation of the B' function from yeast to human. Over-expression of fission yeast B' led to growth defects, a loss of cell shape polarity, septal abnormality and anucleated cell formation. Deltapbp1Deltapbp2 and pbp1 null haploids were hypersensitive to calcineurin inhibitors, cyclosporin A and FK506, with which the mutants underwent arrest at post-anaphase and cell lysis. Double disruption of calcineurin and pbp1+, but not pbp2+, genes led to synthetic lethality. CONCLUSION: The fission yeast B' subunit of PP2A plays critical roles in cell shape control and septum formation, and shares essential functions with calcineurin for viability, possibly through their roles in cytokinesis and cell wall integrity.
Asunto(s)
Proteínas Bacterianas , Calcineurina/metabolismo , Proteínas Portadoras/genética , Hexosiltransferasas , Mitosis/fisiología , Muramoilpentapéptido Carboxipeptidasa/genética , Peptidil Transferasas , Fosfoproteínas Fosfatasas/química , Fosfoproteínas Fosfatasas/fisiología , Proteínas de Saccharomyces cerevisiae , Schizosaccharomyces/genética , Secuencia de Aminoácidos , Secuencia de Bases , Calcineurina/genética , Calcineurina/fisiología , Proteínas Portadoras/fisiología , Dominio Catalítico/genética , Secuencia Conservada , Ciclosporina/metabolismo , Humanos , Immunoblotting , Muramoilpentapéptido Carboxipeptidasa/fisiología , Mutación , Proteínas de Unión a las Penicilinas , Fosfoproteínas Fosfatasas/genética , Reacción en Cadena de la Polimerasa , Pruebas de Precipitina , Proteína Fosfatasa 2 , Subunidades de Proteína , Mapeo Restrictivo , Schizosaccharomyces/citología , Schizosaccharomyces/fisiología , Especificidad por Sustrato , TemperaturaRESUMEN
An internal segment of the penicillin-binding protein gene, pbpA, of Streptomyces griseus was amplified from genomic DNA using the polymerase chain reaction and used as a hybridization probe to isolate the complete gene from a cosmid library. pbpA encodes a 485 amino acid sequence that conserves three motifs of PBPs, SXXK, SXN, and KTG. The pbpA gene was located downstream of a gene homologous to the Bacillus subtilis spoVE gene. The pbpA gene was disrupted by replacing an ApaI fragment of the pbpA gene in S. griseus chromosome with an apramycin resistance gene cassette or directly inserting this apramycin resistance gene cassette at the NcoI site of pbpA penicillin-binding domain. No obvious defects in growth, sporulation, or spore sonication resistance were observed in the constructed pbpA mutants, suggesting that PBPA is not essential for growth and sporulation under normal laboratory conditions in S. griseus.
Asunto(s)
Proteínas Bacterianas , Proteínas Portadoras/genética , Hexosiltransferasas , Muramoilpentapéptido Carboxipeptidasa/genética , Peptidil Transferasas , Streptomyces griseus/genética , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Secuencia de Bases , Southern Blotting , Proteínas Portadoras/química , Proteínas Portadoras/fisiología , Clonación Molecular , Genes Bacterianos , Datos de Secuencia Molecular , Muramoilpentapéptido Carboxipeptidasa/química , Muramoilpentapéptido Carboxipeptidasa/fisiología , Mutagénesis Insercional , Proteínas de Unión a las Penicilinas , Fenotipo , Reacción en Cadena de la Polimerasa , Esporas Bacterianas/fisiología , Streptomyces griseus/química , Streptomyces griseus/fisiologíaRESUMEN
Penicillin-binding proteins (PBPs) are involved in the regulation of beta-lactamase expression by determining the level of anhydromuramylpeptides in the periplasmatic space. It was hypothesized that one or more PBPs act as a sensor in the beta-lactamase induction pathway. We have performed induction studies with Escherichia coli mutants lacking one to four PBPs with DD-carboxypeptidase activity. Therefore, we conclude that a strong beta-lactamase inducer must inhibit all DD-carboxypeptidases as well as the essential PBPs 1a, 1b, and/or 2.
Asunto(s)
Proteínas Bacterianas , Proteínas Portadoras/fisiología , Enterobacter cloacae/enzimología , Hexosiltransferasas , Muramoilpentapéptido Carboxipeptidasa/fisiología , Peptidil Transferasas , beta-Lactamasas/biosíntesis , Carboxipeptidasas/antagonistas & inhibidores , Carboxipeptidasas/fisiología , Enterobacter cloacae/efectos de los fármacos , Inducción Enzimática , Pruebas de Sensibilidad Microbiana , Proteínas de Unión a las PenicilinasRESUMEN
Deletion of the mexAB-oprM multidrug efflux operon substantially compromised the beta-lactam resistance of beta-lactamase-derepressed mutants of Pseudomonas aeruginosa, although it had only a modest impact on resistance of a penicillin-binding protein mutant. This highlights the multifactorial nature of beta-lactam resistance in this organism. Moreover, the contribution of efflux to the net resistance seen in some beta-lactam-resistant mutants suggests that inhibition of MexAB-OprM-mediated drug efflux might be an effective approach to overcoming beta-lactam resistance attributed to efflux as well as to other mechanisms of beta-lactam resistance.
Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas , Proteínas Portadoras/fisiología , Hexosiltransferasas , Muramoilpentapéptido Carboxipeptidasa/fisiología , Operón , Peptidil Transferasas , Pseudomonas aeruginosa/efectos de los fármacos , beta-Lactamasas/fisiología , Farmacorresistencia Microbiana , Mutación , Proteínas de Unión a las Penicilinas , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/metabolismo , beta-LactamasRESUMEN
The penicillin binding proteins (PBPs) synthesize and remodel peptidoglycan, the structural component of the bacterial cell wall. Much is known about the biochemistry of these proteins, but little is known about their biological roles. To better understand the contributions these proteins make to the physiology of Escherichia coli, we constructed 192 mutants from which eight PBP genes were deleted in every possible combination. The genes encoding PBPs 1a, 1b, 4, 5, 6, and 7, AmpC, and AmpH were cloned, and from each gene an internal coding sequence was removed and replaced with a kanamycin resistance cassette flanked by two res sites from plasmid RP4. Deletion of individual genes was accomplished by transferring each interrupted gene onto the chromosome of E. coli via lambda phage transduction and selecting for kanamycin-resistant recombinants. Afterwards, the kanamycin resistance cassette was removed from each mutant strain by supplying ParA resolvase in trans, yielding a strain in which a long segment of the original PBP gene was deleted and replaced by an 8-bp res site. These kanamycin-sensitive mutants were used as recipients in further rounds of replacement mutagenesis, resulting in a set of strains lacking from one to seven PBPs. In addition, the dacD gene was deleted from two septuple mutants, creating strains lacking eight genes. The only deletion combinations not produced were those lacking both PBPs 1a and 1b because such a combination is lethal. Surprisingly, all other deletion mutants were viable even though, at the extreme, 8 of the 12 known PBPs had been eliminated. Furthermore, when both PBPs 2 and 3 were inactivated by the beta-lactams mecillinam and aztreonam, respectively, several mutants did not lyse but continued to grow as enlarged spheres, so that one mutant synthesized osmotically resistant peptidoglycan when only 2 of 12 PBPs (PBPs 1b and 1c) remained active. These results have important implications for current models of peptidoglycan biosynthesis, for understanding the evolution of the bacterial sacculus, and for interpreting results derived by mutating unknown open reading frames in genome projects. In addition, members of the set of PBP mutants will provide excellent starting points for answering fundamental questions about other aspects of cell wall metabolism.
Asunto(s)
Proteínas Bacterianas , Proteínas Portadoras/fisiología , Dipeptidasas , Proteínas de Escherichia coli , Escherichia coli/fisiología , Hexosiltransferasas/fisiología , Complejos Multienzimáticos/fisiología , Muramoilpentapéptido Carboxipeptidasa/fisiología , Mutación , Peptidoglicano/biosíntesis , Peptidil Transferasas/fisiología , beta-Lactamasas/fisiología , Bacteriólisis/genética , Endopeptidasas , Evolución Molecular , Eliminación de Gen , Regulación Bacteriana de la Expresión Génica , Modelos Biológicos , Presión Osmótica , Proteínas de Unión a las PenicilinasRESUMEN
The loss of Bacillus subtilis penicillin-binding protein (PBP) 2a, encoded by pbpA, was previously shown to slow spore outgrowth and result in an increased diameter of the outgrowing spore. Further analyses to define the defect in pbpA spore outgrowth have shown that (i) outgrowing pbpA spores exhibited only a slight defect in the rate of peptidoglycan (PG) synthesis compared to wild-type spores, but PG turnover was significantly slowed during outgrowth of pbpA spores; (ii) there was no difference in the location of PG synthesis in outgrowing wild-type and pbpA spores once cell elongation had been initiated; (iii) outgrowth and elongation of pbpA spores were dramatically affected by the levels of monovalent or divalent cations in the medium; (iv) there was a partial redundancy of function between PBP2a and PBP1 or -4 during spore outgrowth; and (v) there was no difference in the structure of PG from outgrowing wild-type spores or spores lacking PBP2a or PBP2a and -4; but also (vi) PG from outgrowing spores lacking PBP1 and -2a had transiently decreased cross-linking compared to PG from outgrowing wild-type spores, possibly due to the loss of transpeptidase activity.
Asunto(s)
Bacillus subtilis/fisiología , Proteínas Bacterianas , Proteínas Portadoras/fisiología , Hexosiltransferasas , Muramoilpentapéptido Carboxipeptidasa/fisiología , Peptidil Transferasas , Bacillus subtilis/ultraestructura , Conformación de Carbohidratos , Cationes Bivalentes , Cationes Monovalentes , Proteínas de Unión a las Penicilinas , Peptidoglicano/química , Peptidoglicano/metabolismo , Esporas BacterianasRESUMEN
pbpA, a gene encoding penicillin-binding protein (PBP) 1 of Staphylococcus aureus, was cloned in an Escherichia coli MC1061 transformant which grew on a plate containing 512 microg of vancomycin per ml. This gene encodes a 744-amino-acid sequence which conserves three motifs of PBPs, SXXK, SXN, and KTG. The chromosomal copy of pbpA could be disrupted only when RN4220, a methicillin-sensitive S. aureus strain, had additional copies of pbpA in its episome. Furthermore, these episomal copies of pbpA could not be eliminated by an incompatible plasmid when the chromosomal copy of pbpA was disrupted beforehand. Based on these observations, we concluded that pbpA is essential for the growth of methicillin-sensitive S. aureus.
Asunto(s)
Proteínas Bacterianas , Proteínas Portadoras/genética , Hexosiltransferasas , Muramoilpentapéptido Carboxipeptidasa/genética , Peptidil Transferasas , Staphylococcus aureus/crecimiento & desarrollo , Secuencia de Aminoácidos , Secuencia de Bases , Proteínas Portadoras/fisiología , Clonación Molecular , Datos de Secuencia Molecular , Muramoilpentapéptido Carboxipeptidasa/fisiología , Proteínas de Unión a las Penicilinas , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Staphylococcus aureus/genéticaRESUMEN
Methicillin-resistant Staphylococcus aureus (MRSA) strains are among the most common nosocomial pathogens. The most significant mechanism of resistance to methicillin in this-species is the acquisition of a genetic determinant (mecA gene). However, resistance seems to have a more complex molecular basis, since additional chromosomal material is involved in such resistance. Besides, overproduction of penicillinase and/or alterations in the PBPs can contribute to the formation of resistance phenotypes. Genetic and environmental factors leading to MRSA are reviewed.
Asunto(s)
Proteínas Bacterianas/fisiología , Proteínas Portadoras/fisiología , Hexosiltransferasas , Resistencia a la Meticilina/fisiología , Muramoilpentapéptido Carboxipeptidasa/fisiología , Peptidil Transferasas , Staphylococcus aureus/efectos de los fármacos , Proteínas Bacterianas/genética , Proteínas Portadoras/genética , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Resistencia a la Meticilina/genética , Muramoilpentapéptido Carboxipeptidasa/genética , Proteínas de Unión a las Penicilinas , Fenotipo , Staphylococcus aureus/genéticaRESUMEN
Meningococcal moderate resistance to penicillin (MICs 0.12 to 1 mg/l) was rarely reported before the 1980's in Spain. The frequency of isolation of resistant strains increased from 0.4% in 1985 to 42.6% in 1990. In the last few years, these strains have been reported in several countries, which suggests a change in the meningococcal response to penicillin. The resistance is due, at least in part, to a decreased affinity of penicillin binding protein 2 (PBP2) for penicillin. This decreased affinity has also been found in commensal Neisseriae. Population genetic studies demonstrate that recombinational events, replacing parts of the PBP2 gene by the corresponding regions of commensal species, followed by a rapid spread of the clones could be the origin of such resistant strains.