RESUMEN
Background: Interventions are needed to reduce unnecessary antibiotic prescribing for respiratory tract infections (RTIs). Although community antibiotic prescribing appears to be decreasing in the UK, figures for out-of-hours (OOH) prescribing have substantially increased. Understanding the factors influencing prescribing in OOH and any perceived differences between general practitioner (GP) and nurse prescriber (NP) prescribing habits may enable the development of tailored interventions promoting optimal prescribing in this setting. Objectives: To explore UK GP and NP views on and experiences of prescribing antibiotics for RTIs in primary care OOH services. Methods: Thirty semi-structured interviews were conducted with GPs and NPs working in primary care OOH services. Inductive thematic analysis was used to analyse data. Results: The research shows that factors particular to OOH influence antibiotic prescribing, including a lack of patient follow-up, access to patient GP records, consultation time, working contracts and implementation of feedback, audit and supervision. NPs reported perceptions of greater accountability for their prescribing compared with GPs and reported they had longer consultations during which they were able to discuss decisions with patients. Participants agreed that more complex cases should be seen by GPs and highlighted the importance of consistency of decision making, illness explanations to patients as well as a perception that differences in clinical training influence communication with patients and antibiotic prescribing decisions. Conclusions: Environmental and social factors in OOH services and a mixed healthcare workforce provide unique influences on antibiotic prescribing for RTIs, which would need to be considered in tailoring interventions that promote prudent antibiotic prescribing in OOH services.
Asunto(s)
Atención Posterior/estadística & datos numéricos , Antibacterianos/uso terapéutico , Prescripciones de Medicamentos/estadística & datos numéricos , Enfermeras Practicantes/estadística & datos numéricos , Pautas de la Práctica en Enfermería/estadística & datos numéricos , Pautas de la Práctica en Medicina/estadística & datos numéricos , Infecciones del Sistema Respiratorio/tratamiento farmacológico , Adulto , Anciano , Actitud del Personal de Salud , Femenino , Medicina General/métodos , Medicina General/estadística & datos numéricos , Médicos Generales , Humanos , Masculino , Persona de Mediana Edad , Atención Primaria de Salud/métodos , Atención Primaria de Salud/estadística & datos numéricos , Reino UnidoRESUMEN
AIMS: The purpose of this study was to determine the optimal clinical and cost-effective strategy for managing people following ACL rupture. METHODS: A systematic review of the published (AMED, CINAHL, MEDLINE, EMBASE, PubMed, psycINFO and the Cochrane Library) and unpublished literature (OpenGrey, the WHO International Clinical Trials Registry Platform, Current Controlled Trials and the UK National Research Register Archive) was conducted on April 2013. All randomised and non-randomised controlled trials evaluating clinical or health economic outcomes of isolated ligament reconstruction versus non-surgical management following ACL rupture were included. Methodological quality was assessed using the PEDro appraisal tool. When appropriate, meta-analysis was conducted to pool data. RESULTS: From a total of 943 citations, sixteen studies met the eligibility criteria. These included 1397 participants, 825 who received ACL reconstruction versus 592 who were managed non-surgically. The methodological quality of the literature was poor. The findings indicated that whilst reconstructed ACL offers significantly greater objective tibiofemoral stability (p<0.001), there appears limited evidence to suggest a superiority between reconstruction versus non-surgical management in functional outcomes. There was a small difference between the management strategies in respect to the development of osteoarthritis during the initial 20 years following index management strategy (Odds Ratio 1.56; p=0.05). CONCLUSIONS: The current literature is insufficient to base clinical decision-making with respect to treatment opinions for people following ACL rupture. Whilst based on a poor evidence, the current evidence would indicate that people following ACL rupture should receive non-operative interventions before surgical intervention is considered.
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Lesiones del Ligamento Cruzado Anterior , Reconstrucción del Ligamento Cruzado Anterior , Ligamento Cruzado Anterior/cirugía , Traumatismos de la Rodilla/terapia , Plastía con Hueso-Tendón Rotuliano-Hueso , Análisis Costo-Beneficio , Toma de Decisiones , Humanos , Inestabilidad de la Articulación/etiología , Traumatismos de la Rodilla/diagnóstico , Osteoartritis de la Rodilla/etiología , Evaluación del Resultado de la Atención al Paciente , Modalidades de Fisioterapia , Rango del Movimiento Articular , Rotura/terapia , Tendones/trasplanteRESUMEN
The purpose of this study was to assess the effectiveness of such proprioceptive exercise following ankle ligament injury. A systematic review of the databases MEDLINE, EMBASE, CINHAL, AMED, the Cochrane library database and the PEDro database, in addition to unpublished literature databases was conducted to July 2011. When appropriate, meta-analysis was conducted to pool results from homogeneous studies. The methodological quality of the literature was reviewed using the Critical Appraisal Skills Programme tool. The results indicated that there is no statistically significant difference in recurrent injury between the addition of proprioceptive exercises during the rehabilitation of patients following ankle ligament injury (p = 0.68). The addition of proprioceptive training demonstrated a significant reduction in subjective instability and functional outcomes (p < 0.05). There was no consensus on the advantages of including proprioceptive training in the rehabilitation of this population for swelling, postural sway, joint position sense, ankle range of motion or return to sport outcomes. Further study is warranted to develop the rigour of the evidence-base and to determine the optimal proprioceptive training programme following ankle ligament injury with different populations.
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Traumatismos del Tobillo/rehabilitación , Terapia por Ejercicio/métodos , Inestabilidad de la Articulación/rehabilitación , Ligamentos Articulares/lesiones , Propiocepción , Adulto , Traumatismos del Tobillo/diagnóstico , Femenino , Humanos , Puntaje de Gravedad del Traumatismo , Inestabilidad de la Articulación/fisiopatología , Masculino , Pronóstico , Ensayos Clínicos Controlados Aleatorios como Asunto , Rango del Movimiento Articular/fisiología , Resultado del Tratamiento , Adulto JovenRESUMEN
As bacteria need iron from the environment to survive, they have evolved active iron transporter proteins in their outer membranes. In her Perspective, Postle discusses new insights into iron transport revealed by the crystal structure of the iron transporter FecA in E. coli (Ferguson et al.).
Asunto(s)
Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Escherichia coli/metabolismo , Compuestos Férricos/metabolismo , Receptores de Superficie Celular , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Bacterianas/metabolismo , Sitios de Unión , Transporte Biológico Activo , Membrana Celular/metabolismo , Cristalografía por Rayos X , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Activación del Canal Iónico , Ligandos , Proteínas de la Membrana/metabolismo , Modelos Biológicos , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Sideróforos/metabolismoRESUMEN
BACKGROUND: Nurse-led intermediate care units are being set up across the UK primarily as potential solutions to hospital bed crises. AIMS: This paper draws on data collected as part of a comprehensive evaluation of one 10-bedded nurse-led unit (NLU) located in the South of England. It explores the potential for enhanced nursing roles provided by such units by focusing on the views of NLU nursing staff and other professional groups within the Hospital Trust where the unit is located. METHODS: A total of 38 in-depth audio-taped qualitative interviews were conducted with NLU nursing staff and with a range of other professional groups (managers, acute ward nurses and doctors). FINDINGS: These data indicated that models of nurse-led postacute care do provide opportunities for nurses to develop enhanced nursing roles in which care associated with concepts of therapeutic nursing can be provided. However, even though the nurses derived satisfaction from their work on the NLU this model of care was seen by junior and middle grade nurses and other professional groups as being of low status. In contrast to senior nurses' views, they did not equate work on the NLU with the continuing professionalization of nursing. Senior nurses viewed the route to developing nursing on the NLU as involving nurses as doctor substitutes (extended roles) rather than as working in separate but complementary therapeutic domains (enhanced roles). CONCLUSIONS: NLUs provide opportunities for nurses to develop enhanced roles in which they can work autonomously in providing elements of therapeutic nursing aimed at improving patient outcomes at discharge. However, education, training and leadership will be needed to ensure that such opportunities are well understood and are optimized to the benefit of nurses and their patients.
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Actitud del Personal de Salud , Instituciones de Cuidados Intermedios/organización & administración , Personal de Enfermería en Hospital/organización & administración , Inglaterra , Humanos , Satisfacción en el Trabajo , Relaciones Enfermero-Paciente , Relaciones Médico-Enfermero , Autonomía Profesional , Evaluación de Programas y Proyectos de Salud , RolRESUMEN
The cytoplasmic membrane protein TonB couples the proton electrochemical potential of the cytoplasmic membrane to transport events at the outer membrane of Gram-negative bacteria. The amino-terminal signal anchor of TonB and its interaction with the cytoplasmic membrane protein ExbB are essential to this process. The TonB signal anchor is predicted to form an alpha-helix, with a conserved face comprised of residues Ser(16), His(20), Leu(27), and Ser(31). Deletion of either Ser(16) or His(20) or of individual intervening but not flanking residues rendered TonB inactive and unable to assume a proton motive force-dependent conformation. In vivo formaldehyde cross-linking experiments revealed that the ability of this subset of mutants to form a characteristic heterodimer with ExbB was greatly diminished. Replacement of residues 17-19 by three consecutive alanines produced a wild type TonB allele, indicating that the intervening residues (Val, Cys, and Ile) contributed only to spacing. These data indicated that the spatial relationship of Ser(16) to His(20) was essential to function and suggested that the motif HXXXS defines the minimal requirement for the coupling of TonB to the cytoplasmic membrane electrochemical gradient. Deletion of Trp(11) resulted in a TonB that remained active yet was unable to cross-link with ExbB. Because Trp(11) was demonstrably not involved in the actual cross-linking, these results suggest that the TonB/ExbB interaction detected by cross-linking occurred at a step in the energy transduction cycle distinct from the coupling of TonB to the electrochemical gradient.
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Proteínas Bacterianas/química , Proteínas de Escherichia coli , Proteínas de la Membrana/química , Proteínas Bacterianas/fisiología , Proteínas de la Membrana/fisiología , Mutagénesis Sitio-Dirigida , Conformación Proteica , Fuerza Protón-Motriz , Relación Estructura-Actividad , TermodinámicaAsunto(s)
Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/metabolismo , Escherichia coli/química , Escherichia coli/metabolismo , Periplasma/química , Proteínas de la Membrana Bacteriana Externa/genética , Transporte Biológico , Colicinas/metabolismo , Cristalografía por Rayos X , Escherichia coli/genética , Proteínas de Escherichia coli , Proteínas de Transporte de Membrana , Modelos Moleculares , Mutación/genética , Periplasma/metabolismo , Fenotipo , Estructura Secundaria de Proteína , Relación Estructura-ActividadRESUMEN
The objectives of this study were to discover the views of patients about the effects of breast-reduction surgery carried out 2 years previously, to detect any change from perceptions at 3-6 months after surgery, and to determine whether the benefits of this operation are maintained long term. Qualitative research methods were employed, comprising: (a) an open-format survey of opinions; (b) semi-structured telephone interviews with a smaller number of patients; and (c) assessment of self-concept using a well-known scalar measure (the Rosenberg Self-esteem Scale). The subjects were 93 patients treated at the regional Plastic Surgery Service in Salisbury, who had previously participated in a quantitative study at 3-6 months after surgery. Sixty patients responded to the 2-year follow-up. Benefits of breast reduction most valued by patients did not change significantly with time and were: relief of pain and discomfort, which led to increased physical activity and better general health; greatly increased choice and fit of attractive clothes and underwear; improved personal and social life, leading to enhanced relationships with partner or friends; and greatly improved self-confidence in all areas of life. The interaction of all these factors led to improved self-image and improved quality of life. The main disadvantage of the operation for a small number of patients was the persistence of painful, disfiguring scarring which in two cases had a detrimental effect on social and personal relationships and led to a deterioration in quality of life. Improvement in self-esteem after surgery was maintained in 55 out of the 60 2-year responders. The results indicate that breast reduction confers significant health gains which are maintained in the long term.
Asunto(s)
Mamoplastia/rehabilitación , Satisfacción del Paciente , Adolescente , Adulto , Anciano , Peso Corporal , Cicatriz/etiología , Ejercicio Físico , Femenino , Estudios de Seguimiento , Humanos , Relaciones Interpersonales , Mamoplastia/efectos adversos , Mamoplastia/psicología , Persona de Mediana Edad , Periodo Posoperatorio , Calidad de Vida , AutoimagenRESUMEN
TonB couples the cytoplasmic membrane protonmotive force (pmf) to active transport across the outer membrane, potentially through a series of conformational changes. Previous studies of a TonB transmembrane domain mutant (TonB-delta V17) and its phenotypical suppressor (ExbB-A39E) suggested that TonB is conformationally sensitive. Here, two new mutations of the conserved TonB transmembrane domain SHLS motif were isolated, TonB-S16L and -H20Y, as were two new suppressors, ExbB-V35E and -V36D. Each suppressor ExbB restored at least partial function to the TonB mutants, although TonB-delta V17, for which both the conserved motif and the register of the predicted transmembrane domain alpha-helix are affected, was the most refractory. As demonstrated previously, TonB can undergo at least one conformational change, provided both ExbB and a functional TonB transmembrane domain are present. Here, we show that this conformational change reflects the ability of TonB to respond to the cytoplasmic membrane proton gradient, and occurs in proportion to the level of TonB activity attained by mutant-suppressor pairs. The phenotype of TonB-delta V17 was more complex than the -S16L and -H20Y mutations, in that, beyond the inability to be energized efficiently, it was also conditionally unstable. This second defect was evident only after suppression by the ExbB mutants, which allow transmembrane domain mutants to be energized, and presented as the rapid turnover of TonB-delta V17. Importantly, this degradation was dependent upon the presence of a TonB-dependent ligand, suggesting that TonB conformation also changes following the energy transduction event. Together, these observations support a dynamic model of energy transduction in which TonB cycles through a set of conformations that differ in potential energy, with a transition to a higher energy state driven by pmf and a transition to a lower energy state accompanying release of stored potential energy to an outer membrane receptor.
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Proteínas de la Membrana Bacteriana Externa , Proteínas Bacterianas/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Escherichia coli , Proteínas de la Membrana/metabolismo , Fuerza Protón-Motriz/fisiología , Receptores de Superficie Celular , Membrana Celular/metabolismo , Fraccionamiento Químico , Citoplasma/metabolismo , Endopeptidasa K/farmacología , Transferencia de Energía/fisiología , Enterobactina/farmacología , Modelos Biológicos , Mutagénesis , Mutación Puntual , Conformación Proteica , Supresión Genética , Factores de TiempoAsunto(s)
Bacterias/metabolismo , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Proteínas de Escherichia coli , Receptores de Superficie Celular , Receptores Virales/química , Receptores Virales/metabolismo , Secuencia de Aminoácidos , Proteínas de la Membrana Bacteriana Externa/genética , Transporte Biológico Activo , Proteínas Portadoras/genética , Genes Bacterianos , Datos de Secuencia Molecular , Receptores Virales/genéticaRESUMEN
The cytoplasmic membrane proteins ExbB and ExbD support TonB-dependent active transport of iron siderophores and vitamin B12 across the essentially unenergized outer membrane of Escherichia coli. In this study, in vivo formaldehyde cross-linking analysis was used to investigate the interactions of T7 epitope-tagged ExbB or ExbD proteins. ExbB and ExbD each formed two unique cross-linked complexes which were not dependent on the presence of TonB, the outer membrane receptor protein FepA, or the other Exb protein. Cross-linking analysis of ExbB- and ExbD-derived size variants demonstrated instead that these ExbB and ExbD complexes were homodimers and homotrimers and suggested that ExbB also interacted with an unidentified protein(s). Cross-linking analysis of epitope-tagged ExbB and ExbD proteins with TonB antisera afforded detection of a previously unrecognized TonB-ExbD cross-linked complex and confirmed the composition of the TonB-ExbB cross-linked complex. The implications of these findings for the mechanism of TonB-dependent energy transduction are discussed.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa , Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli , Proteínas de la Membrana/metabolismo , Bacteriófago T7 , Proteínas Portadoras/metabolismo , Reactivos de Enlaces Cruzados , Dimerización , Transferencia de Energía , Escherichia coli/metabolismo , Vectores Genéticos , Receptores de Superficie Celular/metabolismo , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
The ferrichrome-iron receptor of Escherichia coli is FhuA, an outer membrane protein that is dependent upon the energy-coupling protein TonB to enable active transport of specific hydroxamate siderophores, infection by certain phages, and cell killing by the protein antibiotics colicin M and microcin 25. In vivo cross-linking studies were performed to establish at the biochemical level the interaction between FhuA and TonB. In an E. coli strain in which both proteins were expressed from the chromosome, a high molecular mass complex was detected when the ferrichrome homologue ferricrocin was added immediately prior to addition of cross-linker. The complex included both proteins; it was absent from strains of E. coli that were devoid of either FhuA or TonB, and it was detected with anti-FhuA and anti-TonB monoclonal antibodies. These results indicate that, in vivo, the binding of ferricrocin to FhuA enhances complex formation between the receptor and TonB. An in vitro system was established with which to examine the FhuA-TonB interaction. Incubation of TonB with histidine-tagged FhuA followed by addition of Ni2+-nitrilotriacetate-agarose led to the specific recovery of both TonB and FhuA. Addition of ferricrocin or colicin M to FhuA in this system greatly increased the coupling between FhuA and TonB. Conversely, a monoclonal antibody that binds near the N terminus of FhuA reduced the retention of TonB by histidine-tagged FhuA. These studies demonstrate the significance of ligand binding at the external surface of the cell to mediate signal transduction across the outer membrane.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Proteínas de la Membrana/metabolismo , Receptores de Superficie Celular , Receptores Virales/metabolismo , Transducción de Señal , Animales , Transporte Biológico , Proteínas Portadoras/metabolismo , Ferricromo/análogos & derivados , Ferricromo/metabolismo , Cabras , Ligandos , Ratones , Unión Proteica , Conformación ProteicaRESUMEN
The transport of Fe(III)-siderophore complexes and vitamin B12 across the outer membrane of Escherichia coli is an active transport process requiring a cognate outer membrane receptor, cytoplasmic membrane-derived proton motive force, and an energy-transducing protein anchored in the cytoplasmic membrane, TonB. This process requires direct physical contact between the outer membrane receptor and TonB. Previous studies have identified an amino-terminally located region (termed the TonB box) conserved in all known TonB-dependent outer membrane receptors as being essential for productive energy transduction. In the present study, a mutation in the TonB box of the ferric enterochelin receptor FepA resulted in the loss of detectable in vivo chemical cross-linking between FepA and TonB. Protease susceptibility studies indicated this effect was due to an alteration of conformation rather than the direct disruption of a specific site of physical contact. This suggested that TonB residue 160, implicated in previous studies as a site of allele-specific suppression of TonB box mutants, also made a conformational rather than a direct contribution to the physical interaction between TonB and the outer membrane receptors. This possibility was supported by the finding that TonB carboxyl-terminal truncations that retained Gln-160 were unable to participate in TonB-FepA complex formation, indicating that this site alone was not sufficient to support the physical interactions involved in energy transduction. These studies indicated that the final 48 residues of TonB were essential to this physical interaction. This region contains a putative amphipathic helix which could facilitate TonB-outer membrane interaction. Amino acid replacements at one site in this region were found to affect energy transduction but did not appear to greatly alter TonB conformation or the formation of a TonB-FepA complex. The effects of amino acid substitutions at several other TonB sites were also examined.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa , Proteínas Bacterianas/química , Proteínas Bacterianas/fisiología , Proteínas Portadoras/química , Proteínas Portadoras/fisiología , Proteínas de Escherichia coli , Proteínas de la Membrana/química , Proteínas de la Membrana/fisiología , Receptores de Superficie Celular , Proteínas Bacterianas/genética , Transporte Biológico , Proteínas Portadoras/genética , Reactivos de Enlaces Cruzados , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/fisiología , Formaldehído , Genes Bacterianos , Isoleucina/genética , Proteínas de la Membrana/genética , Mutación , Prolina/genética , Conformación Proteica , Supresión Genética , Transformación BacterianaRESUMEN
The energy source for active transport of iron-siderophore complexes and vitamin B12 across the outer membrane in Gram-negative bacteria is the cytoplasmic membrane proton-motive force (pmf). TonB protein is required in this process to transduce cytoplasmic membrane energy to the outer membrane. In this study, Escherichia coli TonB was found to be distributed in sucrose density gradients approximately equally between the cytoplasmic membrane and the outer membrane fractions, while two proteins with which it is known to interact, ExbB and ExbD, as well as the NADH oxidase activity characteristic of the cytoplasmic membrane, were localized in the cytoplasmic membrane fraction. Neither the N-terminus of TonB nor the cytoplasmic membrane pmf, both of which are essential for TonB activity, were required for TonB to associate with the outer membrane. When the TonB C-terminus was absent, TonB was found associated with the cytoplasmic membrane, suggesting that the C-terminus was required for outer membrane association. When ExbB and ExbD, as well as their cross-talk-competent homologues ToIQ and ToIR, were absent, TonB was found associated with the outer membrane. TetA-TonB protein, which cannot interact with ExbB/D, was likewise found associated with the outer membrane. These results indicated that the role of ExbB/D in energy transduction is to bring TonB that has reached the outer membrane back to associate with the cytoplasmic membrane. Two possible explanations exist for the observations presented in this study. One possibility is that TonB transduces energy by shuttling between membranes, and, at some stages in the energy-transduction cycle, is associated with either the cytoplasmic membrane or the outer membrane, but not with both at the same time. This hypothesis, together with the alternative interpretation that TonB remains localized in the cytoplasmic membrane and changes its affinity for the outer and cytoplasmic membrane during energy transduction, are incorporated with previous observations into two new models, consistent with the novel aspects of this system, that describe a mechanism for TonB-dependent energy transduction.
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Proteínas Bacterianas/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Proteínas de Escherichia coli , Escherichia coli/química , Escherichia coli/metabolismo , Proteínas de la Membrana/metabolismo , Antiportadores/metabolismo , Proteínas de la Membrana Bacteriana Externa/análisis , Proteínas de la Membrana Bacteriana Externa/aislamiento & purificación , Proteínas Bacterianas/fisiología , Proteínas de la Membrana/fisiología , Complejos Multienzimáticos/metabolismo , NADH NADPH Oxidorreductasas/metabolismo , Fuerza Protón-MotrizRESUMEN
The transport of Fe(III)-siderophore complexes and vitamin B12 across the outer membrane of Escherichia coli requires the TonB-dependent energy transduction system. A set of murine monoclonal antibodies (MAbs) was generated against an E. coli TrpC-TonB fusion protein to facilitate structure and function studies. In the present study, the epitopes recognized by these MAbs were mapped, and their distribution in gram-negative organisms was examined. Cross-species reactivity patterns obtained against TonB homologs of known sequence were used to refine epitope mapping, with some epitopes ultimately confirmed by inhibition experiments using synthetic polypeptides. Epitopes recognized by this set of MAbs were conserved in TonB homologs for 9 of 12 species in the family Enterobacteriaceae (including E. coli), including previously unidentified TonB homologs in Shigella, Citrobacter, Proteus, and Kluyvera species. These homologs were also detected by a polyclonal alpha-TrpC-TonB serum that additionally recognized the known Yersinia enterocolitica TonB homolog and a putative TonB homolog in Edwardsiella tarda. These antibody preparations failed to detect the known TonB homologs of either Pseudomonas putida or Haemophilus influenzae but did identify potential TonB homologs in several other nonenteric gram-negative species. In vivo chemical cross-linking experiments demonstrated that in addition to TonB, auxiliary components of the TonB-dependent energy transduction system are broadly conserved in members of the family Enterobacteriaceae, suggesting that the TonB system represents a common system for high-affinity active transport across the gram-negative outer membrane.
Asunto(s)
Proteínas Bacterianas/inmunología , Metabolismo Energético , Enterobacteriaceae/inmunología , Mapeo Epitopo , Proteínas de Escherichia coli , Proteínas de la Membrana/inmunología , Secuencia de Aminoácidos , Anticuerpos Antibacterianos , Anticuerpos Monoclonales , Especificidad de Anticuerpos , Antígenos Bacterianos , Proteínas Bacterianas/genética , Secuencia de Bases , Secuencia Conservada , Reacciones Cruzadas , Enterobacteriaceae/genética , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Fragmentos de Péptidos/inmunología , Especificidad de la EspecieRESUMEN
TonB protein appears to couple the electrochemical potential of the cytoplasmic membrane to active transport across the essentially unenergized outer membrane of gram-negative bacteria. ExbB protein has been identified as an auxiliary protein in this process. In this paper we show that ExbD protein, encoded by an adjacent gene in the exb cluster at 65', was also required for TonB-dependent energy transduction and, like ExbB, was required for the stability of TonB. The phenotypes of exbB exbD+ strains were essentially indistinguishable from the phenotypes of exbB+ exbD strains. Mutations in either gene resulted in the degradation of TonB protein and in decreased, but not entirely absent, sensitivities to colicins B and Ia and to bacteriophage phi 80. Evidence that the absence of ExbB or ExbD differentially affected the half-lives of newly synthesized and steady-state TonB was obtained. In the absence of ExbB or ExbD, newly synthesized TonB was degraded with a half-life of 5 to 10 min, while the half-life of TonB under steady-state conditions was significantly longer, approximately 30 min. These results were consistent with the idea that ExbB and ExbD play roles in the assembly of TonB into an energy-transducing complex. While interaction between TonB and ExbD was suggested by the effect of ExbD on TonB stability, interaction of ExbD with TonB was detected by neither in vivo cross-linking assays nor genetic tests for competition. Assays of a chromosomally encoded exbD::phoA fusion showed that exbB and exbD were transcribed as an operon, such that ExbD-PhoA levels in an exbB::Tn10 strain were reduced to 4% of the levels observed in an exbB+ strain under iron-limiting conditions. Residual ExbD-PhoA expression in an exbB::Tn10 strain was not iron regulated and may have originated from within the Tn10 element in exbB.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli , Escherichia coli/genética , Proteínas de la Membrana/metabolismo , Operón/genética , Fosfatasa Alcalina/genética , Proteínas Bacterianas/biosíntesis , Transporte Biológico , Semivida , Mutación , Fenotipo , Regiones Promotoras Genéticas/genética , Proteínas Recombinantes de Fusión/biosíntesisRESUMEN
Active transport of vitamin B12 and Fe(III)-siderophore complexes across the outer membrane of Escherichia coli appears to be dependent upon the ability of the TonB protein to couple cytoplasmic membrane-generated protonmotive force to outer membrane receptors. TonB is supported in this role by an auxiliary protein, ExbB, which, in addition to stabilizing TonB against the activities of endogenous envelope proteases, directly contributes to the energy transduction process. The topological partitioning of TonB and ExbB to either side of the cytoplasmic membrane restricts the sites of interaction between these proteins primarily to their transmembrane domains. In this study, deletion of valine 17 within the aminoterminal transmembrane anchor of TonB resulted in complete loss of TonB activity, as well as loss of detectable in vivo crosslinking into a 59 kDa complex believed to contain ExbB. The delta V17 mutation had no effect on TonB export. The loss of crosslinking appeared to reflect conformational changes in the TonB/ExbB pair rather than loss of interaction since ExbB was still required for some stabilization of TonB delta V17. Molecular modeling suggested that the delta V17 mutation caused a significant change in the predicted conserved face of the TonB amino-terminal membrane anchor. TonB delta V17 was unable to achieve the 23 kDa proteinase K-resistant form in lysed sphaeroplasts that is characteristic of active TonB. Wild-type TonB also failed to achieve the proteinase K-resistant configuration when ExbB was absent. Taken together these results suggested that the delta V17 mutation interrupted productive TonB-ExbB interactions. The apparent ability to crosslink to ExbB as well as a limited ability to transduce energy were restored by a second mutation (A39E) in or near the first predicted transmembrane domain of the ExbB protein. Consistent with the weak suppression, a 23 kDa proteinase K-resistant form of TonB delta V17 was not observed in the presence of ExbBA39E. Neither the ExbBA39E allele nor the absence of ExbB affected TonB or TonB delta V17 export. Unlike the tonB delta V17 mutation, the exbBA39E mutation did not greatly alter a modelled ExbB transmembrane domain structure. Furthermore, the suppressor ExbBA39E functioned normally with wild-type TonB, suggesting that the suppressor was not allele specific. Contrary to expectations, the TonB delta V17, ExbBA39E pair resulted in a TonB with a greatly reduced half-life (approximately 10 min). These results together with protease susceptibility studies suggest that ExbB functions by modulating the conformation of TonB.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Proteínas de la Membrana/genética , Supresión Genética , Secuencia de Aminoácidos , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Transporte Biológico , Reactivos de Enlaces Cruzados , Metabolismo Energético , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Reacción en Cadena de la Polimerasa , Unión Proteica , Proteínas Recombinantes/metabolismo , Análisis de Secuencia de ADN , Relación Estructura-Actividad , Valina/genéticaRESUMEN
Escherichia coli TonB protein is an energy transducer, coupling cytoplasmic membrane energy to active transport of vitamin B12 and iron-siderophores across the outer membrane. TonB is anchored in the cytoplasmic membrane by its hydrophobic amino terminus, with the remainder occupying the periplasmic space. In this report we establish several functions for the hydrophobic amino terminus of TonB. A G-26-->D substitution in the amino terminus prevents export of TonB, suggesting that the amino terminus contains an export signal for proper localization of TonB within the cell envelope. Substitution of the first membrane-spanning domain of the cytoplasmic membrane protein TetA for the TonB amino terminus eliminates TonB activity without altering TonB export, suggesting that the amino terminus contains sequence-specific information. Detectable TonB cross-linking to ExbB is also prevented, suggesting that the two proteins interact primarily through their transmembrane domains. In vivo cleavage of the amino terminus of TonB carrying an engineered leader peptidase cleavage site eliminates (i) TonB activity, (ii) detectable interaction with a membrane fraction having a density intermediate to those of the cytoplasmic and outer membranes, and (iii) cross-linking to ExbB. In contrast, the amino terminus is not required for cross-linking to other proteins with which TonB can form complexes, including FepA. Additionally, although the amino terminus clearly is a membrane anchor, it is not the only means by which TonB associates with the cytoplasmic membrane. TonB lacking its amino-terminal membrane anchor still remains largely associated with the cytoplasmic membrane.
Asunto(s)
Proteínas Bacterianas/fisiología , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Proteínas de la Membrana/fisiología , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Secuencia de Bases , Transporte Biológico/fisiología , Membrana Celular/metabolismo , Transferencia de Energía/fisiología , Proteínas de la Membrana/química , Datos de Secuencia Molecular , Vitamina B 12/metabolismoRESUMEN
Although iron is an essential nutrient, its toxicity at high levels necessitates regulated transport. In Gram-negative bacteria a central target for regulation is the TonB protein, an energy transducer that couples the cytoplasmic membrane proton motive force to active transport of (FeIII)-siderophore complexes across the outer membrane. We have previously demonstrated the threefold repression of tonB transcription by excess iron in the presence of Fur repressor protein under aerobic conditions. In this report, we examine tonB regulation under anaerobic conditions where the solubility of iron is not a limiting factor and, presumably, siderophore-mediated transport is not required. Under these conditions, tonB transcription is repressed at least 10-fold by excess iron in the presence of Fur, but can be fully derepressed in the absence of Fur. Based on several lines of evidence, this anaerobic repression is not due to increased negative supercoiling as previously postulated. Our results rule out both supercoiling-mediated decreased promoter function and increased Fur binding as mediators of anaerobic repression. Under iron-limiting anaerobic conditions tonB expression is as high or higher than under iron-limiting aerobic conditions, suggesting that promoter function has not decreased anaerobically. Furthermore, under anaerobic conditions in tonB+ strains, tonB promoter function is insensitive to the gyrase inhibitor novobiocin and to changes in medium osmolarity and temperature, three conditions known to change levels of supercoiling. We also rule out effects of mutations in arcA or fnr as mediators of anaerobic repression. Results from in vivo dimethyl sulphate protection foot-printing indicate that Fur binds to an operator site between the -10 and -35 regions of the promoter, but not to a less homologous operator site centered at +26. The binding is, if anything, weaker under anaerobic conditions, indicating that anaerobic repression is not mediated through Fur. Additional changes in the in vivo footprint upstream from the promoter implicate a second factor in tonB anaerobic repression. Together, these results suggest that the mechanism responsible for this regulation (and, by analogy, that of other anaerobically repressed, iron-regulated genes such as cir, exbB, and fhuA) is a novel one.