RESUMEN
Bacterial conjugation is an example of macromolecular trafficking between cells, based on the translocation of single-stranded DNA across membranes through a type IV secretion system. TrwBDeltaN70 is the soluble domain of TrwB, an essential integral membrane protein that couples the relaxosome (a nucleoprotein complex) to the DNA transport apparatus in plasmid R388 conjugation. TrwBDeltaN70 crystallographic structure revealed a hexamer with six equivalent subunits and a central channel. In this work, we characterize a DNA-dependent ATPase activity for TrwBDeltaN70. The protein displays positive cooperativity for ATP hydrolysis, with at least three catalytic sites involved. The activity is sensitive to pH and salt concentration, being more active at low pH values. The effective oligonucleotide size required for activation of the ATPase function is between 40 and 45 nucleotides, and the same length is required for the formation of high-molecular-weight TrwBDeltaN70-DNA complexes, as observed by gel filtration chromatography. A mutation in a tryptophan residue (W216A), placed in the central pore formed by the hexameric structure, resulted in a protein that did not hydrolyze ATP. In addition, it exerted a dominant negative effect, both on R388 conjugation frequency and ATP hydrolysis, underscoring the multimeric state of the protein. ATP hydrolysis was not coupled to a DNA unwinding activity under the tested conditions, which included forked DNA substrates. These results, together with TrwB structural similarity to F1-ATPase, lead us to propose a mechanism for TrwB as a DNA-translocating motor.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Conjugación Genética/fisiología , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Proteínas de Escherichia coli/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/genética , Adenosina Trifosfato/metabolismo , Transporte Biológico , Cromatografía en Gel , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Genes Dominantes/genética , Concentración de Iones de Hidrógeno , Hidrólisis , Modelos Moleculares , Estructura Cuaternaria de Proteína , Eliminación de Secuencia/genéticaRESUMEN
In mitochondria, the hydrolytic activity of ATP synthase is regulated by an inhibitor protein, IF(1). Its binding to ATP synthase depends on pH, and below neutrality, IF(1) is dimeric and forms a stable complex with the enzyme. At higher pH values, IF(1) forms tetramers and is inactive. In the 2.2 A structure of the bovine IF(1) described here, the four monomers in the asymmetric unit are arranged as a dimer of dimers. Monomers form dimers via an antiparallel alpha-helical coiled coil in the C-terminal region. Dimers are associated into oligomers and form long fibres in the crystal lattice, via coiled-coil interactions in the N-terminal and inhibitory regions (residues 14-47). Therefore, tetramer formation masks the inhibitory region, preventing IF(1) binding to ATP synthase.
Asunto(s)
Mitocondrias/enzimología , ATPasas de Translocación de Protón/metabolismo , Animales , Bovinos , Dimerización , Histidina/metabolismo , Concentración de Iones de Hidrógeno , Unión Proteica , Conformación Proteica , ATPasas de Translocación de Protón/químicaRESUMEN
The transfer of DNA across membranes and between cells is a central biological process; however, its molecular mechanism remains unknown. In prokaryotes, trans-membrane passage by bacterial conjugation, is the main route for horizontal gene transfer. It is the means for rapid acquisition of new genetic information, including antibiotic resistance by pathogens. Trans-kingdom gene transfer from bacteria to plants or fungi and even bacterial sporulation are special cases of conjugation. An integral membrane DNA-binding protein, called TrwB in the Escherichia coli R388 conjugative system, is essential for the conjugation process. This large multimeric protein is responsible for recruiting the relaxosome DNA-protein complex, and participates in the transfer of a single DNA strand during cell mating. Here we report the three-dimensional structure of a soluble variant of TrwB. The molecule consists of two domains: a nucleotide-binding domain of alpha/beta topology, reminiscent of RecA and DNA ring helicases, and an all-alpha domain. Six equivalent protein monomers associate to form an almost spherical quaternary structure that is strikingly similar to F1-ATPase. A central channel, 20 A in width, traverses the hexamer.
Asunto(s)
Conjugación Genética , Proteínas de Unión al ADN/química , Proteínas de Escherichia coli , Proteínas Bacterianas/química , Cristalografía por Rayos X , ADN Helicasas/química , Proteínas de Unión al ADN/fisiología , Modelos Moleculares , Conformación Proteica , ATPasas de Translocación de Protón/químicaRESUMEN
In mitochondria, the hydrolytic activity of ATP synthase is regulated by a natural inhibitor protein, IF(1). The binding of IF(1) to ATP synthase depends on pH values, and below neutrality, IF(1) forms a stable complex with the enzyme. Bovine IF(1) has two oligomeric states, dimer and tetramer, depending on pH values. At pH 6.5, where it is active, IF(1) dimerizes by formation of an antiparallel alpha-helical coiled-coil in its C-terminal region. This arrangement places the inhibitory N-terminal regions in opposition, implying that active dimeric IF(1) can bind two F(1) domains simultaneously. Evidence of dimerization of F(1)-ATPase by binding to IF(1) is provided by gel filtration chromatography, analytical ultracentrifugation, and electron microscopy. At present, it is not known whether IF(1) can bring about the dimerization of the F(1)F(0)-ATPase complex.
Asunto(s)
Inhibidores Enzimáticos/química , ATPasas de Translocación de Protón/química , Animales , Bovinos , Dimerización , Microscopía Electrónica , Peso Molecular , Conformación Proteica , ATPasas de Translocación de Protón/antagonistas & inhibidoresRESUMEN
Bovine IF(1), a basic protein of 84 amino acids, is involved in the regulation of the catalytic activity of the F(1) domain of ATP synthase. At pH 6.5, but not at basic pH values, it inhibits the ATP hydrolase activity of the enzyme. The oligomeric state of bovine IF(1) has been investigated at various pH values by sedimentation equilibrium analytical ultracentrifugation and by covalent cross-linking. Both techniques confirm that the protein forms a tetramer at pH 8, and below pH 6.5, the protein is predominantly dimeric. By covalent cross-linking, it has been found that at pH 8.0 the fragment of IF(1) consisting of residues 44-84 forms a dimer, whereas the fragment from residues 32-84 is tetrameric. Therefore, some or all of the residues between positions 32 and 43 are necessary for tetramer formation and are involved in the pH-sensitive interconversion between dimer and tetramer. One important residue in the interconversion is histidine 49. Mutation of this residue to lysine abolishes the pH-dependent activation-inactivation, and the mutant protein is active and dimeric at all pH values investigated. It is likely from NMR studies that the inhibitor protein dimerizes by forming an antiparallel alpha-helical coiled-coil over its C-terminal region and that at high pH values, where the protein is tetrameric, the inhibitory regions are masked. The mutation of histidine 49 to lysine is predicted to abolish coiled-coil formation over residues 32-43 preventing interaction between two dimers, forcing the equilibrium toward the dimeric state, thereby freeing the N-terminal inhibitory regions and allowing them to interact with F(1).
Asunto(s)
Proteínas/química , Proteínas/metabolismo , Secuencia de Aminoácidos , Aminoácidos/química , Animales , Bovinos , Reactivos de Enlaces Cruzados , Dimerización , Escherichia coli/metabolismo , Histidina/química , Concentración de Iones de Hidrógeno , Lisina/química , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Plásmidos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , ATPasas de Translocación de Protón/antagonistas & inhibidores , ATPasas de Translocación de Protón/metabolismo , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Ultracentrifugación , Proteína Inhibidora ATPasaRESUMEN
TrwB is the conjugative coupling protein of plasmid R388. TrwBDeltaN70 contains the soluble domain of TrwB. It was constructed by deletion of trwB sequences containing TrwB N-proximal transmembrane segments. Purified TrwBDeltaN70 protein bound tightly the fluorescent ATP analogue TNP-ATP (K(s) = 8.7 microM) but did not show measurable ATPase or GTPase activity. A single ATP binding site was found per TrwB monomer. An intact ATP-binding site was essential for R388 conjugation, since a TrwB mutant with a single amino acid alteration in the ATP-binding signature (K136T) was transfer-deficient. TrwBDeltaN70 also bound DNA nonspecifically. DNA binding enhanced TrwC nic cleavage, providing the first evidence that directly links TrwB with conjugative DNA processing. Since DNA bound by TrwBDeltaN70 also showed increased negative superhelicity (as shown by increased sensitivity to topoisomerase I), nic cleavage enhancement was assumed to be a consequence of the increased single-stranded nature of DNA around nic. The mutant protein TrwB(K136T)DeltaN70 was indistinguishable from TrwBDeltaN70 with respect to the above properties, indicating that TrwB ATP binding activity is not required for them. The reported properties of TrwB suggest potential functions for conjugative coupling proteins, both as triggers of conjugative DNA processing and as motors in the transport process.
Asunto(s)
Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/metabolismo , Conjugación Genética , ADN Bacteriano/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Escherichia coli , Proteínas Bacterianas/química , Sitios de Unión , Proteínas de Unión al ADN/química , Plásmidos , Unión ProteicaRESUMEN
We isolated and characterized traD mutants with an altered specificity of interaction with relaxosomes of various conjugative (F and R388) and mobilizable (RSF1010 and ColE1) plasmids. The change in specificity was due to a loss of some amino acids in the carboxyl terminus of TraD that resulted in a broadening of the range of mobilizable relaxosomes at the expense of a decrease in the efficiency of F-plasmid transfer.
Asunto(s)
Proteínas Bacterianas/metabolismo , Conjugación Genética , Proteínas de Escherichia coli , Escherichia coli/genética , Factor F , Proteínas de la Membrana , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , ADN Bacteriano , Datos de Secuencia Molecular , MutagénesisRESUMEN
A 1.7-kilobase pair segment from the conjugative transfer region of plasmid R388 DNA was cloned and sequenced. It contained trwD, a gene essential for plasmid R388 conjugation, for expression of the conjugative W-pilus and for sensitivity to phage PRD1. The deduced amino acid sequence of TrwD showed homology to the PulE/VirB11 superfamily of potential ATPases involved in various types of transport processes. A fusion of trwD with the glutathione S-transferase (GST) was constructed, and the resulting fusion protein was purified from overproducing bacteria. Factor Xa hydrolysis of GST-TrwD and further purification rendered TrwD protein with more than 95% purity. Antibodies raised against TrwD localized it both in the soluble fraction and in the outer membrane of Escherichia coli. TrwD is probably a peripheral outer membrane protein because it could be solubilized by increasing salt concentration to 0.5 M NaCl in the lysis buffer. Both purified GST-TrwD and TrwD could hydrolize ATP. ATPase activity increased 2-fold in the presence of detergent-phospholipid mixed micelles. To study the importance of the nucleotide-binding site, Walker box A (GXXGXGK(T/S)), present in TrwD, the conserved lysine residue was replaced by glutamine. The mutant protein, expressed and purified under the same conditions as the wild type, did not exhibit ATPase activity. TrwD(K203Q) was not able to complement the mutation in trwD of the R388 mutant plasmid, suggesting the essentiality of the ATPase activity of the protein in the conjugative process. Furthermore, the dominant character of this mutation suggested that GST-TrwD(K432Q) was still able to interact either with itself or with other component(s) of the conjugative machinery.
Asunto(s)
Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Proteínas Bacterianas/metabolismo , Conjugación Genética , Proteínas de Escherichia coli , Proteínas de Transporte de Membrana , Adenosina Trifosfatasas/química , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Secuencia de Bases , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Conjugación Genética/fisiología , Secuencia Conservada , ADN Bacteriano/química , Desoxirribonucleasa EcoRI/metabolismo , Desoxirribonucleasa HindIII/metabolismo , Escherichia coli/enzimología , Escherichia coli/genética , Escherichia coli/fisiología , Concentración de Iones de Hidrógeno , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Plásmidos/genéticaRESUMEN
The ability of conjugative plasmids from six different incompatibility groups to mobilize a set of mobilizable plasmids was examined. The mobilization frequencies of plasmids RSF1010, ColE1, ColE3, and CloDF13 varied over seven orders of magnitude, depending on the helper conjugative plasmid used. Mobilization of CloDF13 was unique in that it did not require TrwB, TraG or TraD (all members of the TraG family) for mobilization by R388, RP4 or F, respectively. CloDF13 itself codes for an essential mobilization protein (MobB) which is also a TraG homolog, only requiring a source of the genes for pilus formation. Besides, CloDF13 was mobilized efficiently by all conjugative plasmids, suggesting that TraG homologs are the primary determinants of the mobilization efficiency of a plasmid, interacting differentially with the various relaxosomes. Previous results indicated that TraG and TrwB were interchangeable for mobilization of RSF1010 and ColE1 by PILw (the pilus system of IncW plasmids) but TraG could not complement conjugation of trwB mutants, suggesting that additional interactions were taking place between TrwB and oriT(R388) that were not essential for mobilization. To further test this hypothesis, we analyzed the mobilization frequencies of ColE1 and RSF1010 by the P, W, and F pili in the presence of alternative TraG homologs. The results obtained indicated that the frequency of mobilization was determined both by the particular TraG-like protein used and by the pilus system. Thus, TraG-like proteins are not generally interchangeable for mobilization. Therefore we suggest that the factors that determine the frequencies of transfer of different MOB regions are the differential interactions of TrwB with pilus and relaxosome.
Asunto(s)
Proteínas Bacterianas/metabolismo , Conjugación Genética , Proteínas de Escherichia coli , Escherichia coli/genética , Proteínas de la Membrana , Plásmidos/genética , Proteínas Bacterianas/genética , Plásmidos de Bacteriocinas/genética , Escherichia coli/química , Escherichia coli/metabolismo , Genes Bacterianos/genética , Pili Sexual/genética , Transactivadores/metabolismoRESUMEN
Mobilization of plasmid RSF1010 by the IncW plasmid R388 requires the genes involved in W pilus synthesis plus trwB. traG of the IncP plasmid RP4 can substitute for trwB in RSF1010 mobilization by R388 but not in self-transfer of R388. This result suggests a dual specificity of TrwB-like proteins in conjugation. The same genetic requirements were found for R388 to mobilize the unrelated plasmid ColE1.
Asunto(s)
Proteínas Bacterianas/genética , Plásmidos de Bacteriocinas , Conjugación Genética , Proteínas de Escherichia coli , Escherichia coli/genética , Proteínas de la Membrana , Plásmidos , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Genes Bacterianos , Datos de Secuencia MolecularRESUMEN
Fusion of mouse melanoma cells grown in monolayers has been directly monitored by fluorescence resonance energy transfer between fluorescein and rhodamine probes attached to octadecanoic acid. Various poly(ethylene glycol)s (PEG), either alone or in combination with amphipathic molecules, have been used as fusogens. Fusion starts at a maximum rate as soon as PEG is removed from the medium and reaches a plateau after 20-30 min. Both the initial rate and extent of fusion have been recorded for each experiment. The extent of fusion shows in general a positive correlation with the initial rate, although PEGs with different molar masses appear to induce fusion at different rates, but to a similar extent. A good correlation has been found between the extent of fusion, as measured by fluorescence, and the 'fusion index' computed from cell and nucleus counting; a calibration curve is provided for the interconversion of both parameters. Optimum fusion values are obtained with 50% (w/v) PEG 1500. The effect of pre-treatments with surfactants (Triton X-100, sodium dodecylsulphate) on PEG-induced fusion has also been tested. Sodium dodecylsulphate, but not Triton, enhances considerably both the rate and extent of cell fusion. The in situ generation of the amphipathic molecule diacylglycerol, through the catalytic activity of a phospholipase C, also enhances significantly the fusion parameters. These results are in good agreement with previous studies based on syncytia counting.
Asunto(s)
Fusión Celular/efectos de los fármacos , Polietilenglicoles/farmacología , Animales , Transferencia de Energía , Colorantes Fluorescentes , Fusión de Membrana/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Células Tumorales Cultivadas , Fosfolipasas de Tipo C/farmacologíaRESUMEN
Children with sickle cell disease have a greatly increased potential for developing rapid and at times fatal sepsis from Streptococcus pneumoniae. Hospitalization and parenteral antibiotic treatment in all febrile children with sickle cell disease have thus become the standard of care at most sickle cell centers. As an alternative approach, we managed selected febrile children with sickle cell disease on an ambulatory basis with parenteral ceftriaxone to determine its safety and effectiveness in preventing sepsis and reducing the number of days of hospitalization. Twenty of 40 children who presented with significant fever met the study criteria and received ceftriaxone on an ambulatory basis. Three were subsequently hospitalized. Compared with a previous year, when all febrile children were admitted, ceftriaxone use reduced the days of hospitalization from 214 (6.3 +/- 1.6 days/patient) to 111 days (2.8 +/- 0.7 days/patient). The empiric use of ceftriaxone appears safe and effective, but it requires an expanded study over an extended period.