RESUMEN
The PulE component of the pullulanase secretion pathway, a typical main terminal branch of the general secretory pathway, has a tetracysteine motif (4Cys) that is also present in almost all of the many PulE homologues, including those involved in type-IV piliation and conjugal DNA transfer. The 4Cys resembles a zinc-binding motif found in other proteins such as adenylate kinases, which may be pertinent in view of the fact that PulE has a consensus ATP-binding motif and since at least one PulE homologue has been reported to have kinase activity. In PulE, the Cys residues of this motif form scrambled intra- and intermolecular disulfide bonds when cells are disrupted. Replacement of one or more Cys of this motif by Ser reduces PulE function, but at least two adjacent Cys must be replaced to prevent intramolecular disulfide bond formation.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas Portadoras/metabolismo , Cisteína/química , Proteínas de Escherichia coli , Glicósido Hidrolasas/metabolismo , Bacterias Gramnegativas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Western Blotting , Proteínas Portadoras/genética , Secuencia Conservada , Disulfuros/química , Disulfuros/metabolismo , Escherichia coli/genética , Fimbrias Bacterianas/química , Prueba de Complementación Genética , Bacterias Gramnegativas/enzimología , Bacterias Gramnegativas/genética , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Unión Proteica , Conformación ProteicaRESUMEN
The main terminal branch (MTB) of the general secretory pathway is used by a wide variety of Gram- bacteria to transport exoproteins from the periplasm to the outside milieu. Recent work has led to the identification of the function of two of its 14 (or more) components: an enzyme with type-IV prepilin peptidase activity and a chaperone-like protein required for the insertion of another of the MTB components into the outer membrane. Despite these important discoveries, little tangible progress has been made towards identifying MTB components that determine secretion specificity (presumably by binding to cognate exoproteins) or which form the putative channel through which exoproteins are transported across the outer membrane. However, the idea that the single integral outer membrane component of the MTB could line the wall of this channel, and the intriguing possibility that other components of the MTB form a rudimentary type-IV pilus-like structure that might span the periplasm both deserve more careful examination. Although Escherichia coli K-12 does not normally secrete exoproteins, its chromosome contains an apparently complete set of genes coding for MTB components. At least two of these genes code for functional proteins, but the operon in which twelve of the genes are located does not appear to be expressed. We are currently searching for conditions which allow these genes to be expressed with the eventual aim of identifying the protein(s) that E. coli K-12 can secrete.
Asunto(s)
Proteínas Bacterianas/metabolismo , Endopeptidasas , Bacterias Gramnegativas/metabolismo , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Portadoras/metabolismo , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Glicósido Hidrolasas/metabolismoRESUMEN
Pullulanase of Klebsiella oxytoca is one of a wide variety of extracellular proteins that are secreted by Gram-negative bacteria by the complex main terminal branch (MTB) of the general secretory pathway. The roles of some of the 14 components of the MTB are now becoming clear. In this review it is proposed that most of these proteins form a complex, the secretion, that spans the cell envelope to control the opening and closing of channel in the outer membrane. Progress toward the goal of testing this model is reviewed.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas Portadoras/metabolismo , Glicósido Hidrolasas/metabolismo , Bacterias Gramnegativas/metabolismo , Proteínas de la Membrana Bacteriana Externa/metabolismo , Transporte Biológico Activo , Genes Bacterianos , Klebsiella/enzimología , Klebsiella/metabolismo , Modelos BiológicosRESUMEN
The energy requirement for the second step in pullulanase secretion by the general secretory pathway was studied in Escherichia coli. In order to uncouple the two steps in the secretion pathway (across the cytoplasmic and outer membranes, respectively) and to facilitate kinetic analysis of secretion, a variant form of pullulanase lacking its N-terminal fatty acid membrane anchor was used. The transport of the periplasmic secretion intermediate form of this protein across the outer membrane was not inhibited by concentrations of sodium arsenate in excess of those required to reduce ATP levels to < or = 10% of their normal value. Pullulanase secretion was inhibited by the protonophore carbonyl cyanide m-chlorophenyl hydrazone at concentrations which were similar to those reported by others to be required to prevent solute uptake or the export and processing of preproteins across the cytoplasmic membrane, but which were in excess of those required to fully dissipate the proton-motive force and to reduce lactose uptake to a significant extent.
Asunto(s)
Escherichia coli/enzimología , Glicósido Hidrolasas/metabolismo , Adenosina Trifosfato/metabolismo , Transporte Biológico Activo/efectos de los fármacos , Carbonil Cianuro m-Clorofenil Hidrazona/farmacología , Membrana Celular/metabolismo , Citoplasma/metabolismo , Metabolismo Energético , Escherichia coli/genética , Escherichia coli/metabolismo , Glicósido Hidrolasas/química , Glicósido Hidrolasas/genética , Concentración de Iones de Hidrógeno , Desacopladores/farmacologíaRESUMEN
The PulC component of the Klebsiella oxytoca pullulanase secretion machinery (the secreton) was found by subcellular fractionation to be associated with both the cytoplasmic (inner) and outer membranes. Association with the outer membrane was independent of other secreton components, including the outer membrane protein PulD (secretin). The association of PulC with the inner membrane is mediated by the signal anchor sequence located close to its N terminus. These results suggest that PulC forms a bridge between the two membranes that is disrupted when bacteria are broken open for fractionation. Neither the signal anchor sequence nor the cytoplasmic N-terminal region that precedes it was found to be required for PulC function, indicating that PulC does not undergo sequence-specific interactions with other cytoplasmic membrane proteins. Cross-linking of whole cells resulted in the formation of a ca. 110-kDa band that reacted with PulC-specific serum and whose detection depended on the presence of PulD. However, antibodies against PulD failed to react with this band, suggesting that it could be a homo-PulC trimer whose formation requires PulD. The data are discussed in terms of the possible role of PulC in energy transduction for exoprotein secretion.
Asunto(s)
Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/metabolismo , Membrana Celular/enzimología , Glicósido Hidrolasas/metabolismo , Klebsiella/enzimología , Proteínas de la Membrana/aislamiento & purificación , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Fraccionamiento Celular , Clonación Molecular , Reactivos de Enlaces Cruzados , Klebsiella/genética , Proteínas de la Membrana/metabolismo , Mutación , Unión Proteica , Conformación Proteica , Señales de Clasificación de ProteínaRESUMEN
We report attempts to analyze interactions between components of the pullulanase (Pul) secreton (type II secretion machinery) from Klebsiella oxytoca encoded by a multiple-copy-number plasmid in Escherichia coli. Three of the 15 Pul proteins (B, H, and N) were found to be dispensable for pullulanase secretion. The following evidence leads us to propose that PulE, PulL, and PulM form a subcomplex with which PulC and PulG interact. The integral cytoplasmic membrane protein PulL prevented proteolysis and/or aggregation of PulE and mediated its association with the cytoplasmic membrane. The cytoplasmic, N-terminal domain of PulL interacted directly with PulE, and both PulC and PulM were required to prevent proteolysis of PulL. PulM and PulL could be cross-linked as a heterodimer whose formation in a strain producing the secreton required PulG. However, PulL and PulM produced alone could also be cross-linked in a 52-kDa complex, indicating that the secreton exerts subtle effects on the interaction between PulE and PulL. Antibodies against PulM coimmunoprecipitated PulL, PulC, and PulE from detergent-solubilized cell extracts, confirming the existence of a complex containing these four proteins. Overproduction of PulG, which blocks secretion, drastically reduced the cellular levels of PulC, PulE, PulL, and PulM as well as PulD (secretin), which probably interacts with PulC. The Pul secreton components E, F, G, I, J, K, L, and M could all be replaced by the corresponding components of the Out secretons of Erwinia chrysanthemi and Erwinia carotovora, showing that they do not play a role in secretory protein recognition and secretion specificity.