RESUMEN
Extremophiles and their products have been a major focus of research interest for over 40 years. Through this period, studies of these organisms have contributed hugely to many aspects of the fundamental and applied sciences, and to wider and more philosophical issues such as the origins of life and astrobiology. Our understanding of the cellular adaptations to extreme conditions (such as acid, temperature, pressure and more), of the mechanisms underpinning the stability of macromolecules, and of the subtleties, complexities and limits of fundamental biochemical processes has been informed by research on extremophiles. Extremophiles have also contributed numerous products and processes to the many fields of biotechnology, from diagnostics to bioremediation. Yet, after 40 years of dedicated research, there remains much to be discovered in this field. Fortunately, extremophiles remain an active and vibrant area of research. In the third decade of the twenty-first century, with decreasing global resources and a steadily increasing human population, the world's attention has turned with increasing urgency to issues of sustainability. These global concerns were encapsulated and formalized by the United Nations with the adoption of the 2030 Agenda for Sustainable Development and the presentation of the seventeen Sustainable Development Goals (SDGs) in 2015. In the run-up to 2030, we consider the contributions that extremophiles have made, and will in the future make, to the SDGs.
Asunto(s)
Extremófilos , Extremófilos/metabolismo , Extremófilos/fisiología , Desarrollo Sostenible , Adaptación Fisiológica , Ambientes Extremos , BiotecnologíaRESUMEN
Virus-like particles (VLPs), i.e. molecular assemblies that resemble the geometry and organization of viruses, are promising platforms for therapeutics and imaging. Understanding the assembly and cellular uptake pathways of VLPs can contribute to the development of new antiviral drugs and new virus-based materials for the delivery of drugs or nucleic acid-based therapies. Here we report the assembly of capsid proteins of the cowpea chlorotic mottle virus (CCMV) around DNA into defined structures at neutral pH. Depending on the type of DNA used, we are able to create spherical structures of various diameters and rods of various lengths. In order to determine the shape dependency, the cellular uptake routes and intracellular positioning of these formed polymorphic VLPs in RAW264.7, HeLa and HEK 293 cells are evaluated using flow cytometry analysis with specific chemical inhibitors for different uptake routes. We observed particular uptake routes for the various CCMV-based nanostructures, but the experiments point to clathrin-mediated endocytosis as the major route for cell entry for the studied VLPs. Confocal microscopy reveals that the formed VLPs enter the cells, with clear colocalization in the endosomes. The obtained results provide insight in the cargo dependent VLP morphology and increase the understanding of shape dependent uptake into cells, which is relevant in the design of new virus-based structures with applications in drug and gene delivery.
Asunto(s)
Bromovirus , Proteínas de la Cápside/administración & dosificación , ADN/administración & dosificación , Nanoestructuras/administración & dosificación , Animales , Clorpromazina/administración & dosificación , Citocalasina D/administración & dosificación , Endocitosis , Células HEK293 , Células HeLa , Humanos , Ratones , Células RAW 264.7RESUMEN
AIMS: Optimizing D-xylose transport in Saccharomyces cerevisiae is essential for efficient bioethanol production from cellulosic materials. We have used a gene shuffling approach of hexose (Hxt) transporters in order to increase the affinity for D-xylose. METHODS AND RESULTS: Various libraries were transformed to a hexose transporter deletion strain, and shuffled genes were selected via growth on low concentrations of D-xylose. This screening yielded two homologous fusion proteins (fusions 9,4 and 9,6), both consisting of the major central part of Hxt2 and various smaller parts of other Hxt proteins. Both chimeric proteins showed the same increase in D-xylose affinity (8·1 ± 3·0 mmol l-1 ) compared with Hxt2 (23·7 ± 2·1 mmol l-1 ). The increased D-xylose affinity could be related to the C terminus, more specifically to a cysteine to proline mutation at position 505 in Hxt2. CONCLUSIONS: The Hxt2C505P mutation increased the affinity for D-xylose for Hxt2, thus providing a way to increase D-xylose transport flux at low D-xylose concentration. SIGNIFICANCE AND IMPACT OF THE STUDY: The gene shuffling protocol using the highly homologues hexose transporters family provides a powerful tool to enhance the D-xylose affinity of Hxt transporters in S. cerevisiae, thus providing a means to increase the D-xylose uptake flux at low D-xylose concentrations.
Asunto(s)
Proteínas Facilitadoras del Transporte de la Glucosa/genética , Proteínas de Transporte de Membrana/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Xilosa/metabolismo , Transporte Biológico , Barajamiento de ADN , Glucosa/metabolismo , Proteínas Facilitadoras del Transporte de la Glucosa/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Mutación Missense , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
CRISPR/Cas9 based systems have emerged as versatile platforms for precision genome editing in a wide range of organisms. Here we have developed powerful CRISPR/Cas9 tools for marker-based and marker-free genome modifications in Penicillium chrysogenum, a model filamentous fungus and industrially relevant cell factory. The developed CRISPR/Cas9 toolbox is highly flexible and allows editing of new targets with minimal cloning efforts. The Cas9 protein and the sgRNA can be either delivered during transformation, as preassembled CRISPR-Cas9 ribonucleoproteins (RNPs) or expressed from an AMA1 based plasmid within the cell. The direct delivery of the Cas9 protein with in vitro synthesized sgRNA to the cells allows for a transient method for genome engineering that may rapidly be applicable for other filamentous fungi. The expression of Cas9 from an AMA1 based vector was shown to be highly efficient for marker-free gene deletions.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica/métodos , Penicillium chrysogenum/genética , Proteínas Bacterianas/genética , Proteína 9 Asociada a CRISPR , Reparación del ADN , Endonucleasas/genética , Eliminación de Gen , Marcación de Gen/métodos , Marcadores Genéticos , Vectores Genéticos , Genoma Fúngico , Oligonucleótidos/genética , ARN Guía de KinetoplastidaRESUMEN
AIMS: Saccharomyces cerevisiae does not express any xylose-specific transporters. To enhance the xylose uptake of S. cerevisiae, directed evolution of the Gal2 transporter was performed. METHODS AND RESULTS: Three rounds of error-prone PCR were used to generate mutants with improved xylose-transport characteristics. After developing a fast and reliable high-throughput screening assay based on flow cytometry, eight mutants were obtained showing an improved uptake of xylose compared to wild-type Gal2 out of 41 200 single yeast cells. Gal2 variant 2·1 harbouring five amino acid substitutions showed an increased affinity towards xylose with a faster overall sugar metabolism of glucose and xylose. Another Gal2 variant 3·1 carrying an additional amino acid substitution revealed an impaired growth on glucose but not on xylose. CONCLUSIONS: Random mutagenesis of the S. cerevisiae Gal2 led to an increased xylose uptake capacity and decreased glucose affinity, allowing improved co-consumption. SIGNIFICANCE AND IMPACT OF THE STUDY: Random mutagenesis is a powerful tool to evolve sugar transporters like Gal2 towards co-consumption of new substrates. Using a high-throughput screening system based on flow-through cytometry, various mutants were identified with improved xylose-transport characteristics. The Gal2 variants in this work are a promising starting point for further engineering to improve xylose uptake from mixed sugars in biomass.
Asunto(s)
Proteínas de Transporte de Monosacáridos/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/enzimología , Xilosa/metabolismo , Transporte Biológico , Evolución Molecular Dirigida , Glucosa/metabolismo , Ensayos Analíticos de Alto Rendimiento , Proteínas de Transporte de Monosacáridos/metabolismo , Mutagénesis , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Bacteria use a communication system, called quorum sensing (QS), to organize into communities and synchronize gene expression to promote virulence and secure survival. Here we report on a proof-of-principle for externally interfering with this bacterial communication system, using light. By employing photoswitchable small molecules, we were able to photocontrol the QS-related bioluminescence in an Escherichia coli reporter strain, and the expression of target QS genes and pyocyanin production in Pseudomonas aeruginosa.
RESUMEN
Penicillium chrysogenum is widely used as an industrial antibiotic producer, in particular in the synthesis of ß-lactam antibiotics such as penicillins and cephalosporins. In industrial processes, oxalic acid formation leads to reduced product yields. Moreover, precipitation of calcium oxalate complicates product recovery. We observed oxalate production in glucose-limited chemostat cultures of P. chrysogenum grown with or without addition of adipic acid, side-chain of the cephalosporin precursor adipoyl-6-aminopenicillinic acid (ad-6-APA). Oxalate accounted for up to 5% of the consumed carbon source. In filamentous fungi, oxaloacetate hydrolase (OAH; EC3.7.1.1) is generally responsible for oxalate production. The P. chrysogenum genome harbours four orthologs of the A. niger oahA gene. Chemostat-based transcriptome analyses revealed a significant correlation between extracellular oxalate titers and expression level of the genes Pc18g05100 and Pc22g24830. To assess their possible involvement in oxalate production, both genes were cloned in Saccharomyces cerevisiae, yeast that does not produce oxalate. Only the expression of Pc22g24830 led to production of oxalic acid in S. cerevisiae. Subsequent deletion of Pc22g28430 in P. chrysogenum led to complete elimination of oxalate production, whilst improving yields of the cephalosporin precursor ad-6-APA.
Asunto(s)
Hidrolasas/genética , Hidrolasas/metabolismo , Oxalatos/metabolismo , Penicillium chrysogenum/metabolismo , beta-Lactamas/metabolismo , Medios de Cultivo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Perfilación de la Expresión Génica , Ingeniería Genética/métodos , Microbiología Industrial/métodos , Penicillium chrysogenum/enzimología , Penicillium chrysogenum/genética , Penicillium chrysogenum/crecimiento & desarrolloRESUMEN
Many systems are available for the production of recombinant proteins in bacterial and eukaryotic model organisms, which allow us to study proteins in their native hosts and to identify protein-protein interaction partners. In contrast, only a few transformation systems have been developed for archaea, and no system for high-level gene expression existed for hyperthermophilic organisms. Recently, a virus-based shuttle vector with a reporter gene was developed for the crenarchaeote Sulfolobus solfataricus, a model organism of hyperthermophilic archaea that grows optimally at 80 degrees C (M. Jonuscheit, E. Martusewitsch, K. M. Stedman, and C. Schleper, Mol. Microbiol. 48:1241-1252, 2003). Here we have refined this system for high-level gene expression in S. solfataricus with the help of two different promoters, the heat-inducible promoter of the major chaperonin, thermophilic factor 55, and the arabinose-inducible promoter of the arabinose-binding protein AraS. Functional expression of heterologous and homologous genes was demonstrated, including production of the cytoplasmic sulfur oxygenase reductase from Acidianus ambivalens, an Fe-S protein of the ABC class from S. solfataricus, and two membrane-associated ATPases potentially involved in the secretion of proteins. Single-step purification of the proteins was obtained via fused His or Strep tags. To our knowledge, these are the first examples of the application of an expression vector system to produce large amounts of recombinant and also tagged proteins in a hyperthermophilic archaeon.
Asunto(s)
Proteínas Arqueales/metabolismo , Vectores Genéticos , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/metabolismo , Proteínas Recombinantes/metabolismo , Sulfolobus solfataricus/metabolismo , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Proteínas Arqueales/genética , Regulación de la Expresión Génica Arqueal , Proteínas Hierro-Azufre/genética , Proteínas Hierro-Azufre/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/genética , Regiones Promotoras Genéticas , Proteínas Recombinantes/genética , Sulfolobus solfataricus/genéticaRESUMEN
LmrP from Lactococcus lactis is a 45-kDa membrane protein that confers resistance to a wide variety of lipophilic compounds by acting as a proton motive force-driven efflux pump. This study shows that both the proton motive force and ligand interaction alter the accessibility of cytosolic tryptophan residues to a hydrophilic quencher. The proton motive force mediates an increase of LmrP accessibility toward the external medium and results in higher drug binding. Residues Asp128 and Asp68, from cytosolic loops, are involved in the proton motive force-mediated accessibility change. Ligand binding does not modify the protein accessibility, but the proton motive force-mediated restructuring is prerequisite for a subsequent accessibility change mediated by ligand binding. Asp142 cooperates with other membrane-embedded carboxylic residues to promote a conformational change that increases LmrP accessibility toward the hydrophilic quencher. This drug binding-mediated reorganization may be related to the transition between the high- and low-affinity drug-binding sites and is crucial for drug release in the extracellular medium.
Asunto(s)
Proteínas Bacterianas/fisiología , Proteínas de Transporte de Membrana/fisiología , Acrilamida/farmacología , Ácido Aspártico/química , Proteínas Bacterianas/química , Bencimidazoles/farmacología , Transporte Biológico , Membrana Celular/metabolismo , Citosol/química , Relación Dosis-Respuesta a Droga , Resistencia a Múltiples Medicamentos , Concentración de Iones de Hidrógeno , Lactococcus lactis/metabolismo , Ligandos , Liposomas/metabolismo , Proteínas de Transporte de Membrana/química , Unión Proteica , Estructura Terciaria de Proteína , Proteolípidos/química , Protones , Sefarosa/química , Espectroscopía Infrarroja por Transformada de Fourier , Tetraciclina/química , Factores de Tiempo , Triptófano/químicaRESUMEN
Classical strain improvement of beta-lactam producing organisms by random mutagenesis has been a powerful tool during the last century. Current insights in the biochemistry and genetics of beta-lactam production, in particular in the filamentous fungus Penicillium chrysogenum, however, make a more directed and rational approach of metabolic pathway engineering possible. Besides the need for efficient genetic methods, a thorough understanding is needed of the metabolic fluxes in primary, intermediary and secondary metabolism. Controlling metabolic fluxes can be achieved by adjusting enzyme activities and metabolite levels in such a way that the main flow is directed towards the desired product. In addition, compartmentalization of specific parts of the beta-lactam biosynthesis pathways provides a way to control this pathway by clustering enzymes with their substrates inside specific membrane bound structures sequestered from the cytosol. This compartmentalization also requires specific membrane transport steps of which the details are currently uncovered.
Asunto(s)
Acremonium/metabolismo , Transporte Biológico Activo/fisiología , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica/fisiología , Mejoramiento Genético/métodos , Factores de Transcripción/metabolismo , beta-Lactamas/metabolismo , Acremonium/clasificación , Acremonium/genética , Antibacterianos/biosíntesis , Antibacterianos/química , Antibacterianos/clasificación , Proteínas Fúngicas/genética , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismo , Transducción de Señal/fisiología , Especificidad de la Especie , Factores de Transcripción/genética , beta-Lactamas/químicaRESUMEN
The major route of protein translocation in bacteria is the so-called general secretion pathway (Sec-pathway). This route has been extensively studied in Escherichia coli and other bacteria. The movement of preproteins across the cytoplasmic membrane is mediated by a multimeric membrane protein complex called translocase. The core of the translocase consists of a proteinaceous channel formed by an oligomeric assembly of the heterotrimeric membrane protein complex SecYEG and the peripheral adenosine triphosphatase (ATPase) SecA as molecular motor. Many secretory proteins utilize the molecular chaperone SecB for targeting and stabilization of the unfolded state prior to translocation, while most nascent inner membrane proteins are targeted to the translocase by the signal recognition particle and its membrane receptor. Translocation is driven by ATP hydrolysis and the proton motive force. In the last decade, genetic and biochemical studies have provided detailed insights into the mechanism of preprotein translocation. Recent crystallographic studies on SecA, SecB and the SecYEG complex now provide knowledge about the structural features of the translocation process. Here, we will discuss the mechanistic and structural basis of the translocation of proteins across and the integration of membrane proteins into the cytoplasmic membrane.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Bacterias/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Partícula de Reconocimiento de Señal/metabolismo , Transporte de Proteínas/fisiología , Canales de Translocación SEC , Proteína SecARESUMEN
The Escherichia coli inner membrane protein (IMP) YidC is involved in the membrane integration of IMPs both in concert with and independently from the Sec translocase. YidC seems to be dispensable for the assembly of Sec-dependent IMPs, and so far it has been shown to be essential only for the proper Sec-independent integration of some phage coat proteins. Here, we studied the physiological consequences of YidC depletion in an effort to understand the essential function of YidC. The loss of YidC rapidly and specifically induced the Psp stress response, which is accompanied by a reduction of the proton-motive force. This reduction is due to defects in the functional assembly of cytochrome o oxidase and the F(1)F(o) ATPase complex, which is reminiscent of the effects of mutations in the yidC homologue OXA1 in the yeast mitochondrial inner membrane. The integration of CyoA (subunit II of the cytochrome o oxidase) and F(o)c (membrane subunit of the F(1)F(o) ATPase) appeared exceptionally sensitive to depletion of YidC, suggesting that these IMPs are natural substrates of a membrane integration and assembly pathway in which YidC plays an exclusive or at least a pivotal role.
Asunto(s)
Membrana Celular/metabolismo , Membrana Celular/ultraestructura , Proteínas de Escherichia coli/biosíntesis , Escherichia coli/metabolismo , Proteínas de Transporte de Membrana/biosíntesis , Consumo de Oxígeno/fisiología , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Cinética , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/metabolismo , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Canales de Translocación SECRESUMEN
The ion and particularly the proton and sodium ion permeabilities of cytoplasmic membranes play crucial roles in the bioenergetics of microorganisms. The proton and sodium permeabilities of membranes increase with temperature. Psychrophilic and mesophilic bacteria and mesophilic, (hyper)thermophilic, and halophilic archaea are capable of adjusting the lipid composition of their membranes in such a way that the proton permeability at the respective growth temperature remains constant (homeoproton permeability). Thermophilic bacteria are an exception. They rely on the less permeable sodium ions to generate a sodium motive force, which is subsequently used to drive energy-requiring membrane-bound processes. Transport of solutes across bacterial and archaeal membranes is mainly catalyzed by primary ATP-driven transport systems or by proton- or sodium-motive-force-driven secondary transport systems. Unlike most bacteria, hyperthermophilic bacteria and archaea prefer primary uptake systems. Several high-affinity ATP-binding cassette (ABC) transporters for sugars from hyperthermophiles have been identified and characterized. The activities of these ABC transporters allow these organisms to thrive in their nutrient-poor environments.
Asunto(s)
Archaea/metabolismo , Bacterias/metabolismo , Metabolismo Energético , Transportadoras de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfato/metabolismo , Transporte Biológico Activo , Proteínas Portadoras/metabolismo , Permeabilidad de la Membrana Celular , Ambiente , Concentración de Iones de Hidrógeno , TemperaturaRESUMEN
SecDFyajC of Escherichia coli is required for efficient export of proteins in vivo. However, the functional role of SecDFyajC in protein translocation is unclear. We evaluated the postulated function of SecDFyajC in the maintenance of the proton motive force. As previously reported, inner membrane vesicles (IMVs) lacking SecDFyajC are defective in the generation of a stable proton motive force when energized with succinate. This phenomenon is, however, not observed when NADH is used as an electron donor. Moreover, the proton motive force generated in SecDFyajC-depleted vesicles stimulated translocation to the same extent as seen with IMVs containing SecDFyajC. Further analysis demonstrates that the reduced proton motive force with succinate in IMVs lacking SecDFyajC is due to a lower amount of the enzyme succinate dehydrogenase. The expression of this enzyme complex is repressed by growth on glucose media, the condition used to deplete SecDFyajC. These results demonstrate that SecDFyajC is not required for proton motive force-driven protein translocation.
Asunto(s)
Antígenos Bacterianos , Proteínas Bacterianas/metabolismo , Membrana Celular/metabolismo , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Proteínas de Transporte de Membrana , Transporte de Proteínas , Fuerza Protón-Motriz , Proteínas Bacterianas/química , Escherichia coli/enzimología , Escherichia coli/genética , Sustancias Macromoleculares , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Precursores de Proteínas/metabolismo , Succinato Deshidrogenasa/metabolismo , Vesículas Transportadoras/química , Vesículas Transportadoras/metabolismoRESUMEN
Proteins imported into the mitochondrial matrix are synthesized in the cytosol with an N-terminal presequence and are translocated through hetero-oligomeric translocase complexes of the outer and inner mitochondrial membranes. The channel across the inner membrane is formed by the presequence translocase, which consists of roughly six distinct subunits; however, it is not known which subunits actually form the channel. Here we report that purified Tim23 forms a hydrophilic, approximately 13-24 A wide channel characteristic of the mitochondrial presequence translocase. The Tim23 channel is cation selective and activated by a membrane potential and presequences. The channel is formed by the C-terminal domain of Tim23 alone, whereas the N-terminal domain is required for selectivity and a high-affinity presequence interaction. Thus, Tim23 forms a voltage-sensitive high-conductance channel with specificity for mitochondrial presequences.
Asunto(s)
Activación del Canal Iónico , Proteínas de Transporte de Membrana/química , Proteínas de Transporte de Membrana/metabolismo , Mitocondrias/química , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial , Precursores de Proteínas/metabolismo , Señales de Clasificación de Proteína/fisiología , Proteínas Represoras , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Electrofisiología , Membranas Intracelulares/química , Membranas Intracelulares/enzimología , Membranas Intracelulares/metabolismo , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Liposomas/química , Liposomas/metabolismo , Sustancias Macromoleculares , Potenciales de la Membrana , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana/genética , Mitocondrias/enzimología , Mitocondrias/genética , Proteínas del Complejo de Importación de Proteínas Precursoras Mitocondriales , Mutación/genética , Permeabilidad , Unión Proteica , Precursores de Proteínas/química , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Subunidades de Proteína , Transporte de Proteínas , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Especificidad por SustratoRESUMEN
Bacteria and archaea usually divide symmetrically by formation of a septum in the middle of the cell. A key event in cell division is the assembly of the FtsZ ring. FtsZ is the prokaryotic homolog of tubulin and forms polymers in the presence of guanine nucleotides. Here, we specifically address the polymerization of FtsZ and the role of nucleotide hydrolysis in polymer formation and stabilization. Recent structural and biochemical results are discussed and a model for FtsZ polymerization, similar to that for tubulin, is presented.
Asunto(s)
Proteínas Bacterianas/fisiología , Proteínas del Citoesqueleto , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Biopolímeros , Guanosina Trifosfato/metabolismo , Hidrólisis , Modelos Moleculares , Conformación Proteica , Tubulina (Proteína)/químicaRESUMEN
In contrast to Gram-negative bacteria, secretory proteins of Gram-positive bacteria only need to traverse a single membrane to enter the extracellular environment. For this reason, Gram-positive bacteria (e.g. various Bacillus species) are often used in industry for the commercial production of extracellular proteins that can be produced in yields of several grams per liter culture medium. The central components of the main protein translocation system (Sec system) of Gram-negative and Gram-positive bacteria show a high degree of conservation, suggesting similar functions and working mechanisms. Despite this fact, several differences can be identified such as the absence of a clear homolog of the secretion-specific chaperone SecB in Gram-positive bacteria. The now available detailed insight into the organization of the Gram-positive protein secretion system and how it differs from the well-characterized system of Escherichia coli may in the future facilitate the exploitation of these organisms in the high level production of heterologous proteins which, so far, is sometimes very inefficient due to one or more bottlenecks in the secretion pathway. In this review, we summarize the current knowledge on the various steps of the protein secretion pathway of Gram-positive bacteria with emphasis on Bacillus subtilis, which during the last decade, has arisen as a model system for the study of protein secretion in this industrially important class of microorganisms.
Asunto(s)
Proteínas Bacterianas/metabolismo , Membrana Celular/metabolismo , Bacterias Grampositivas/citología , Bacterias Grampositivas/metabolismo , Bacillus subtilis/citología , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Portadoras/metabolismo , Pared Celular/metabolismo , Bacterias Grampositivas/genética , Señales de Clasificación de Proteína/genética , Señales de Clasificación de Proteína/fisiología , Transporte de Proteínas , Partícula de Reconocimiento de Señal/metabolismoRESUMEN
The hyperthermophilic archaeon Pyrococcus furiosus can utilize different beta-glucosides, like cellobiose and laminarin. Cellobiose uptake occurs with high affinity (K(m) = 175 nM) and involves an inducible binding protein-dependent transport system. The cellobiose binding protein (CbtA) was purified from P. furiosus membranes to homogeneity as a 70-kDa glycoprotein. CbtA not only binds cellobiose but also cellotriose, cellotetraose, cellopentaose, laminaribiose, laminaritriose, and sophorose. The cbtA gene was cloned and functionally expressed in Escherichia coli. cbtA belongs to a gene cluster that encodes a transporter that belongs to the Opp family of ABC transporters.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/aislamiento & purificación , Transportadoras de Casetes de Unión a ATP/metabolismo , Celobiosa/metabolismo , Pyrococcus/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Escherichia coli , Cinética , Peso MolecularRESUMEN
In Escherichia coli, the SecYEG complex mediates the translocation and membrane integration of proteins. Both genetic and biochemical data indicate interactions of several transmembrane segments (TMSs) of SecY with SecE. By means of cysteine scanning mutagenesis, we have identified intermolecular sites of contact between TMS7 of SecY and TMS3 of SecE. The cross-linking of SecY to SecE demonstrates that these subunits are present in a one-to-one stoichiometry within the SecYEG complex. Sites in TMS3 of SecE involved in SecE dimerization are confined to a specific alpha-helical interface and occur in an oligomeric SecYEG complex. Although cross-linking reversibly inactivates translocation, the contact between TMS7 of SecY and TMS3 of SecE remains unaltered upon insertion of the preprotein into the translocation channel. These data support a model for an oligomeric translocation channel in which pairs of SecYEG complexes contact each other via SecE.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Cisteína , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Proteínas Bacterianas/genética , Sitios de Unión , Membrana Celular/metabolismo , Dimerización , Disulfuros/análisis , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Estructura Secundaria de Proteína , Subunidades de Proteína , Transporte de Proteínas , Canales de Translocación SECRESUMEN
The inner membrane protein YidC is associated with the preprotein translocase of Escherichia coli and contacts transmembrane segments of nascent inner membrane proteins during membrane insertion. YidC was purified to homogeneity and co-reconstituted with the SecYEG complex. YidC had no effect on the SecA/SecYEG-mediated translocation of the secretory protein proOmpA; however, using a crosslinking approach, the transmembrane segment of nascent FtsQ was found to gain access to YidC via SecY. These data indicate the functional reconstitution of the initial stages of YidC-dependent membrane protein insertion via the SecYEG complex.