RESUMEN
Cell surface display of proteins can be used for several biotechnological applications such as the screening of protein libraries, whole cell biocatalysis and live vaccine development. Amongst all secretion systems and surface appendages of Gram-negative bacteria, the autotransporter secretion pathway holds great potential for surface display because of its modular structure and apparent simplicity. Autotransporters are polypeptides made up of an N-terminal signal peptide, a secreted or surface-displayed passenger domain and a membrane-anchored C-terminal translocation unit. Genetic replacement of the passenger domain allows for the surface display of heterologous passengers. An autotransporter-based surface expression module essentially consists of an application-dependent promoter system, a signal peptide, a passenger domain of interest and the autotransporter translocation unit. The passenger domain needs to be compatible with surface translocation although till now no general rules have been determined to test this compatibility. The autotransporter technology for surface display of heterologous passenger domains is critically discussed for various applications.
Asunto(s)
Sistemas de Secreción Bacterianos , Biotecnología/métodos , Técnicas de Visualización de Superficie Celular/métodos , Bacterias Gramnegativas , Proteínas de la Membrana Bacteriana Externa , Biodegradación Ambiental , Modelos Moleculares , Proteínas Recombinantes , Vacunas SintéticasRESUMEN
BACKGROUND: Solanum lycopersicum or tomato is extensively studied with respect to the ethylene metabolism during climacteric ripening, focusing almost exclusively on fruit pericarp. In this work the ethylene biosynthesis pathway was examined in all major tomato fruit tissues: pericarp, septa, columella, placenta, locular gel and seeds. The tissue specific ethylene production rate was measured throughout fruit development, climacteric ripening and postharvest storage. All ethylene intermediate metabolites (1-aminocyclopropane-1-carboxylic acid (ACC), malonyl-ACC (MACC) and S-adenosyl-L-methionine (SAM)) and enzyme activities (ACC-oxidase (ACO) and ACC-synthase (ACS)) were assessed. RESULTS: All tissues showed a similar climacteric pattern in ethylene productions, but with a different amplitude. Profound differences were found between tissue types at the metabolic and enzymatic level. The pericarp tissue produced the highest amount of ethylene, but showed only a low ACC content and limited ACS activity, while the locular gel accumulated a lot of ACC, MACC and SAM and showed only limited ACO and ACS activity. Central tissues (septa, columella and placenta) showed a strong accumulation of ACC and MACC. These differences indicate that the ethylene biosynthesis pathway is organized and regulated in a tissue specific way. The possible role of inter- and intra-tissue transport is discussed to explain these discrepancies. Furthermore, the antagonistic relation between ACO and E8, an ethylene biosynthesis inhibiting protein, was shown to be tissue specific and developmentally regulated. In addition, ethylene inhibition by E8 is not achieved by a direct interaction between ACO and E8, as previously suggested in literature. CONCLUSIONS: The Ethylene biosynthesis pathway and E8 show a tissue specific and developmental differentiation throughout tomato fruit development and ripening.
Asunto(s)
Etilenos/metabolismo , Solanum lycopersicum/metabolismo , Aminoácido Oxidorreductasas/metabolismo , Aminoácidos Cíclicos/metabolismo , Regulación de la Expresión Génica de las Plantas , Liasas/metabolismo , Solanum lycopersicum/fisiologíaRESUMEN
BACKGROUND: Autotransporters represent a widespread family of secreted proteins in Gram-negative bacteria. Their seemingly easy secretion mechanism and modular structure make them interesting candidates for cell surface display of heterologous proteins. The most widely applied host organism for this purpose is Escherichia coli. Pseudomonas stutzeri A15 is an interesting candidate host for environmentally relevant biotechnological applications. With the recently characterized P. stutzeri A15 EstA autotransporter at hand, all tools for developing a surface display system for environmental use are available. More general, this system could serve as a case-study to test the broad applicability of autotransporter based surface display. RESULTS: Based on the P. stutzeri A15 EstA autotransporter ß-domain, a surface display expression module was constructed for use in P. stutzeri A15. Proof of concept of this module was presented by successful surface display of the original EstA passenger domain, which retained its full esterase activity. Almost all of the tested heterologous passenger domains however were not exposed at the cell surface of P. stutzeri A15, as assessed by whole cell proteinase K treatment. Only for a beta-lactamase protein, cell surface display in P. stutzeri A15 was comparable to presentation of the original EstA passenger domain. Development of expression modules based on the full-length EstA autotransporter did not resolve these problems. CONCLUSIONS: Since only one of the tested heterologous passenger proteins could be displayed at the cell surface of P. stutzeri A15 to a notable extent, our results indicate that the EstA autotransporter cannot be regarded as a broad spectrum cell surface display system in P. stutzeri A15.
Asunto(s)
Proteínas Bacterianas/genética , Hidrolasas de Éster Carboxílico/genética , Membrana Celular/enzimología , Pseudomonas stutzeri/enzimología , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Hidrolasas de Éster Carboxílico/química , Hidrolasas de Éster Carboxílico/metabolismo , Membrana Celular/química , Membrana Celular/genética , Expresión Génica , Estructura Terciaria de Proteína , Transporte de Proteínas , Pseudomonas stutzeri/química , Pseudomonas stutzeri/genéticaRESUMEN
Autotransporters are a widespread family of proteins, generally known as virulence factors produced by Gram-negative bacteria. In this study, the esterase A (EstA) autotransporter of the rice root-colonizing beneficial bacterium Pseudomonas stutzeri A15 was characterized. A multiple sequence alignment identified EstA as belonging to clade II of the GDSL esterase family. Autologous overexpression allowed the investigation of several features of both autotransporter proteins and GDSL esterases. First, the correctly folded autotransporter was shown to be present in the membrane fraction. Unexpectedly, after separation of the membrane fraction, EstA was detected in the N-laurylsarcosine soluble fraction. However, evidence is presented for the surface exposure of EstA based on fluorescent labeling with EstA specific antibodies. Another remarkable feature is the occurrence of a C-terminal leucine residue instead of the canonical phenylalanine or tryptophan residue. Replacement of this residue with a phenylalanine residue reduced the stability of the ß-barrel. Regarding the esterase passenger domain, we show the importance of the catalytic triad residues, with the serine and histidine residues being more critical than the aspartate residue. Furthermore, the growth of an estA-negative mutant was not impaired and cell mobility was not disabled compared to the wild type. No specific phenotype was detected for an estA-negative mutant. Overall, P. stutzeri A15 EstA is a new candidate for the surface display of proteins in environmentally relevant biotechnological applications.