RESUMEN
This study investigated the structural and biophysical characteristics of GumB and GumC, two Xanthomonas campestris membrane proteins that are involved in xanthan biosynthesis. Xanthan is an exopolysaccharide that is thought to be a virulence factor that contributes to bacterial in planta growth. It also is one of the most important industrial biopolymers. The first steps of xanthan biosynthesis are well understood, but the polymerization and export mechanisms remain unclear. For this reason, the key proteins must be characterized to better understand these processes. Here we characterized, by biochemical and biophysical techniques, GumB, the outer membrane polysaccharide export protein, and GumC, the polysaccharide co-polymerase protein of the xanthan biosynthesis system. Our results suggested that recombinant GumB is a tetrameric protein in solution. On the other hand, we observed that both native and recombinant GumC present oligomeric conformation consistent with dimers and higher-order oligomers. The transmembrane segments of GumC are required for GumC expression and/or stability. These initial results provide a starting point for additional studies that will clarify the roles of GumB and GumC in the xanthan polymerization and export processes and further elucidate their functions and mechanisms of action.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/genética , Proteínas Bacterianas/metabolismo , Proteínas Portadoras/genética , Proteínas de Transporte de Membrana/metabolismo , Xanthomonas campestris/enzimología , Secuencia de Aminoácidos , Proteínas de la Membrana Bacteriana Externa/análisis , Proteínas de la Membrana Bacteriana Externa/química , Proteínas Bacterianas/genética , Proteínas Portadoras/análisis , Proteínas Portadoras/química , Proteínas de Transporte de Membrana/genética , Polisacáridos Bacterianos/biosíntesis , Proteolisis , Proteínas Recombinantes de Fusión/análisis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Xanthomonas campestris/genética , Xanthomonas campestris/metabolismoRESUMEN
The homogeneous reaction between glucose oxidase and osmium bipyridine-pyridine carboxylic acid in the presence of glucose has been studied in detail by cyclic voltammetry and digital simulation. Combination of the analytical equations that describe the dependence of the amperometric response on enzyme, substrate and co-substrate concentrations for the limiting cases with digital simulation of the coupled enzyme reaction diffusion problem allows us to extract kinetic parameters for the substrate-enzyme reaction: K(MS)=10.8 mM, k(cat)=254 s(-1) and for the redox mediator-enzyme reaction, k=2.2x10(5) M(-1) s(-1). The accurate determination of the kinetic parameters at low substrate concentrations (<7 mM) is limited by depletion of the substrate close to the electrode surface. At high substrate concentrations (>20 mM) inactivation of the reduced form of glucose oxidase in the bulk solution must be taken into account in the analysis of the results.
Asunto(s)
Electroquímica/métodos , Glucosa Oxidasa/química , Compuestos Organometálicos/química , Osmio/química , Activación Enzimática , Cinética , Oxidación-ReducciónRESUMEN
The possibility of detection of incipient cellular alterations is central to early diagnosis and to clinician's capacity to discriminate between samples that appear similar on routine preparations. We examined the value of silver-stained nucleolar organizer regions (AgNOR) in detecting radio-induced alterations in a model of squamous epithelium biologically similar to oral mucosa. Morphometry of AgNOR has been proven to be of value in the detection of incipient cellular alterations. This method allows for the quantitative evaluation of lesions induced by high doses of radiation long before they become apparent in routine preparations. We herein examine the capacity of AgNOR to reveal the response to low doses of radiation, closer to the therapeutic or accidental dose to which the epithelium of oral mucosa may be exposed.
Asunto(s)
Células Epiteliales/efectos de la radiación , Región Organizadora del Nucléolo/efectos de la radiación , Análisis de Varianza , Animales , Relación Dosis-Respuesta en la Radiación , Ratas , Ratas Wistar , Tinción con Nitrato de PlataRESUMEN
We describe useful vectors to select double-crossover events directly in site-directed marker exchange mutagenesis in gram-negative bacteria. These vectors contain the gusA marker gene, providing colorimetric screens to identify bacteria harboring those sequences. The applicability of these vectors was shown by mapping the 3' end of the Xanthomonas campestris gum operon, involved in biosynthesis of xanthan.
Asunto(s)
Genes Bacterianos , Vectores Genéticos , Bacterias Gramnegativas/genética , Operón , Xanthomonas campestris/genética , Secuencia de Bases , Mapeo Cromosómico , Cartilla de ADN/genética , ADN Bacteriano/genética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Xanthan is an industrially important exopolysaccharide produced by the phytopathogenic, gram-negative bacterium Xanthomonas campestris pv. campestris. It is composed of polymerized pentasaccharide repeating units which are assembled by the sequential addition of glucose-1-phosphate, glucose, mannose, glucuronic acid, and mannose on a polyprenol phosphate carrier (L. Ielpi, R. O. Couso, and M. A. Dankert, J. Bacteriol. 175:2490-2500, 1993). A cluster of 12 genes in a region designated xpsI or gum has been suggested to encode proteins involved in the synthesis and polymerization of the lipid intermediate. However, no experimental evidence supporting this suggestion has been published. In this work, from the biochemical analysis of a defined set of X. campestris gum mutants, we report experimental data for assigning functions to the products of the gum genes. We also show that the first step in the assembly of the lipid-linked intermediate is severely affected by the combination of certain gum and non-gum mutations. In addition, we provide evidence that the C-terminal domain of the gumD gene product is sufficient for its glucosyl-1-phosphate transferase activity. Finally, we found that alterations in the later stages of xanthan biosynthesis reduce the aggressiveness of X. campestris against the plant.