RESUMEN
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.
Asunto(s)
Genes Bacterianos , Enfermedades de las Plantas/etiología , Polisacáridos Bacterianos/biosíntesis , Xanthomonas campestris/genética , Secuencia de Bases , Datos de Secuencia Molecular , Mutación , Uridina Difosfato Glucosa/metabolismo , Virulencia , Xanthomonas campestris/metabolismo , Xanthomonas campestris/patogenicidadRESUMEN
The primary structure of several chloroplast fructose-1,6-bisphosphatase (CFBPase) was deduced from DNA sequences, but only spinach, pea and rapeseed enzymes have been characterized structurally. We analyzed whether CFBPases from different phylogenetic origin contain a common epitope. To this end a DNA fragment of 1200 base pairs encoding 338 amino acid residues of wheat CFBPase (38 kDa) was cloned in the expression plasmid pGEX-1 in frame with the gene coding for glutathione S-transferase (GT) of Schistosoma japonicun (26.5 kDa). Upon transformation of Escherichia coli and induction with isopropyl-beta-D-thiogalactopyranoside, centrifugation of the lysate partitioned 10% of the fusion protein in the supernatant fraction and the remaining 90% in the precipitate. The expected 65 kDa protein was purified from both the soluble and the particulate fraction by affinity chromatography on columns of glutathione-agarose. This fusion protein was successfully used to produce a monoclonal antibody that specifically recognized the CFBPase region of the fusion protein but not the GT moiety. Moreover, the monoclonal antibody immunoreacted not only with polypeptides (ca. 40 kDa) present in leaf crude extracts of other varieties of wheat (Triticum spelta, T. aestivum and T. durum), but also with homogeneous preparations of the spinach (Spinacia oleracea) and rapeseed (Brassica napus) enzymes. Thus, the cross reaction of this monoclonal antibody with counterparts from different plant species indicates the persistency of a common epitope through biological evolution.
Asunto(s)
Anticuerpos Monoclonales , Cloroplastos/enzimología , Fructosa-Bifosfatasa/inmunología , Triticum/enzimología , Secuencia de Aminoácidos , Animales , Especificidad de Anticuerpos , Cloroplastos/genética , Cloroplastos/inmunología , Clonación Molecular , Reacciones Cruzadas , Epítopos/genética , Epítopos/inmunología , Escherichia coli/genética , Evolución Molecular , Fructosa-Bifosfatasa/genética , Fructosa-Bifosfatasa/aislamiento & purificación , Glutatión Transferasa/genética , Ratones , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/aislamiento & purificación , Plantas/enzimología , Plantas/genética , Plantas/inmunología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/aislamiento & purificación , Schistosoma japonicum/enzimología , Schistosoma japonicum/genética , Especificidad de la Especie , Triticum/genética , Triticum/inmunologíaRESUMEN
The Xanthomonas campestris gum gene cluster is composed of 12 genes designated gumB, -C, -D, -E, -F, -G, -H, -I, -J, -K, -L, and -M. The transcriptional organization of this gene cluster was analyzed by the construction of gum-lacZ transcriptional fusions in association with plasmid integration mutagenesis. This analysis, coupled with primer extension assays, indicated that the gum region was mainly expressed as an operon from a promoter located upstream of the first gene, gumB.