title: Cloning and heterologous overexpression of three gap genes encoding different glyceraldehyde-3-phosphate dehydrogenases from the plant pathogenic bacterium Pseudomonas syringae pv. tomato strain DC3000.
abstract: The gammaproteobacterium Pseudomonas syringae pv. tomato DC3000 is the causal agent of bacterial speck, a common disease of tomato. The mode of infection of this pathogen is not well understood, but according to molecular biological, genomic and proteomic data it produces a number of proteins that may promote infection and draw nutrients from the plant. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a major enzyme of carbon metabolism that was reported to be a surface antigen and virulence factor in other pathogenic microorganisms, but its possible role in the infection process of P. syringae has so far not been studied. Whole-genome sequence analyses revealed the occurrence in this phytopathogenic bacterium of three paralogous gap genes encoding distinct GAPDHs, namely two class I enzymes having different molecular mass subunits and one class III bifunctional D-erythrose-4-phosphate dehydrogenase/GAPDH enzyme. By using genome bioinformatics data, as well as alignments of both DNA and deduced protein sequences, the three gap genes of P. syringae were one-step cloned with a His-Tag in pET21a vector using a PCR-based strategy, and its expression optimized in Escherichia coli BL21 to achieve high yield of the heterologous proteins. In accordance with their distinct molecular phylogenies, these bacterial gap genes encode functional GAPDHs of diverse molecular masses and nicotinamide-coenzyme specificities, suggesting specific metabolic and/or cellular roles.
authors.affiliation: Laboratory of Physiology & Genetics Molecular, Faculty of Sciences Ain Chock, Casablanca, Morocco.
authors.forename: Bouchra
authors.initials: B
authors.lastname: Elkhalfi
authors.forename: José Miguel
authors.initials: JM
authors.lastname: Araya-Garay
authors.forename: Jorge
authors.initials: J
authors.lastname: Rodríguez-Castro
authors.forename: Manuel
authors.initials: M
authors.lastname: Rey-Méndez
authors.forename: Abdelaziz
authors.initials: A
authors.lastname: Soukri
authors.forename: Aurelio
authors.initials: A
authors.lastname: Serrano Delgado
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D005798:Genes, Bacterial; D005822:Genetic Vectors / Q000235:genetics; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D018551:Solanum lycopersicum / Q000382:microbiology*; D008969:Molecular Sequence Data; D010802:Phylogeny; D044224:Pseudomonas syringae / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D015854:Up-Regulation
chemical_list: D011994:Recombinant Proteins; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases