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.

A homolog of glyceraldehyde-3-phosphate dehydrogenase from Riemerella anatipestifer is an extracellular protein and exhibits biological activity

“…In EHEC, gapA and gapC genes encode GAPDH proteins with highly similar sequences, and the gapA gene accounted for the exported GAPDH enzyme (Egea et al, 2007). In the Pseudomonas syringae genome, there are three paralogous gapdh genes encoding distinct GAPDHs, namely two class I enzymes having different molecular mass subunits and one class III biofunctional D-erythose-4-phosphate dehydrogenase/GAPDH enzyme (Elkhalfi et al, 2013). However, our results showed that there were several R. anatipestifer strains which did not have a homologous amplicon of GAPDH and showed no extracellular GAPDH activity.…”

Identification of an extracellular infection-induced glyceraldehyde-3-phosphate dehydrogenase of the phytopathogenic proteobacterium <i>Pseudomonas syringae</i> pv tomato DC3000

“…1.41 (gap1), 2.28 (gap2) and 0.43 (gap3) Mb from the replication origin. Moreover, these paralogous genes are predicted to encode three GAPDH enzymatic proteins with distinct molecular and catalytic features, namely two Class I enzymes having different molecular mass subunits and one class III D-erythrose-4-phosphate dehydrogenase/GAPDH bifunctional enzyme [25].…”

“…tomato DC3000 [25], it remains to be established whether any of these GAPDHs are actually extracellular proteins involved in the plant infection process. As described, the pathogenicity of DC3000 resembles those of most animal and plant microbial pathogens of the Gammaproteobacteria, which in the infective state secrete a number of proteins across their cell envelopes [30][31][32].…”

“…The apparent molecular masses of the proteins were estimated using the Precision Plus Protein Standard (BioRad) and the analysis software Quantity One (Bio-Rad). Western blots after SDS-PAGE and transfer to nitrocellulose membranes were carried out as previously described [27] using anti-GAPDH rabbit antibodies [25].…”

“…Three gap genes encoding distinct GAPDH proteins are present in the genome of P. syringae pv. tomato DC3000 [25]. Whether any of these bacterial GAPDHs are extracellular proteins involved in the plant infection process remains to be established.…”