Cloning, purification, crystallization and 1.57 Å resolution X-ray data analysis of AmsI, the tyrosine phosphatase controlling amylovoran biosynthesis in the plant pathogen Erwinia amylovora

Abstract

The Gram-negative bacterium Erwinia amylovora is a destructive pathogen of plants belonging to the Rosaceae family. Amongst its pathogenicity factors, E. amylovora produces the exopolysaccharide amylovoran, which contributes to the occlusion of plant vessels, causing wilting of shoots and eventually resulting in plant death. Amylovoran biosynthesis requires the presence of 12 genes (from amsA to amsL) clustered in the ams region of the E. amylovora genome. They mostly encode glycosyl transferases (AmsG, AmsB, AmsD, AmsE, AmsJ and AmsK), proteins involved in amylovoran translocation and assembly (AmsH, AmsL and AmsC), and also a tyrosine kinase (AmsA) and a tyrosine phosphatase (AmsI), which are both involved in the regulation of amylovoran biosynthesis. The low-molecular-weight protein tyrosine phosphatase AmsI was overexpressed as a His6-tagged protein in Escherichia coli, purified and crystallized. X-ray diffraction data were collected to a maximum resolution of 1.57 Å in space group P3121.

Keywords: AmsI; Erwinia; amylovoran; exopolysaccharides; tyrosine phosphatase.

Figure 1

Elution profile of the size-exclusion chromatography carried out as a second step of the purification using a Sephadex S75 16/60 column (1.6 × 60 cm; GE Healthcare, Sweden). The injection volume was 5 ml protein solution (the blue line represents the UV absorption at 280 nm). This step was used as a desalting step as well (the brown line represents the conductivity of the solution).

Figure 2

Diffraction image of AmsI collected on a PILATUS 6M detector. The insets show magnifications of the low-resolution and high-resolution regions.