Abstract:
Pseudomonas syringae pv. Actnidiae (Psa), a gram-negative bacterium, is the causative agent of bacterial kiwifruit vine canker disease. An extremely virulent global outbreak of kiwifruit vine canker occurred from 2008 to 2010, affecting a number of kiwifruit growing nations. The disease was first identified in New Zealand in 2010 at a kiwifruit orchard in Te Puke. From the initial site of infection, the disease spread rapidly throughout New Zealand affecting ‘Hort16A’ plantings especially. Analysis of the strains involved in the global outbreak found that they had evolved from an independent evolutionary lineage to the previously identified strains of Psa; and also found a high level of diversity within the accessory genome. A genomic analysis of the New Zealand strain of Psa (Psa ICMP18884) identified a 74.4 kb plasmid that contained a number of genes not found in other geographic isolates. Two adjacent gene clusters with orthologues in vascular pathogens were of special interest, the first gene cluster was annotated as an anthranilate synthase and the second had the hallmarks of a secreted secondary metabolite pathway. The aim of this project was to characterize the structure and activity of the putative anthranilate synthase component I, especially to identify the product produced by this enzyme (Psa CUE). The Enzyme was over expressed in Escherichia coli (E. coli) and purified using immobilized metal affinity chromatography, anion exchange chromatography, and size exclusion chromatography Structural characterization of Psa CUE using mass spectrometry, size exclusion multi angle laser light scattering (SEC-MALLS), and small angle x-ray scattering (SAXS) showed that it existed as a homogenous monomer in solution with an overall shape that is consistent with known anthranilate synthase monomers. Analysis of enzymatic activity by nuclear magnetic resonance (NMR) spectroscopy and fluorimetric assays. Results showed that Psa CUE was accumulating the intermediary product 4-amino-4-deoxyisochorismate (ADIC) in solution, but also producing anthranilate at much lower rate of turnover. This behavior is atypical of anthranilate synthase and suggests the Psa CUE may be an ADIC synthase with weak anthranilate production. Further work studying the ASI-ASII complex is needed to confirm this.