1887

Abstract

The genome of the pathovar () strain 8004 encodes three uncharacterized proteins, XC1166, XC1223 and XC1976, annotated as glucose kinase (Glk) by bioinformatic studies. Here we have investigated the biochemical characteristics and physiological roles of these proteins with particular reference to the synthesis of extracellular polysaccharide (EPS). XC1166, XC1223 and XC1976 were overexpressed as fusion proteins with a His affinity tag and purified by nickel affinity chromatography. The standard Glk activity assay revealed that all three proteins possessed apparent Glk activity, with XC1976-His being the most active; the specific activity values were 1.16×10 U mg for XC1166-His, 4.36×10 U mg for XC1223-His and 2.63×10 U mg for XC1976-His. TLC analysis showed, however, that only XC1976-His could phosphorylate glucose. Insertional mutants of , and were generated using the suicide plasmid pK18. Although mutant strains with insertions in or had Glk activity similar to that of the wild-type strain, the mutant had only about 6 % of the wild-type activity. Mutation in had complex effects on EPS production. In media containing arabinose, glucose, galactose, sucrose or maltose, the mutant produced about 40–75 % of the wild-type level of EPS, whereas in medium containing fructose, the mutant showed a 30 % increase in EPS production compared to the wild-type strain. The mutant also showed attenuated virulence on the host plant Chinese radish (). The results indicate that XC1976 has the most significant role for the parameters tested.

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2007-12-01
2020-04-07
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