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The role of glucose kinase in carbohydrate utilization and extracellular polysaccharide production in Xanthomonas campestris pathovar campestris

Guangxi Key Laboratory of Subtropical Bioresources Conservation and Utilization, The Key Laboratory of Ministry of Education for Microbial and Plant Genetic Engineering, and College of Life Science and Technology, Guangxi University, 100 Daxue Road, Nanning, Guangxi 530004, China

Correspondence
Ji-Liang Tang
jltang{at}gxu.edu.cn

The genome of the Xanthomonas campestris pathovar campestris (Xcc) 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 His₆ affinity tag and purified by nickel affinity chromatography. The standard Glk activity assay revealed that all three proteins possessed apparent Glk activity, with XC1976-His₆ being the most active; the specific activity values were 1.16x10⁶ U mg^–1 for XC1166-His₆, 4.36x10⁷ U mg^–1 for XC1223-His₆ and 2.63x10⁸ U mg^–1 for XC1976-His₆. TLC analysis showed, however, that only XC1976-His₆ could phosphorylate glucose. Insertional mutants of XC1166, XC1223 and XC1976 were generated using the suicide plasmid pK18mob. Although mutant strains with insertions in XC1166 or XC1223 had Glk activity similar to that of the wild-type strain, the XC1976 mutant had only about 6 % of the wild-type activity. Mutation in XC1976 had complex effects on EPS production. In media containing arabinose, glucose, galactose, sucrose or maltose, the XC1976 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 XC1976 mutant also showed attenuated virulence on the host plant Chinese radish (Raphanus sativus). The results indicate that XC1976 has the most significant role for the parameters tested.

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