Gluconeogenesis Is Required For Virulence In Xanthomonas Campestris Pathovar Campestris

The most obvious reason why bacteria seek to interact with plants and animals is for acquisition of nutrients more efficiently, and carbon is one of the most basic elements. Glucose is one of the most utilization of carbon source by microorganisms. Gluconeogenesis is a metabolic pathway for the biosynthesis of new glucose or 6-phosphoglucose from non-sugar compounds when there is not sufficient hexose in their niches. The formation of phosphoenolpyruvate (PEP) is the key step in the gluconeogenic pathway in which tricarboxylic acid (TCA) cycle intermediates are converted to hexose sugars and in bacteria there exist two routes to synthesize PEP: the phosphoenolpyruvate carboxykinase (PCK) route and the malic enzyme-phosphoenolpyruvate synthase (PpsA) route. We demonstrated genetically that Xanthomonas campestris pathovar campestris (Xcc 8004) possesses only the malic enzyme-PpsA route in gluconeogenesis. To identify the function of the malic enzyme-PpsA route in gluconeogenesis in Xcc 8004, the nonpolar mutants of ppsA gene was generated by homologous suicide vector integration into target gene by a single crossover event, named NK1880. The wild type strain 8004 and the complemented strains CNK1880 grew normally on NCM medium supplemented with malate, succinate, fumarate, pyruvate or acetate as the sole carbon source but the ppsA mutant NK1880 was unable to grow in such medium. These results demonstrated that the PckA route is functionally lacked and the malic enzyme-PpsA route is the only route to synthesize PEP from C4-dicarboxylate in gluconeogenesis of Xcc, which is consist with genomic annotation. Virulence of the NK1880 was determined on Chinese radish (Raphanus sativus L. var. niger) by the leaf clippingmethod, Ten days post-inoculation, lesion length with a mean of 4.3 mm was observed on leaves inoculated with the NK1880, while a mean lesion length of 14.96 mm and 13.95 mm were observed with wild type strain 8004 and complemented strain CNK1880, respectively. The cell numbers of the ppsA mutant in the infected leaves were about 100-fold fewer than that of the wild type strain and the complemented strains from day 4 to day 10 after inoculation.These results demonstrated that an intact gluconeogenic pathway is required for full virulence and in planta multiplication mXcc.