The metabolic context for virulence in Pseudomonas syringae

The apoplast is the site of infection for many important bacterial crop pathogens, including the model pathogen <em>Pseudomonas syringae</em> pv. tomato DC3000 (Pst DC3000). The chemical environment within the plant apoplast can determine the outcome of bacterial infection and the composition of this compartment is known to change in response to the presence of invading organisms. However, this metabolically dynamic environment has received little attention in the literature, and even less is known about how metabolites in the apoplast influence the expression of virulence genes. In this study, several aspects of the metabolic context of virulence were assessed. First, a broad-scale analysis of the tomato apoplast was undertaken, which identified metal ions, sugars, organic acids and amino acids, the most abundant of which was the non-protein amino acid gamma-aminobutyric acid (GABA). The impact these components had on the expression of virulence genes and metabolism in Pst DC3000 were then tested. Components such as fructose and aspartate caused high levels of virulence gene expression which correlated with the accumulation of intracellular glutamate, whereas repressive components, such as GABA and threonine, resulted in lower glutamate levels. Second, metabolic flux analysis showed that Pst DC3000 underwent major changes in central carbon metabolism in response to virulence gene inducing conditions. The identification of altered internal metabolism in Pst DC3000 cells expressing virulence genes led to the conclusion that Pst DC3000 may understand its external environment by sensing intracellular metabolites or metabolic fluxes. Third, the role of GABA assimilation in virulence was explored, and it was found that high internal GABA levels resulted in virulence gene repression. In addition, previously unidentified mechanisms for GABA uptake and transport were detected by the use of a novel ‘unlabelling’ experiment.