Metabolic Adaptation of Ralstonia solanacearum during Plant Infection: A Methionine Biosynthesis Case Study

MetE and MetH are two distinct enzymes that catalyze a similar biochemical reaction during the last step of methionine biosynthesis, MetH being a cobalamin-dependent enzyme whereas MetE activity is cobalamin-independent. In this work, we show that the last step of methionine synthesis in the plant pathogen Ralstonia solanacearum is under the transcriptional control of the master pathogenicity regulator HrpG. This control is exerted essentially on metE expression through the intermediate regulator MetR. Expression of metE is strongly and specifically induced in the presence of plant cells in a hrpG- and metR-dependent manner. metE and metR mutants are not auxotrophic for methionine and not affected for growth inside the plant but produce significantly reduced disease symptoms on tomato whereas disruption of metH has no impact on pathogenicity. The finding that the pathogen preferentially induces metE expression rather than metH in the presence of plant cells is indicative of a probable metabolic adaptation to physiological host conditions since this induction of metE occurs in an environment in which cobalamin, the required co-factor for MetH, is absent. It also shows that MetE and MetH are not functionally redundant and are deployed during specific stages of the bacteria lifecycle, the expression of metE and metH being controlled by multiple and distinct signals

Contribution de l'INRA à la réflexion sur l'avenir de la recherche - Synthèse des débats internes.

document présenté devant le collège de direction, les présidents de Centre et les Chefs de Département, le 7 juillet 2004

Pathways for methionine biosynthesis in <i>E. coli</i>, <i>P. putida</i> and <i>R. solanacearum</i>.

<p>The MetE and MetH methionine synthases catalyze the methylation of homocysteine to produce methionine, with methyltetrahydrofolate [5-methyl tetrahydropteroyltri-L-glutamate] being the methyl group donor <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036877#pone.0036877-Figge1" target="_blank">[13]</a>.</p

Expression of <i>metE</i> and <i>metHab</i> is differentially regulated in the presence of plant cells.

<p>Gene expression of <i>metE</i> (A) and <i>metHab</i> (B) was monitored after 16 h of growth in complete medium, minimal medium supplemented with glutamate at 20 mM final concentration, in Gamborg medium containing <i>Arabidopsis thaliana</i> cells, in Gamborg medium without plant cells and in Gamborg medium supplemented with homocysteine at 100 µM final concentration. β-galactosidase activity is expressed in Miller units. Each measurement corresponds to the average of three replicates and bars indicate standard deviations.</p

Expression of <i>metE</i>, <i>metHab</i> and <i>metR</i> in different genetic backgrounds.

<p>Expression of <i>metE</i> (A), <i>metHab</i> (B) and <i>metR</i> (C) was determined after 16 h of growth in minimal medium supplemented with glutamate at a 20 mM final concentration (A and B) and in the presence of plant cells (C). β-galactosidase activity is expressed in Miller units. Each measurement corresponds to the average of three replicates and bars indicate standard deviations.</p

Disease progress curves of the various <i>R. solanacearum</i> methionine mutants on tomato plants.

<p>(A) For each strain 24 plants were inoculated using 50 mL of a bacterial suspension at 10⁷ CFU/mL for each plant. (B) For each strain 12 plants were inoculated by injection of 10⁴ CFU directly into the stem of the plant. Disease progress was rated using a disease index where 0 indicates healthy plants and 4 indicates a 100% wilted plant. For both tests 4 week old tomato plants were used. Results are representative of at least three independent experiments.</p