P-389 - Tolerance to the benzoxazinoid MBOA in Pseudomonadaceae

Benzoxazinoids (BXs) are the specialized and bioactive metabolites of maize root exudates and contribute to structuring of the root microbiota. The most abundant and selective BX in the maize rhizosphere is 6-methoxy-benzoxazolin-2-one (MBOA). In vitro tolerance to MBOA has been shown to correlate with abundance on BX-producing roots. Here, we used random barcode transposon-site sequencing (RB-TnSeq) to identify tolerance mechanisms in Pseudomonadaceae, incl. plant-growth promoting Pseudomonas putida KT2440 and P. simiae WCS417. KT2440 was very tolerant to MBOA with growth not affected even at high MBOA concentrations (2500 µM) whereas WCS417 showed intermediate tolerance. Both strains did not degrade or detoxify MBOA. We grew replicated cultures containing up to 100k different barcoded transposon mutants in presence of varying MBOA concentrations and monitored mutant abundances using sequencing. Based on changes in mutant abundances, genes affecting tolerance were identified. Genes contributing to fitness in P. putida in presence of MBOA included an RND-type efflux pump and the toluene tolerance protein as well as an LPS-modifying enzyme. Genes whose functions were reducing strain fitness in presence of MBOA were mostly linked to the outer membrane (e.g. LPS biosynthesis). These results suggest that tolerance is linked to reduced accumulation of BXs by active efflux as well as cell envelope structure. In how far these mechanisms affect root colonization remains to be tested.