A proteomic study of Methylobacterium extorquens reveals a response regulator essential for epiphytic growth

Aerial plant surfaces are colonized by diverse bacteria such as the ubiquitous Methylobacterium spp. The specific physiological traits as well as the underlying regulatory mechanisms for bacterial plant colonization are largely unknown. The purpose of this study was to identify proteins produced specifically in the phyllosphere by comparing the proteome of Methylobacterium extorquens colonizing the leaves either with that of bacteria colonizing the roots or with that of bacteria growing on synthetic medium. We identified 45 proteins that were more abundant in M. extorquens present on plant surfaces as compared with bacteria growing on synthetic medium, including 9 proteins that were more abundant on leaves compared with roots. Among the proteins induced during epiphytic growth, we found enzymes involved in methanol utilization, prominent stress proteins, and proteins of unknown function. In addition, we detected a previously undescribed type of two-domain response regulator, named PhyR, that consists of an N-terminal sigma factor (RpoE)-like domain and a C-terminal receiver domain and is predicted to be present in essentially all Alphaproteobacteria. The importance of PhyR was demonstrated through phenotypic tests of a deletion mutant strain shown to be deficient in plant colonization. Among PhyR-regulated gene products, we found a number of general stress proteins and, in particular, proteins known to be involved in the oxidative stress response such as KatE, SodA, AhpC, Ohr, Trx, and Dps. The PhyR-regulated gene products partially overlap with the bacterial in planta-induced proteome, suggesting that PhyR is a key regulator for adaptation to epiphytic life of M. extorquens.