Optimized fluorescent proteins for the rhizosphere-associated bacterium Bacillus mycoides with endophytic and...

Tracking of fluorescent protein (FP)-labelled rhizobacteria is a key prerequisite to gain insights into plant-bacteria interaction mechanisms. However, the performance of FPs mostly has to be optimized for the bacterial host and for the environment of intended application. We report on the construction of mutational libraries of the superfolder green fluorescent protein sfGFP and the red fluorescent protein mKate2 in the bacterium B. mycoides, which next to its potential as plant-biocontrol agent occasionally enters an endophytic lifestyle. By fluorescence-activated cell sorting and comparison of signal intensities at the colony and single-cell level, the variants sfGFP(SPS6) and mKate (KPS12) with significantly increased brightness were isolated. Their high applicability for plant-bacteria interaction studies was shown by confocal laser scanning microscopy tracking of FP-tagged B. mycoides strains after inoculation to Chinese cabbage plants in a hydroponic system. During the process of colonization, strain EC18 rapidly attached to plant roots and formed a multicellular matrix, especially at the branching regions of the root hair, which probably constitute entrance sites to establish an endophytic lifestyle. The universal applicability of the novels FPs was proven by expression from a weak promoter, dual-labelling of B. mycoides, and by excellent expression and detectability in additional soil- and rhizosphere-associated Bacillus species.