Phenotypic heterogeneity during heterologous protein production in Bacillus megaterium

During the last decade Bacillus megaterium was systemically developed for the gram per liter production of recombinant proteins. In contrast to the usual bacterial gene expression scenario, recombinant protein production systems often rely on multicopy plasmid encoded promoters, repressors and target genes. During production of the green fluorescent model protein (GFP) as a model culture heterogeneity was observed at the single cell level using time-lapse microscopy. The two subpopulations (producing versus low-producing) were separated by cell sorting and initially analyzed for limiting components of the expression system via quantitative RT-PCR. Further cultivation of both subpopulations revealed a systematic reajustment of their subpopulation distributions, indicationg bistability. An initial model was deduced as working hypothesis to identify the molecular basis for the observed phenotype and to improve our mathematical model.

Fluorescence microscopic image of a growing B. megaterium microcolony and colony heterogeneity of plated cells. The same image is shown as fluorescent image (A) and bright image overlaid with green-colored fluorescent image (B). Arrows indicate low-producing cells. Colony heterogeneity is shown in C) with small green-colored high GFP producing colonies and large white low GFP producing colonies. In the enlarged detail of image (C) three large white colonies are marked indicating low-producing cells.