Measurement of Macromolecular Crowding in Rhodobacter sphaeroides under Different Growth Conditions

The bacterial cytoplasm is a very crowded environment, and changes in crowding are thought to have an impact on cellular processes including protein folding, molecular diffusion and complex formation. Previous studies on the effects of crowding have generally compared cellular activity after imposition of stress. In response to different light intensities, in unstressed conditions, Rhodobacter sphaeroides changes the number of 50-nm intracytoplasmic membrane (ICM) vesicles, with the number varying from a few to over a thousand per cell. In this work, the effects of crowding induced by ICM vesicles in photoheterotrophic R. sphaeroides were investigated using a fluorescence resonance energy transfer (FRET) sensor and photoactivated localization microscopy (PALM). In low light grown cells where the cytoplasm has large numbers of ICM vesicles, the FRET probe adopts a more condensed conformation, resulting in higher FRET ratio readouts compared to high light cells with fewer ICM vesicles. The apparent diffusion coefficients of different sized proteins, PAmCherry, PAmCherry-CheY(6), and L1-PAmCherry, measured via PALM showed that diffusion of protein molecules >27kDa decreased as the number of ICM vesicles increased. In low light R. sphaeroides where the crowding level is high, protein molecules were found to diffuse more slowly than in aerobic and high light cells. This suggests that some physiological activities might show different kinetics in bacterial species whose intracellular membrane organization can change with growth conditions. IMPORTANCE The bacterial cytoplasm is known to be crowded, with that crowding suggested to change with growth, with chromosome replication, and under stress conditions. Many physiological activities depend on proteins and substrates diffusing through the cytoplasm; in some cases, large complexes need to diffuse from pole to pole. It is unclear how increases in crowding might affect cellular functions. We investigated whether we could naturally change the crowded state of the Rhodobacter sphaeroides cytoplasm by growing under different growth conditions. We show that increasing the number of intracytoplasmic vesicles by growing photosynthetically does change the crowded state of the cytoplasm and also alters the diffusion rates of different sized proteins measured. As many other cellular processes require protein movement, these findings could have broader implications for bacterial growth and responses under changing conditions that could alter cytoplasmic crowding.

Automated summary

Crowding in bacterial cytoplasm can affect cellular processes such as protein folding and molecular diffusion. This study investigates the effects of crowding induced by intracytoplasmic membrane vesicles on Rhodobacter sphaeroides using fluorescence resonance energy transfer (FRET) and photoactivated localization microscopy (PALM). The results show that increased crowding leads to more condensed protein conformation and decreased protein diffusion rates. The findings suggest that physiological activities in bacteria can exhibit different kinetics depending on intracellular membrane organization.