Abstract
ABSTRACTThe 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-CheY6, and L1-PAmCherry, measured via PALM showed that diffusion of protein molecules >27 kDa 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.
Highlights
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
We show that the presence of intracytoplasmic membrane (ICM) vesicles in photoheterotrophic R. sphaeroides leads to macromolecular crowding effects that can be detected in vivo by a genetically encoded fluorescence resonance energy transfer (FRET) sensor and reflected through the reduction of protein diffusion rates when compared to high light grown cells
Using a genetically encoded FRET probe and single molecule tracking, we show that ICM vesicles in photoheterotrophic R. sphaeroides increase crowding in the cytoplasm
Summary
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. 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. Macromolecular crowding in live cells has been estimated by studying the motions of fluorescently labeled molecules in the cytoplasm and the relationship between crowding and diffusion studied by inducing cellular changes that promote or reduce the formation of cytoplasmic barriers and might restrict molecular diffusion Most of these studies involved the use of stressed conditions such as osmotic upshift [1,2,3], antibiotic treatments [4,5], and nutrient limitation [6] to allow comparisons between the perturbed and the native state of the bacterial population. These physiological responses to growth conditions could all have effects on the crowded state of the cytoplasm
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