细菌细胞质是一个非常拥挤的环境,拥挤的变化被认为对包括蛋白质折叠在内的细胞过程有影响,分子扩散和复合物形成。先前关于拥挤影响的研究通常比较了施加压力后的细胞活性。为了响应不同的光强度,在无压力条件下,球形红杆菌属改变了50-nm胞质内膜(ICM)囊泡的数量,每个细胞的数量从几到一千多不等。在这项工作中,使用荧光共振能量转移(FRET)传感器和光激活定位显微镜(PALM)研究了ICM囊泡在光异养的R.spaeroides中引起的拥挤效应。在低光生长的细胞中,细胞质中有大量的ICM囊泡,FRET探针采用更凝聚的构象,与具有较少ICM囊泡的高光细胞相比,导致更高的FRET比率读数。不同大小蛋白质的表观扩散系数,PAmCherry,PAmCherry-CheY6和L1-PAmCherry,通过PALM测量表明,随着ICM囊泡数量的增加,>27kDa的蛋白质分子的扩散减少。在拥挤程度高的低光R.sphaeroides中,蛋白质分子的扩散速度比有氧和强光细胞慢。这表明,某些生理活动可能在细菌物种中显示出不同的动力学,这些细菌的胞内膜组织可以随生长条件而变化。重要性已知细菌细胞质很拥挤,随着增长,人们认为拥挤会随着增长而变化,染色体复制,在压力条件下。许多生理活动依赖于蛋白质和底物通过细胞质扩散;在某些情况下,大型复合物需要从极点扩散到极点。目前尚不清楚拥挤的增加如何影响细胞功能。我们调查了是否可以通过在不同的生长条件下生长来自然地改变球形红细菌细胞质的拥挤状态。我们表明,通过光合作用增加胞质内囊泡的数量确实会改变细胞质的拥挤状态,也会改变测得的不同大小蛋白质的扩散速率。由于许多其他细胞过程需要蛋白质运动,这些发现可能对细菌在改变细胞质拥挤条件下的生长和反应产生更广泛的影响.
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-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. 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.