Macro-Level and Genetic-Level Responses of Bacillus subtilis to Shear Stress

Responses of bacterial (Bacillus subtilis) cells under different shear levels, from both the macro and genetic viewpoints, have been presented. The responses were studied using a novel, couette flow bioreactor (CFB), in which the entire cultivation can be performed under defined shear conditions. Oxygen supply, the normal limiting factor for entire cultivations under defined shear conditions, has been achieved by passing air through a poly(tetrafluoroethylene) (PTFE) membrane fixed on the inner cylinder of the CFB. More importantly, analyses of the oxygen transfer capabilities as well as the shear rates show that in this CFB, the effects of defined shear can be studied without interference from the effects of oxygen supply. Further, the shake flask can be used as a proper control for studying the shear effects, mainly because the shear rate in the shake flask under normal shaker operating conditions of 190 rpm has been estimated to be a negligible 0.028 s^-1 compared to a value of 445 s^-1 at the lowest rpm employed in the CFB. At the macro level the cell size decreased by almost 50% at 1482 s^-1 compared to that at 0.028 s^-1, the growth rate increased by 245%, and the maximum cell concentration increased by 190% when the shear rate was increased from 0.028 to 1482 s^-1. The specific intracellular catalase level increased by 335% and protease by 87% at 1482 s^-1 as compared to the control cultures at a shear rate 0.028 s^-1. In addition, the specific intracellular reactive oxygen species level (siROS) at the highest shear rate was 9.3-fold compared to the control conditions. At the genetic level we have established the involvement of the transcription factor, σ^B, in the bacterial responses to shear stress, which was unknown in the literature thus far; the σ^B expression correlated inversely with the siROS. Further, through experiments with ROS quenchers, we showed that ROS regulated σ^B expression under shear.