Objective: To study the repair mechanisms of frozen sublethally damaged Staphylococcus aurous cells.
Methods: We resuscitated frozen sublethally damaged S. aureus at 37 degrees C for different time within 3 h. Meanwhile, we compared the morphological changes of the frozen sublethally damaged cells after 1 h of resuscitation using transmission electron microscopy assay (TEM). The expressions of the transcriptional attenuator MsrR (msrR), iron (Fe3+) ABC transporter ATP-binding protein (fhuC), and cytochrome b (cytB) genes were quantitatively analyzed by real-time fluorescence quantitative PCR (Real-time PCR) method. The content of cells outside leakage, active oxygen (ROS), and superoxide dismutase (SOD) activity were also determined by ultraviolet spectrophotometry.
Results: More than 99% of the frozen sublethally damaged S. aureus repaired after 3 h. The resuscitated cells expressed an equal resistance to high concentration of NaCl. Real-time PCR results showed that the msrR and fhuC genes expressions were down-regulated, whereas the cytB gene expression was up-regulated significantly. The frozen sublethally damaged S. aureus cellar surface ultrastructure significant changed during resuscitation. The cell surface became compact and sturdy from smooth and transparent. The cell leakage rate of ultraviolet absorption material gradually decreased. Meanwhile, the intracellular ROS level declined along with the decrease of SOD activity.
Conclusion: Frozen sublethally damaged cells may regain the capability of resistance to high salt stress by repairing cell membrane integrity, reducing the content of ROS through gene regulation, inhibiting the toxicity of active oxygen to the cells. Meanwhile, the regulation of metabolism related genes (cytB) provides the energy for the requirement of cells, therefore, the frozen sublethally damaged cells were repaired finally.