Transcriptome analysis of B. subtilis cells during recovery from injury after high hydrostatic pressure treatment

Vegetative cells of B. subtilis can recover from injury caused by high hydrostatic pressure (HHP) treatment at 250 MPa. DNA microarray analysis revealed that many ribosomal genes and translation relating factors (e.g. translation initiation factors) were induced during a growth-arrested phase after the HHP treatment. Expression of cold shock-responsive genes, whose products play key roles for efficient translation, and heat shock-responsive genes, whose product mediates the correct protein folding or degrades misfolded proteins, were also induced. In contrast, the expression of hpf, whose product (Hpf) is involved in ribosome inactivation through dimerization of 70S ribosomes, was repressed. Sucrose density gradient sedimentation analysis showed that ribosomes were dissociated in a pressure-dependent manner and then reconstructed during the growth-arrested phase. These results suggested that the translational machinery can be preferentially reconstructed in the HHP-injured cells. We also found that cell growth after HHP-induced injury was apparently inhibited by Mn2+ or Zn2+ supplemented to the recovery medium. Ribosome reconstruction in HHP-injured cells was significantly delayed in the presence of Mn2+ or Zn2+. Moreover, Zn2+ but not Mn2+ stimulated dimer formation of ribosomes in HHP-injured cells. This Zn2+-dependent accumulation of ribosome dimer was no longer observed in a Δhpf mutant lacking the functional Hpf. Furthermore, the growth recovery of the Δhpf mutant in the Zn2+-supplemented medium was faster than that of the parent strain. Thus, our results indicate that Zn2+ can prevent ribosome reconstruction by stimulating the Hpf-dependent ribosome dimerization, thereby inhibiting the growth recovery of the HHP-injured B. subtilis cells. The B. subtilis strain TI465 were grown in NaCl-free L medium (10 g tryptone, 5 g yeast extract, per liter) for 6 h at 37°C with vigorous shaking. Then, the culture was packed into a sterile polyethylene bag that was heat-sealed after exclusion of air bubbles and subjected to HHP treatment (250 MPa) using a Dr. CHEF (Kobe Steel, Kobe, Japan) at 25°C for 10 min as described previously (Inaoka, T., et al., Biosci Biotechnol Biochem 81 (2017) 1235-1240). The rate of increasing and decreasing of pressure was set at 200 MPa/min. The cells treated at 250 MPa were transferred into a recovery medium (NaCl-free L medium) and incubated at 37°C with vigorous shaking. Total RNAs were extracted from the cells incubated for 0 h (immediately after HHP treatment), 2 h (growth arrest phase), or 4 h (re-growing phase) and then subjected to DNA microarray analysis.