Direct high-pressure NMR observation of dipicolinic acid leaking from bacterial spore: A crucial step for thermal inactivation

• Published in: Biophysical chemistry Vol. 231; pp. 10 - 14
• Main Authors: Akasaka, Kazuyuki, Maeno, Akihiro, Yamazaki, Akira
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.12.2017
• Subjects: Bacillus subtilis - physiology; Dipicolinic acid; High-pressure NMR; Hot Temperature; Magnetic Resonance Spectroscopy; Picolinic Acids - metabolism; Pressure; Pressure response; Protein thermal denaturation; Solubility; Spore inactivation; Spore suspension; Spores, Bacterial - physiology; High-pressure NMR; Dipicolinic acid; Protein thermal denaturation; Spore inactivation; Pressure response; Spore suspension
• ISSN: 0301-4622; 1873-4200
• DOI: 10.1016/j.bpc.2017.04.008

A bacterial spore protects itself with an unusually high concentration (~10% in dry weight of spore) of dipicolinic acid (DPA), the release of which is considered the crucial step for inactivating it under mild pressure and temperature conditions. However, the process of how the spore releases DPA in response to pressure remains obscure. Here we apply 1H high-resolution high-pressure NMR spectroscopy, for the first time, to the spore suspension of Bacillus subtilis natto and monitor directly and in real-time the leaking process of DPA in response to pressure of 200MPa at 20°C. We find that about one third of the total DPA leaks immediately upon applying pressure, but that the rest leaks slowly in hrs upon decreasing the pressure. Once DPA is fully released from the spore, the proteins of the spore become easily denatured at a mild temperature, e.g., 80°C, much below the temperature commonly used to inactivate spores (121°C). The success of the present experiment opens a new avenue for studying bacterial spores and cells at the molecular level in response to pressure, temperature and other perturbations. [Display omitted] •High-pressure NMR is used to monitor leaking of DPA from Bacillus subtilis spore by applying 200 MPa at 20°C.•One-third of DPA leaks immediately upon pressurizing at 200 MPa, while the rest leaks slowly only upon depressurizing.•Once DPA is depleted from the core, their proteins become easily denatured at ~80°C, leading to the spore inactivation.