Tanaka Kyosuke, Oketani Ryosuke, Terada Takeshi, Leproux Philippe, Morono Yuki, Kano Hideaki
Department of Chemistry, Faculty of Science, Kyushu University, 744, Motooka, Nishi-ku, Fukuoka 819-0395, Japan.
Marine Works Japan, 3-54-1 Oppamahigashi, Yokosuka, Kanagawa 237-0063, Japan.
J Phys Chem B. 2023 Mar 9;127(9):1940-1946. doi: 10.1021/acs.jpcb.2c07291. Epub 2023 Feb 23.
Spore-forming bacteria accumulate dipicolinic acid (DPA) to form spores to survive in extreme environments. Vibrational spectroscopy is widely used to detect DPA and elucidate the existence of the bacteria, while vegetative cells, another form of spore-forming bacteria, have not been studied extensively. Herein, we applied coherent anti-Stokes Raman scattering (CARS) microscopy to spectroscopically identify both spores and vegetative cells without staining or molecular tagging. The spores were identified by the strong CARS signals due to DPA. Furthermore, we observed bright spots in the vegetative cells in the CARS image at 1735 cm. The vegetative cells contained molecular species with C=O bonds because this vibrational mode was associated with the carbonyl group. One of the candidate molecular species is diketopimelic acid (DKP), a DPA precursor. This hypothesis was verified by comparing the spectrum obtained by the vegetative cells with that of the DKP analogue (ketopimelic acid) and with the result obtained by DFT calculation. The results indicate that the observed vegetative cell is in the sporulation process. CARS spectra can be used to monitor the maturation and preformation of spores.
形成芽孢的细菌积累吡啶二羧酸（DPA）以形成孢子，从而在极端环境中生存。振动光谱法被广泛用于检测DPA并阐明细菌的存在，而营养细胞作为形成芽孢细菌的另一种形式，尚未得到广泛研究。在此，我们应用相干反斯托克斯拉曼散射（CARS）显微镜在不进行染色或分子标记的情况下对孢子和营养细胞进行光谱鉴定。由于DPA，孢子通过强烈的CARS信号得以识别。此外，我们在1735 cm处的CARS图像中观察到营养细胞中的亮点。营养细胞含有具有C=O键的分子种类，因为这种振动模式与羰基相关。候选分子种类之一是二酮哌嗪酸（DKP），一种DPA前体。通过将营养细胞获得的光谱与DKP类似物（酮基哌嗪酸）的光谱以及密度泛函理论（DFT）计算结果进行比较，验证了这一假设。结果表明观察到的营养细胞正处于芽孢形成过程中。CARS光谱可用于监测孢子的成熟和预形成。
