Raman spectroscopy and resonance Raman spectroscopy are widely used to study bacteria and their responses to different environmental conditions. In the present study, the identification of a novel resonance Raman peak for Escherichia coli, recorded with 633 nm laser excitation is discussed. A peak at 740 cm⁻¹ is observed exclusively with 633 nm excitation but not with 514 nm or 785 nm excitation. This peak is absent in the lag phase but appears in the log phase of bacterial growth. The intensity of the peak increases at high temperature (45 °C) compared with growth at low temperature (25 °C) or the physiological temperature (37 °C). Although osmotic stress lowered bacterial growth, the intensity of this peak was unaffected. However, treatment with chemical uncouplers of oxidative phosphorylation resulted in significantly lower intensity of this Raman band, indicating its possible involvement in respiration. Cytochromes, a component of bacterial respiration‚ can show resonance enhancement at 633 nm due to the presence of a shoulder in that region depending on the type and conformation of cytochrome. Therefore, the peak intensity was monitored in different genetic mutants of E. coli lacking cytochromes. This peak is absent in the Escherichia coli mutant lacking cydB, but not ccmE, demonstrating the contribution of cytochrome bd subunit II in the peak’s origin. In future, this newly found cytochrome marker can be used for biochemical assessment of bacteria exposed to various conditions. Overall, this finding opens the scope for use of red laser excitation in resonance Raman in monitoring stress and respiration in bacteria.

Graphical abstract

拉曼光谱和共振拉曼光谱被广泛用于研究细菌及其对不同环境条件的响应。在本研究中，讨论了用633 nm激光激发记录的大肠杆菌新共振拉曼峰的鉴定。740 cm ^-1处的峰仅在633 nm激发下观察到，但在514 nm或785 nm激发下观察不到。该峰在滞后阶段不存在，但在细菌生长的对数阶段出现。与在低温（25°C）或生理温度（37°C）下的增长相比，在高温（45°C）下峰的强度增加。尽管渗透胁迫降低了细菌的生长，但该峰的强度并未受到影响。但是，用化学解偶联剂进行氧化磷酸化处理后，该拉曼谱带的强度明显降低，表明其可能参与了呼吸作用。细胞色素是细菌呼吸的组成部分，由于细胞色素的类型和构象，在该区域存在肩部，因此在633 nm处可显示共振增强。因此，大肠杆菌缺乏细胞色素。缺少cydB的大肠杆菌突变体中没有该峰，但没有ccmE，表明该峰起源中细胞色素bd亚基II的贡献。将来，这种新发现的细胞色素标记物可用于对暴露于各种条件的细菌进行生化评估。总体而言，这一发现为共振拉曼中的红色激光激发用于监测细菌的压力和呼吸打开了广阔的空间。

图形概要