Identification of a resonance Raman marker for cytochrome to monitor stress responses inEscherichia coli

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 forEscherichia 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 degrees C) compared with growth at low temperature (25 degrees C) or the physiological temperature (37 degrees 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 ofE. colilacking cytochromes. This peak is absent in theEscherichia colimutant lackingcydB, but notccmE, 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.