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

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Abstract

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.

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Acknowledgments

The authors acknowledge Ms. Harshitha A for helping in some experiments. In addition, the infrastructural support from DBT-IISc program, DST-FIST, and UGC CAS/SAP is greatly appreciated. The authors acknowledge central facility, Department of Biochemistry, for the use of some instruments.

Funding

The authors thank the Department of Science and Technology (DST) and Department of Biotechnology (DBT), Government of India, for financial support. SU acknowledges the J. C. Bose Fellowship from DST. RM and TV thank the Centre for Science and Industrial Research (CSIR, India) and IISc for research fellowships respectively.

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Authors and Affiliations

  1. Department of Inorganic and Physical Chemistry, Indian Institute of Science, Bangalore, Karnataka, 560012, India

    Ria Mukherjee & Siva Umapathy

  2. Centre for BioSystems Science and Engineering, Indian Institute of Science, Bangalore, Karnataka, 560012, India

    Taru Verma, Dipankar Nandi & Siva Umapathy

  3. Department of Biochemistry, Indian Institute of Science, Bangalore, Karnataka, 560012, India

    Dipankar Nandi

  4. Department of Instrumentation and Applied Physics, Indian Institute of Science, Bangalore, Karnataka, 560012, India

    Siva Umapathy

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  1. Ria Mukherjee
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Correspondence to Dipankar Nandi or Siva Umapathy.

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Mukherjee, R., Verma, T., Nandi, D. et al. Identification of a resonance Raman marker for cytochrome to monitor stress responses in Escherichia coli. Anal Bioanal Chem 412, 5379–5388 (2020). https://doi.org/10.1007/s00216-020-02753-y

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