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

Understanding the effects of culture conditions in bacterial growth: A biochemical perspective using Raman microscopy.

Rapid, sensitive and label-free methods to probe bacterial growth irrespective of the culture conditions can shed light on the mechanisms by which bacteria adapt to different environmental stimuli. Raman spectroscopy can rapidly and continuously monitor the growth of bacteria under varied conditions. In this study, the growth of Escherichia coli in Luria broth (nutrient rich conditions) and minimal media with either glucose or glycerol as carbon source (nutrient limiting conditions) is profiled using Raman spectroscopy. Moreover, the study also gives insights into the altered bacterial biochemistry upon exposure to low- (25°C) and high-temperature (45°C) stress. Raman spectral measurement was performed on bulk bacteria cultured under laboratory conditions. A detailed analysis of the spectra as a function of bacterial growth reveals changes in Raman band intensities/area of biomolecules such as DNA, proteins and lipids. We also report five novel ratiometric markers (I830 /I810 , I1126 /I1100 , I1340 /I1440 , I1207 /I1240 and I1580 /I1440 ) that can identify the phase of growth, independent of the culture condition. Unsupervised multivariate methods like Principal Component Analysis also corroborate the aforementioned markers of growth. Altogether, our findings highlight the potential of Raman spectroscopy in yielding universal biochemical signatures that may be indicative of stress and aging in a growth milieu.

Platinum-bearing chromite layers are caused by pressure reduction during magma ascent.

Platinum-bearing chromitites in mafic-ultramafic intrusions such as the Bushveld Complex are key repositories of strategically important metals for human society. Basaltic melts saturated in chromite alone are crucial to their generation, but the origin of such melts is controversial. One concept holds that they are produced by processes operating within the magma chamber, whereas another argues that melts entering the chamber were already saturated in chromite. Here we address the problem by examining the pressure-related changes in the topology of a Mg2SiO4-CaAl2Si2O8-SiO2-MgCr2O4 quaternary system and by thermodynamic modelling of crystallisation sequences of basaltic melts at 1-10 kbar pressures. We show that basaltic melts located adjacent to a so-called chromite topological trough in deep-seated reservoirs become saturated in chromite alone upon their ascent towards the Earth's surface and subsequent cooling in shallow-level chambers. Large volumes of these chromite-only-saturated melts replenishing these chambers are responsible for monomineralic layers of massive chromitites with associated platinum-group elements.

Raman and infra-red microspectroscopy: towards quantitative evaluation for clinical research by ratiometric analysis.

Biomolecular structure elucidation is one of the major techniques for studying the basic processes of life. These processes get modulated, hindered or altered due to various causes like diseases, which is why biomolecular analysis and imaging play an important role in diagnosis, treatment prognosis and monitoring. Vibrational spectroscopy (IR and Raman), which is a molecular bond specific technique, can assist the researcher in chemical structure interpretation. Based on the combination with microscopy, vibrational microspectroscopy is currently emerging as an important tool for biomedical research, with a spatial resolution at the cellular and sub-cellular level. These techniques offer various advantages, enabling label-free, biomolecular fingerprinting in the native state. However, the complexity involved in deciphering the required information from a spectrum hampered their entry into the clinic. Today with the advent of automated algorithms, vibrational microspectroscopy excels in the field of spectropathology. However, researchers should be aware of how quantification based on absolute band intensities may be affected by instrumental parameters, sample thickness, water content, substrate backgrounds and other possible artefacts. In this review these practical issues and their effects on the quantification of biomolecules will be discussed in detail. In many cases ratiometric analysis can help to circumvent these problems and enable the quantitative study of biological samples, including ratiometric imaging in 1D, 2D and 3D. We provide an extensive overview from the recent scientific literature on IR and Raman band ratios used for studying biological systems and for disease diagnosis and treatment prognosis.