Resonance Raman analysis of intracellular vitamin B12 analogs in methanogenic archaea.

Abstract

Methanogenic archaea (methanogens) are microorganisms that can synthesize methane. They are found in diverse environments ranging from paddy fields to animal digestive tracts to deep-sea hydrothermal vents. Investigating their distribution and physiological activity is crucial for the detailed analysis of the dynamics of greenhouse gas generation and the search for the environmental limits of life. In methanogens, cobamide cofactors (vitamin B12 analogs) play a key role in methane synthesis and carbon fixation, thus serving as a marker compound that metabolically characterizes them. Here, we report on resonance Raman detection of cobamides in methanogenic cells without destroying cells and provide structural insights into those cobamides. We succeeded in detecting cobamides in four representative methanogens Methanosarcina mazei, Methanosarcina barkeri, Methanopyrus kandleri, and Methanocaldococcus jannaschii. The former two are mesophilic, cytochrome-containing methanogens, whereas the latter two are hyperthermophilic, non-cytochrome-containing methanogens. The 532nm-excited Raman spectra of single or multiple cells of the four species all showed resonance Raman bands of cobamides arising mainly from the corrin ring, with the most intense one at ∼1500cm-1. We envision that resonance Raman microspectroscopy could be useful for in situ, nondestructive identification of methanogenic cells that produce high levels of cobamides.