Steady-State and Time-Resolved Spectroscopy of F420 Extracted from Methanogen Cells and Its Utility as a Marker for Fecal Contamination

Abstract

Methanogenic bacteria, which are common inhabitants of the animal digestive tract, contain the fluorescent compound F420 (coenzyme 420), a 7,8-didemethyl-8-hydroxy-5-deazariboflavin chromophore. F420 was characterized as an initial step in determining if this compound would be useful as a fluorescent marker for the detection of fecal and ingesta contamination. Using a single anion exchange chromatographic process, F420 was separated from other cell components of a Methanobrevibacter sp. cell culture. The extent of separation was determined spectroscopically. To aid in the development of possible techniques for the detection of fecal contamination using F420 as a marker, further spectroscopic investigation of F420 was conducted using steady-state and time-resolved fluorescence methods. The fluorescence lifetime of F420 in an elution buffer of pH 7.5 was found to be 4.2 ns. At higher pH values, the fluorescence decay, F(t), was best described by a sum of two exponentials: at pH 13, F(t) = 0.31 exp(−t/4.20 ns) + 0.69 exp(−t/1.79 ns). Further investigation using front-faced fluorescence techniques has shown that emission from F420 can be collected efficiently from samples of methanogen cell cultures as well as from fecal material. KeywordsFluorescence spectroscopy, fluorescent marker, F420, methanogen, methanogenic bacteria, meat contamination Methanogenic bacteria, which are common inhabitants of the animal digestive tract, contain the fluorescent compound F420 (coenzyme 420), a 7,8-didemethyl-8-hydroxy-5-deazariboflavin chromophore. F420 was characterized as an initial step in determining if this compound would be useful as a fluorescent marker for the detection of fecal and ingesta contamination. Using a single anion exchange chromatographic process, F420 was separated from other cell components of a Methanobrevibacter sp. cell culture. The extent of separation was determined spectroscopically. To aid in the development of possible techniques for the detection of fecal contamination using F420 as a marker, further spectroscopic investigation of F420 was conducted using steady-state and timeresolved fluorescence methods. The fluorescence lifetime of F420 in an elution buffer of pH 7.5 was found to be 4.2 ns. At higher pH values, the fluorescence decay, F(t), was best described by a sum of two exponentials: at pH 13, F(t) ) 0.31 exp(-t/4.20 ns) + 0.69 exp(-t/1.79 ns). Further investigation using front-faced fluorescence techniques has shown that emission from F420 can be collected efficiently from samples of methanogen cell cultures as well as from fecal material.

Physiology, Biochemistry, and Applications of F420- and Fo-Dependent Redox Reactions

“…As elaborated below, this is central to the mechanism of the F o -utilizing DNA photolyases (57,58). The autofluorescence of F 420 has also been used for detecting methanogens (59)(60)(61)(62)(63)(64)(65)(66) and mycobacteria (67, 68).…”

Methanogens: Methane Producers of the Rumen and Mitigation Strategies

“…Unlike Bacteria , methanogens lack peptidoglycan in the cell wall, replaced by pseudomurein in Methanobrevibacter and Methanobacterium , heteropolysaccharide in Methanosarcina, and protein in Methanomicrobium [16]. All methanogens have coenzyme F 420 , which is a cofactor necessary for enzymes such as hydrogenase and formate dehydrogenase, and received its name due to its absorbance at 420 nm, which allows it to fluoresce blue-green at 470 nm [17]. Another coenzyme characteristic of methanogens is coenzyme M, which is either produced by the methanogens, such as Methanobacterium , or is required from an external source, which is the case for Methanobrevibacter ruminantium [18].…”

Rapid detection of phylloplane bacterium Enterobacter cloacae based on chitinase gene transformation and lytic infection by specific bacteriophages

“…or blue-light irradiation but might be indistinguishable from pale-green, fluorescent methanogenic bacteria when illuminated under a u.v. lamp (Doddema and Vogels 1978;Ashby et al 2001). In the present report, we propose that a chitinase gene can be effectively used as a reporter to establish an easy and sensitive monitoring method for identifying a target strain of E. cloacae.…”