Abstract
This protocol provides a simple and fast method of quantification for intracellular flavin content, and for flavin secretion by bacteria. Intracellular flavins are extracted from bacterial pellets, and secreted flavins are examined in the cell growth medium. Flavins are separated and measured using HPLC with fluorescence detection, and quantified based on a comparison to standards.
Keywords: Riboflavin Biosynthetic Pathway, FMN, FAD, Alpha-proteobacteria, HPLC
Materials and Reagents
Equipment
Procedure
Representative data
Figure 1. Flavin secretion and accumulation by the S. meliloti strains. S. meliloti strains were grown in MMNH4 media. 1 ml of cell cultures was taken after 3 days of growth. The cells were spun down and the mass of the pellets was measured. Lumichrome (LMC), FMN, FAD and riboflavin (RF) concentration in the supernatants and the pellets were measured using HPLC. The flavin concentration was normalized against the protein concentration. Rm1021 - wild type strain; Rm1021ΔribA and Rm1021ΔribBA - Rm1021 mutants with decreased ability to secret flavins; Rm1021ΔribBAxpCPP30ribBA - Rm1021 mutant with restored ability to secret flavins. Data are averaged from at least three independent experiments.
Recipes
Acknowledgments
This protocol was adapted from Yurgel et al., 2014. This work was supported by the Agricultural Research Center (WNP-00773) at Washington State University and grant DE-FG0396ER20225 Vol. 27, No. 5, 2014 / 445 from the Energy Biosciences Program at the United States Department of Energy, and by grant NSF-MCB 1052492 to S. Rajamani. This activity was funded, in part, with an Emerging Research Issues Internal Competitive grant from the Agricultural Research Center at Washington State University, College of Agricultural, Human, and Natural Resource Sciences and Biologically-Intensive Agriculture and Organic Farming (BIOAg) Internal Competitive grant from The Center for Sustaining Agriculture and Natural Resources (CSANR) at Washington State University to S. Yurgel. We thank M. Kahn for discussions and the Washington State University Laboratory for Biotechnology and Bioanalysis for sequencing support.
References
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