A bioassay for the detection of benzimidazoles reveals their presence in a range of environmental samples.

Terence S Crofts,Lisa Alvarez-Cohen,Michiko E Taga,Yujie Men

doi:10.3389/fmicb.2014.00592

Terence S Crofts, Lisa Alvarez-Cohen + Show 2 more

Open Access

https://doi.org/10.3389/fmicb.2014.00592

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Abstract

Cobamides are a family of enzyme cofactors that include vitamin B12 (cobalamin) and are produced solely by prokaryotes. Structural variability in the lower axial ligand has been observed in cobamides produced by diverse organisms. Of the three classes of lower ligands, the benzimidazoles are uniquely found in cobamides, whereas the purine and phenolic bases have additional biological functions. Many organisms acquire cobamides by salvaging and remodeling cobamides or their precursors from the environment. These processes require free benzimidazoles for incorporation as lower ligands, though the presence of benzimidazoles in the environment has not been previously investigated. Here, we report a new purification method and bioassay to measure the total free benzimidazole content of samples from microbial communities and laboratory media components. The bioassay relies on the “calcofluor-bright” phenotype of a bluB mutant of the model cobalamin-producing bacterium Sinorhizobium meliloti. The concentrations of individual benzimidazoles in these samples were measured by liquid chromatography-tandem mass spectrometry. Several benzimidazoles were detected in subpicomolar to subnanomolar concentrations in host-associated and environmental samples. In addition, benzimidazoles were found to be common contaminants of laboratory media components. These results suggest that benzimidazoles present in the environment and in laboratory media have the potential to influence microbial metabolic activities.

Highlights

Cobamides are cofactors that function in a variety of metabolic processes in animals, protists, and prokaryotes
We report the development of a bioassay for the quantification of benzimidazoles based on the “calcofluor-bright” (CFB) phenotype of Sinorhizobium meliloti mutants that lack a functional bluB gene, which encodes the enzyme responsible for the oxygen-dependent biosynthesis of DMB (Campbell et al, 2006; Taga et al, 2007)
We hypothesized that the CFB phenotype could be a result of reduced activity of the Class II ribonucleotide reductase (RNR) encoded by nrdJ, a cobalamin-dependent enzyme that catalyzes the synthesis of deoxyribonucleotides

Summary

Introduction

Cobamides are cofactors that function in a variety of metabolic processes in animals, protists, and prokaryotes. Cobamides belong to a broader class of molecules called corrinoids that share a common cobalt-containing corrin ring. Cobamides are corrinoids that contain a lower axial ligand covalently bound to the corrin ring via the nucleotide loop (Roth et al, 1996; Escalante-Semerena, 2007). The essential nutrient cobalamin (Figure 1A, known as vitamin B12 when taken in its cyanated form as a supplement) is the best studied cobamide and is well known for its importance in human health. Cobalamin and other cobamides function as cofactors for several ecologically important processes in prokaryotes. Cobamides are found in host-associated and environmental microbial communities including the human intestine, bovine rumen, wood-feeding insects, and TCE-dechlorinating communities (Kräutler et al, 2003; Allen and Stabler, 2008; Girard et al, 2009; Yi et al, 2012)

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