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

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.

Guided cobamide biosynthesis for heterologous production of reductive dehalogenases

“…Strain DCB-2 was routinely cultivated in medium containing 0.2% YE (purchased from Sigma-Aldrich, Munich, Germany). Crofts et al (2014b) reported the presence of benzimidazoles (pmol g À1 ) in YE. To unravel whether 5-azaBza or purine or a precursor thereof was taken up from YE by the cells, a 15 N-isotopically labelled YE ( 15 N-enriched YE) was prepared and fed to D. hafniense strain DCB-2 cultures.…”

Specificity of cobamide remodeling, uptake and utilization in Vibrio cholerae

“…Cyanobacteria are abundant inhabitants of the same aquatic environments as Vibrio species and are major producers of pseudocobalamin in marine environments. The natural source of benzimidazole bases for cobamide biosynthesis in V. cholerae remains unclear, but analysis of soil and freshwater samples indicates that benzimidazole bases are present in the environment (Crofts et al , ). While phenolylcobamides are abundant in some environments (Degnan et al , ; Men et al , ), their concentration in marine environments remains to be determined.…”

Metagenomics-guided analysis of microbial chemolithoautotrophic phosphite oxidation yields evidence of a seventh natural CO 2 fixation pathway

“…Na 2 HPO 3 (5 H 2 O) and Na 2 MoO 4 (2 H 2 O) (Sigma Aldrich) were added from sterile, anaerobic stocks to the basal media as needed. Rumen fluid from fistulated cows fed a high forage diet was obtained from the University of California (UC), Davis Department of Animal Science (37). Rumen fluid stocks were prepared anaerobically, autoclaved at 121°C for 20 min, and added to the basal media as needed.…”