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

Crofts TS, Men Y, Alvarez-Cohen L, Taga ME - Front Microbiol (2014)

Bottom Line: Of the three classes of lower ligands, the benzimidazoles are uniquely found in cobamides, whereas the purine and phenolic bases have additional biological functions.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant and Microbial Biology, University of California at Berkeley Berkeley, CA, USA.

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.

No MeSH data available.


Efficiency of extraction of benzimidazoles and cobalamin. The percent recovery following extraction and purification of the indicated compounds is shown. Bars represent the average of three replicates, and error bars represent the SE. ND, not detected, limit of detection was 5 pmol.
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Figure 5: Efficiency of extraction of benzimidazoles and cobalamin. The percent recovery following extraction and purification of the indicated compounds is shown. Bars represent the average of three replicates, and error bars represent the SE. ND, not detected, limit of detection was 5 pmol.

Mentions: The bluB bioassay was used to measure the concentrations of benzimidazoles present in environmental samples. Because environmental samples may contain compounds such as DNA damaging agents or cobamides that could interfere with the CFB response to benzimidazoles, and because the concentrations of benzimidazoles could be below our detection limit, we first developed a method to purify benzimidazoles from complex mixtures and concentrate them by up to 3000-fold. This method consists of a liquid–liquid extraction with ethyl acetate followed by C18 solid phase extraction (see Materials and Methods for details). The recovery of each benzimidazole varied following this purification method, with the highest yield observed for DMB and the lowest for 5-OHBza; Figure 5). Importantly, cobalamin could not be detected after this procedure, indicating that the bioassay results would not be influenced by cobalamin (or other corrinoids) that may have been present prior to purification (Figure 5).


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

Crofts TS, Men Y, Alvarez-Cohen L, Taga ME - Front Microbiol (2014)

Efficiency of extraction of benzimidazoles and cobalamin. The percent recovery following extraction and purification of the indicated compounds is shown. Bars represent the average of three replicates, and error bars represent the SE. ND, not detected, limit of detection was 5 pmol.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4230183&req=5

Figure 5: Efficiency of extraction of benzimidazoles and cobalamin. The percent recovery following extraction and purification of the indicated compounds is shown. Bars represent the average of three replicates, and error bars represent the SE. ND, not detected, limit of detection was 5 pmol.
Mentions: The bluB bioassay was used to measure the concentrations of benzimidazoles present in environmental samples. Because environmental samples may contain compounds such as DNA damaging agents or cobamides that could interfere with the CFB response to benzimidazoles, and because the concentrations of benzimidazoles could be below our detection limit, we first developed a method to purify benzimidazoles from complex mixtures and concentrate them by up to 3000-fold. This method consists of a liquid–liquid extraction with ethyl acetate followed by C18 solid phase extraction (see Materials and Methods for details). The recovery of each benzimidazole varied following this purification method, with the highest yield observed for DMB and the lowest for 5-OHBza; Figure 5). Importantly, cobalamin could not be detected after this procedure, indicating that the bioassay results would not be influenced by cobalamin (or other corrinoids) that may have been present prior to purification (Figure 5).

Bottom Line: Of the three classes of lower ligands, the benzimidazoles are uniquely found in cobamides, whereas the purine and phenolic bases have additional biological functions.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant and Microbial Biology, University of California at Berkeley Berkeley, CA, USA.

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.

No MeSH data available.