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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.


Calcofluor response of S. meliloti bluB to other benzimidazoles and cobalamin. The CF fluorescence of cultures grown with the indicated concentrations of (A) DMB, (B) 5-OHBza, (C) 5-MeBza, (D) Bza, (E) 5-OMeBza, and (F) cobalamin was assayed as shown in Figure 3D. The EC50 of each compound and curve-fit error are indicated on each graph. Error bars represent the SE of three independent experiments.
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Figure 4: Calcofluor response of S. meliloti bluB to other benzimidazoles and cobalamin. The CF fluorescence of cultures grown with the indicated concentrations of (A) DMB, (B) 5-OHBza, (C) 5-MeBza, (D) Bza, (E) 5-OMeBza, and (F) cobalamin was assayed as shown in Figure 3D. The EC50 of each compound and curve-fit error are indicated on each graph. Error bars represent the SE of three independent experiments.

Mentions: To determine whether the CFB phenotype showed a reproducible, dose-dependent relationship with DMB concentration, cultures of the S. meliloti bluB mutant were grown with DMB at concentrations ranging from 0.24 to 250 nM and incubated with CF. The results in Figure 3D show a relationship between fluorescence, and to a lesser extent the final O.D.600 of the cultures, with the concentration of DMB added. The linear range of the curve was between 4 and 40 nM, and the concentration of DMB that resulted in half maximal fluorescence (EC50) was calculated to be 13 nM. The average and SD of the EC50 values were 7.37 and 5.59 nM, respectively, in 26 independent experiments. Replicates performed in a single experiment gave results with lower variation (Figures 3D and 4A), demonstrating that this method can be used to detect DMB in unknown samples and quantify DMB concentrations if DMB standards are measured in the same experiment.


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)

Calcofluor response of S. meliloti bluB to other benzimidazoles and cobalamin. The CF fluorescence of cultures grown with the indicated concentrations of (A) DMB, (B) 5-OHBza, (C) 5-MeBza, (D) Bza, (E) 5-OMeBza, and (F) cobalamin was assayed as shown in Figure 3D. The EC50 of each compound and curve-fit error are indicated on each graph. Error bars represent the SE of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Calcofluor response of S. meliloti bluB to other benzimidazoles and cobalamin. The CF fluorescence of cultures grown with the indicated concentrations of (A) DMB, (B) 5-OHBza, (C) 5-MeBza, (D) Bza, (E) 5-OMeBza, and (F) cobalamin was assayed as shown in Figure 3D. The EC50 of each compound and curve-fit error are indicated on each graph. Error bars represent the SE of three independent experiments.
Mentions: To determine whether the CFB phenotype showed a reproducible, dose-dependent relationship with DMB concentration, cultures of the S. meliloti bluB mutant were grown with DMB at concentrations ranging from 0.24 to 250 nM and incubated with CF. The results in Figure 3D show a relationship between fluorescence, and to a lesser extent the final O.D.600 of the cultures, with the concentration of DMB added. The linear range of the curve was between 4 and 40 nM, and the concentration of DMB that resulted in half maximal fluorescence (EC50) was calculated to be 13 nM. The average and SD of the EC50 values were 7.37 and 5.59 nM, respectively, in 26 independent experiments. Replicates performed in a single experiment gave results with lower variation (Figures 3D and 4A), demonstrating that this method can be used to detect DMB in unknown samples and quantify DMB concentrations if DMB standards are measured in the same experiment.

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.