Roseoflavin is a natural antibacterial compound that binds to FMN riboswitches and regulates gene expression - PubMed (original) (raw)
Roseoflavin is a natural antibacterial compound that binds to FMN riboswitches and regulates gene expression
Elaine R Lee et al. RNA Biol. 2009 Apr-Jun.
Abstract
Riboswitches in messenger RNAs carry receptor domains called aptamers that can bind to metabolites and control expression of associated genes. The Gram-positive bacterium Bacillus subtilis has two representatives of a class of riboswitches that bind flavin mononucleotide (FMN). These riboswitches control genes responsible for the biosynthesis and transport of riboflavin, a precursor of FMN. We found that roseoflavin, a chemical analog of FMN and riboflavin that has antimicrobial activity, can directly bind to FMN riboswitch aptamers and downregulate the expression of an FMN riboswitch-lacZ reporter gene in B. subtilis. A role for the riboswitch in the antimicrobial mechanism of roseoflavin is supported by our observation that some previously identified roseoflavin-resistant bacteria have mutations within an FMN aptamer. Riboswitch mutations in these resistant bacteria disrupt ligand binding and derepress reporter gene expression in the presence of either riboflavin or roseoflavin. If FMN riboswitches are a major target for roseoflavin antimicrobial action, then future efforts to develop compounds that trigger FMN riboswitch function could lead to the identification of new antimicrobial drugs.
Figures
Figure 1
Ligand binding by an FMN riboswitch from B. subtilis. (A) RNA sequence and predicted secondary structure model of the 165 ribD 5′ UTR from B. subtilis The two G residues at the 5′ end were added to increase the yield of transcription by T7 RNA polymerase. (B) Chemical structures of FMN, riboflavin, and roseoflavin. (C) RNA products generated by incubating 5′ 32P-labeled 165 ribD RNA under in-line probing conditions with concentrations of roseoflavin ranging from 0 to 50 µM. Products were separated by denaturing PAGE. Several bands corresponding to RNase T1 cleavage (3′ of G residues) are annotated. Pre identifies the uncleaved precursor RNA. NR, T1 and −OH identify lanes containing RNA subjected to no reaction, partial digest with RNase T1, and partial alkaline digestion, respectively. (D) Plot of the normalized fraction of 165 ribD RNA modulated versus the concentration of roseoflavin. The sites of modulation that were quantitated are indicated in (C). Theoretical binding curves for FMN and riboflavin (dashed lines) are based on the _K_D values reported previously.
Figure 2
FMN riboswitches control genes essential for optimal bacterial growth in B. subtilis. For the Pxyl-RS variant, the natural ribD promoter was replaced with a xylose-inducible and glucose-repressible promoter, and the FMN riboswitch was deleted. WT and Pxyl-RS bacteria were grown in the presence or absence of 0.5% xylose or glucose and absorbance at 595 nm was measured.
Figure 3
Riboflavin and roseoflavin yield similar levels of expression from a reporter gene controlled by wild-type or mutant ribD FMN riboswitches from B. subtilis. Wild-type and three selected roseoflavin-resistant mutant_ribD_ FMN riboswitches from B. subtilis were fused to an E. coli lacZ gene and inserted into the amyE locus of B. subtilis. Bacteria were grown in glucose minimal medium with 100 µM riboflavin or roseoflavin for approximately two hours before β-galactosidase assays were performed. Mutants have a single G to A substitution at position 41 (M1), 60 (M2), or 133 (M3), where the nucleotide numbers correspond to those in Figure 1A. Maximum Miller units measured were 68, 75, 105, and 50 for cells carrying WT and M1 through M3 RNAs, respectively.
Figure 4
Mutations to the FMN riboswitch that confer roseoflavin resistance disrupt ligand binding. (A) Wild-type and mutant 5′-labeled ribD RNAs were subjected to in-line probing in the presence of no compound (–) or 10 µM FMN, riboflavin or roseoflavin and the resulting products were separated by PAGE. Mutants are as described in the legend of Figure 3. Additional details are as described in the legend of Fig. 1C. (B) Plot of the band intensities at three sites where modulation is indicative of ligand binding as identified in (A) and in Fig. 1C. Site 4 was not included in the analysis due to the large signal from the nearby Pre band. Band intensities at each site were normalized to the NR lane so that no modulation is set to equal 1.
Figure 5
Ligand binding by an FMN riboswitch from S. davawensis. (A) RNA sequence and predicted secondary structure of the 149 ribB 5′ UTR from S. davawensis. (B) Plot of the normalized fraction of 149 ribB RNA modulated versus ligand concentration derived by in-line probing.
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References
- Mandal M, Breaker RR. Gene regulation by riboswitches. Nat Rev Mol Cell Biol. 2004;5:451–463. - PubMed
- Soukup JK, Soukup GA. Riboswitches exert genetic control through metabolite-induced conformational change. Curr Opin Struct Biol. 2004;14:344–349. - PubMed
- Winkler WC, Breaker RR. Regulation of bacterial gene expression by riboswitches. Annu Rev Microbiol. 2005;59:487–517. - PubMed
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