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Borrelia burgdorferi EbfC defines a newly-identified, widespread family of bacterial DNA-binding proteins.

Riley SP, Bykowski T, Cooley AE, Burns LH, Babb K, Brissette CA, Bowman A, Rotondi M, Miller MC, DeMoll E, Lim K, Fried MG, Stevenson B - Nucleic Acids Res. (2009)

Bottom Line: Two high-affinity EbfC-binding sites are located immediately 5' of B. burgdorferi erp transcriptional promoters, and binding of EbfC was found to alter the conformation of erp promoter DNA.These and other features of EbfC suggest that this small protein and its orthologs may represent a distinctive type of bacterial nucleoid-associated protein.EbfC was shown to bind DNA as a homodimer, and site-directed mutagenesis studies indicated that EbfC and its orthologs appear to bind DNA via a novel alpha-helical 'tweezer'-like structure.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky College of Medicine, Lexington, KY 40536-0298, USA.

ABSTRACT
The Lyme disease spirochete, Borrelia burgdorferi, encodes a novel type of DNA-binding protein named EbfC. Orthologs of EbfC are encoded by a wide range of bacterial species, so characterization of the borrelial protein has implications that span the eubacterial kingdom. The present work defines the DNA sequence required for high-affinity binding by EbfC to be the 4 bp broken palindrome GTnAC, where 'n' can be any nucleotide. Two high-affinity EbfC-binding sites are located immediately 5' of B. burgdorferi erp transcriptional promoters, and binding of EbfC was found to alter the conformation of erp promoter DNA. Consensus EbfC-binding sites are abundantly distributed throughout the B. burgdorferi genome, occurring approximately once every 1 kb. These and other features of EbfC suggest that this small protein and its orthologs may represent a distinctive type of bacterial nucleoid-associated protein. EbfC was shown to bind DNA as a homodimer, and site-directed mutagenesis studies indicated that EbfC and its orthologs appear to bind DNA via a novel alpha-helical 'tweezer'-like structure.

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Identification of EbfC amino acid residues involved with binding DNA. (A) Alignment of B. burgdorferi EbfC and H. influenzae YbaB, with identical residues boxed in dark blue, and similar amino acids in light blue. Residues that were individually mutated to alanine are indicated. (B) Model of B. burgdorferi EbfC based on the solved structure of its H. influenzae ortholog. Residues specifically mutated to alanine in this work are indicated. (C) EMSA of DNA probe b-WT with wild-type (WT) and each mutant EbfC protein. The wild-type recombinant EbfC was analyzed at a final concentration of 3 mM, and each mutant at 19.5 mM.
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Figure 7: Identification of EbfC amino acid residues involved with binding DNA. (A) Alignment of B. burgdorferi EbfC and H. influenzae YbaB, with identical residues boxed in dark blue, and similar amino acids in light blue. Residues that were individually mutated to alanine are indicated. (B) Model of B. burgdorferi EbfC based on the solved structure of its H. influenzae ortholog. Residues specifically mutated to alanine in this work are indicated. (C) EMSA of DNA probe b-WT with wild-type (WT) and each mutant EbfC protein. The wild-type recombinant EbfC was analyzed at a final concentration of 3 mM, and each mutant at 19.5 mM.

Mentions: The EbfC ortholog of H. influenzae KW20 Rd (YbaB, HI_0442) shares 27.5% identity and 59.3% similarity with B. burgdorferi EbfC (Figure 7A). As noted in the Introduction section, both the H. influenzae and E. coli EbfC orthologs have been crystallized, with both forming nearly identical structures with a ‘tweezer’-like appearance (18). The predicted gap between the ‘tweezer’ arms of EbfC ranges between 15 and 22 Å, comparable with the diameter of B-DNA, suggesting that the protruding α-helices could form the DNA-binding domain (18,34). To address that hypothesis, site directed mutagenesis of B. burgdorferi EbfC was performed to individually replace nine of the amino acids in those α-helices (Figure 7A and B). Each recombinant protein was expressed and purified two separate times, to guard against preparation artifacts. None of the mutated variants of EbfC was able to detectably bind PerpAB, even when tested at protein concentrations 6.5-fold greater than the wild-type EbfC (Figure 7C).Figure 7.


Borrelia burgdorferi EbfC defines a newly-identified, widespread family of bacterial DNA-binding proteins.

Riley SP, Bykowski T, Cooley AE, Burns LH, Babb K, Brissette CA, Bowman A, Rotondi M, Miller MC, DeMoll E, Lim K, Fried MG, Stevenson B - Nucleic Acids Res. (2009)

Identification of EbfC amino acid residues involved with binding DNA. (A) Alignment of B. burgdorferi EbfC and H. influenzae YbaB, with identical residues boxed in dark blue, and similar amino acids in light blue. Residues that were individually mutated to alanine are indicated. (B) Model of B. burgdorferi EbfC based on the solved structure of its H. influenzae ortholog. Residues specifically mutated to alanine in this work are indicated. (C) EMSA of DNA probe b-WT with wild-type (WT) and each mutant EbfC protein. The wild-type recombinant EbfC was analyzed at a final concentration of 3 mM, and each mutant at 19.5 mM.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2665219&req=5

Figure 7: Identification of EbfC amino acid residues involved with binding DNA. (A) Alignment of B. burgdorferi EbfC and H. influenzae YbaB, with identical residues boxed in dark blue, and similar amino acids in light blue. Residues that were individually mutated to alanine are indicated. (B) Model of B. burgdorferi EbfC based on the solved structure of its H. influenzae ortholog. Residues specifically mutated to alanine in this work are indicated. (C) EMSA of DNA probe b-WT with wild-type (WT) and each mutant EbfC protein. The wild-type recombinant EbfC was analyzed at a final concentration of 3 mM, and each mutant at 19.5 mM.
Mentions: The EbfC ortholog of H. influenzae KW20 Rd (YbaB, HI_0442) shares 27.5% identity and 59.3% similarity with B. burgdorferi EbfC (Figure 7A). As noted in the Introduction section, both the H. influenzae and E. coli EbfC orthologs have been crystallized, with both forming nearly identical structures with a ‘tweezer’-like appearance (18). The predicted gap between the ‘tweezer’ arms of EbfC ranges between 15 and 22 Å, comparable with the diameter of B-DNA, suggesting that the protruding α-helices could form the DNA-binding domain (18,34). To address that hypothesis, site directed mutagenesis of B. burgdorferi EbfC was performed to individually replace nine of the amino acids in those α-helices (Figure 7A and B). Each recombinant protein was expressed and purified two separate times, to guard against preparation artifacts. None of the mutated variants of EbfC was able to detectably bind PerpAB, even when tested at protein concentrations 6.5-fold greater than the wild-type EbfC (Figure 7C).Figure 7.

Bottom Line: Two high-affinity EbfC-binding sites are located immediately 5' of B. burgdorferi erp transcriptional promoters, and binding of EbfC was found to alter the conformation of erp promoter DNA.These and other features of EbfC suggest that this small protein and its orthologs may represent a distinctive type of bacterial nucleoid-associated protein.EbfC was shown to bind DNA as a homodimer, and site-directed mutagenesis studies indicated that EbfC and its orthologs appear to bind DNA via a novel alpha-helical 'tweezer'-like structure.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Immunology, and Molecular Genetics, University of Kentucky College of Medicine, Lexington, KY 40536-0298, USA.

ABSTRACT
The Lyme disease spirochete, Borrelia burgdorferi, encodes a novel type of DNA-binding protein named EbfC. Orthologs of EbfC are encoded by a wide range of bacterial species, so characterization of the borrelial protein has implications that span the eubacterial kingdom. The present work defines the DNA sequence required for high-affinity binding by EbfC to be the 4 bp broken palindrome GTnAC, where 'n' can be any nucleotide. Two high-affinity EbfC-binding sites are located immediately 5' of B. burgdorferi erp transcriptional promoters, and binding of EbfC was found to alter the conformation of erp promoter DNA. Consensus EbfC-binding sites are abundantly distributed throughout the B. burgdorferi genome, occurring approximately once every 1 kb. These and other features of EbfC suggest that this small protein and its orthologs may represent a distinctive type of bacterial nucleoid-associated protein. EbfC was shown to bind DNA as a homodimer, and site-directed mutagenesis studies indicated that EbfC and its orthologs appear to bind DNA via a novel alpha-helical 'tweezer'-like structure.

Show MeSH
Related in: MedlinePlus