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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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Context-independent DNA-binding by EbfC. (A) Interactions between EbfC and erpAB variant DNAs: wild-type b-WT without added protein (lane 1); probe b-C2, which contains a consensus sequence at site I and non-consensus sequences at sites II and III (lane 2); probe b-C20, which lacks consensus sequences at sites I, II or III (lane 3); probe b-C2+3, which lacks a consensus sequence at site II but contains consensus sequences at site I and III (lane 4); probe b-C1/2, which lacks consensus sequences at sites I, II or III (lane 5); and probe b-C1/2+3, which contains a consensus binding sequence at site III but lacks consensus sequences at site I and II (lane 6). All lanes contained 2 μM EbfC and 1 ng/μl poly-dI-dC. (B) Binding of EbfC to DNA probes derived from insertion of small DNAs containing consensus EbfC-binding sites into the TA-cloning site of pCR2.1. Lanes 1–3 contained b-SRK-A, a 34 bp insert containing two EbfC-binding sites. Lanes 4–6 contained b-SRK-B, which contains the same insert as does b-SRK-A except with mutations in both binding sites such that EbfC does not specifically interact (negative control). Lanes 7–9 contained b-SRK-C, which consists of a 12 bp insert containing one consensus EbfC-binding sequence. Lanes 10–12 contained b-SRK-D, which consists of a 7 bp insert with one consensus binding sequence. Lanes 1, 4, 7 and 10 lacked EbfC protein, lanes 2, 5, 8 and 11 contained 4 μM EbfC, while lanes 3, 6, 9 and 12 contained 12 μM EbfC. All lanes contained 1 ng/μl poly-dI-dC.
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Figure 5: Context-independent DNA-binding by EbfC. (A) Interactions between EbfC and erpAB variant DNAs: wild-type b-WT without added protein (lane 1); probe b-C2, which contains a consensus sequence at site I and non-consensus sequences at sites II and III (lane 2); probe b-C20, which lacks consensus sequences at sites I, II or III (lane 3); probe b-C2+3, which lacks a consensus sequence at site II but contains consensus sequences at site I and III (lane 4); probe b-C1/2, which lacks consensus sequences at sites I, II or III (lane 5); and probe b-C1/2+3, which contains a consensus binding sequence at site III but lacks consensus sequences at site I and II (lane 6). All lanes contained 2 μM EbfC and 1 ng/μl poly-dI-dC. (B) Binding of EbfC to DNA probes derived from insertion of small DNAs containing consensus EbfC-binding sites into the TA-cloning site of pCR2.1. Lanes 1–3 contained b-SRK-A, a 34 bp insert containing two EbfC-binding sites. Lanes 4–6 contained b-SRK-B, which contains the same insert as does b-SRK-A except with mutations in both binding sites such that EbfC does not specifically interact (negative control). Lanes 7–9 contained b-SRK-C, which consists of a 12 bp insert containing one consensus EbfC-binding sequence. Lanes 10–12 contained b-SRK-D, which consists of a 7 bp insert with one consensus binding sequence. Lanes 1, 4, 7 and 10 lacked EbfC protein, lanes 2, 5, 8 and 11 contained 4 μM EbfC, while lanes 3, 6, 9 and 12 contained 12 μM EbfC. All lanes contained 1 ng/μl poly-dI-dC.

Mentions: All of the probes used in the above studies contained EbfC binding site(s) in the context of PerpAB (Figure 1). As mentioned above, all erp promoters contain two EbfC binding sites. In the strain B31 erpHY locus, EbfC binding site I is identical to that of PerpAB and all other known erp promoters, but site II is altered to a non-binding 5′-GTAAT-3′ (16,17). However, other sequence changes in PerpHY created a new EbfC-binding consensus immediately 5′ of site II, which we designate site III (Figure 1). This alternative site is particularly noteworthy, because the different spacing of the two sites in PerpHY could influence EbfC–DNA interactions. To study that possibility, PerpAB was mutated to resemble PerpHY by disruption of site II and creation of site III, both with and without a consensus site I. As anticipated, in the absence of a consensus site I, changing the site II sequence from that of PerpAB to the non-consensus sequence of PerpHY eliminated specific binding (Figure 5A, lane 5 [b-C1/2]). However, in that same context, creation of a consensus sequence at site III was sufficient for EbfC binding (Figure 5A, lane 6 [b-C1/2+3]). Notably, mobilities of EbfC–DNA complexes formed with probes containing a consensus sequence at site III were comparable to those with wild type PerpAB sequences.Figure 5.


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)

Context-independent DNA-binding by EbfC. (A) Interactions between EbfC and erpAB variant DNAs: wild-type b-WT without added protein (lane 1); probe b-C2, which contains a consensus sequence at site I and non-consensus sequences at sites II and III (lane 2); probe b-C20, which lacks consensus sequences at sites I, II or III (lane 3); probe b-C2+3, which lacks a consensus sequence at site II but contains consensus sequences at site I and III (lane 4); probe b-C1/2, which lacks consensus sequences at sites I, II or III (lane 5); and probe b-C1/2+3, which contains a consensus binding sequence at site III but lacks consensus sequences at site I and II (lane 6). All lanes contained 2 μM EbfC and 1 ng/μl poly-dI-dC. (B) Binding of EbfC to DNA probes derived from insertion of small DNAs containing consensus EbfC-binding sites into the TA-cloning site of pCR2.1. Lanes 1–3 contained b-SRK-A, a 34 bp insert containing two EbfC-binding sites. Lanes 4–6 contained b-SRK-B, which contains the same insert as does b-SRK-A except with mutations in both binding sites such that EbfC does not specifically interact (negative control). Lanes 7–9 contained b-SRK-C, which consists of a 12 bp insert containing one consensus EbfC-binding sequence. Lanes 10–12 contained b-SRK-D, which consists of a 7 bp insert with one consensus binding sequence. Lanes 1, 4, 7 and 10 lacked EbfC protein, lanes 2, 5, 8 and 11 contained 4 μM EbfC, while lanes 3, 6, 9 and 12 contained 12 μM EbfC. All lanes contained 1 ng/μl poly-dI-dC.
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Figure 5: Context-independent DNA-binding by EbfC. (A) Interactions between EbfC and erpAB variant DNAs: wild-type b-WT without added protein (lane 1); probe b-C2, which contains a consensus sequence at site I and non-consensus sequences at sites II and III (lane 2); probe b-C20, which lacks consensus sequences at sites I, II or III (lane 3); probe b-C2+3, which lacks a consensus sequence at site II but contains consensus sequences at site I and III (lane 4); probe b-C1/2, which lacks consensus sequences at sites I, II or III (lane 5); and probe b-C1/2+3, which contains a consensus binding sequence at site III but lacks consensus sequences at site I and II (lane 6). All lanes contained 2 μM EbfC and 1 ng/μl poly-dI-dC. (B) Binding of EbfC to DNA probes derived from insertion of small DNAs containing consensus EbfC-binding sites into the TA-cloning site of pCR2.1. Lanes 1–3 contained b-SRK-A, a 34 bp insert containing two EbfC-binding sites. Lanes 4–6 contained b-SRK-B, which contains the same insert as does b-SRK-A except with mutations in both binding sites such that EbfC does not specifically interact (negative control). Lanes 7–9 contained b-SRK-C, which consists of a 12 bp insert containing one consensus EbfC-binding sequence. Lanes 10–12 contained b-SRK-D, which consists of a 7 bp insert with one consensus binding sequence. Lanes 1, 4, 7 and 10 lacked EbfC protein, lanes 2, 5, 8 and 11 contained 4 μM EbfC, while lanes 3, 6, 9 and 12 contained 12 μM EbfC. All lanes contained 1 ng/μl poly-dI-dC.
Mentions: All of the probes used in the above studies contained EbfC binding site(s) in the context of PerpAB (Figure 1). As mentioned above, all erp promoters contain two EbfC binding sites. In the strain B31 erpHY locus, EbfC binding site I is identical to that of PerpAB and all other known erp promoters, but site II is altered to a non-binding 5′-GTAAT-3′ (16,17). However, other sequence changes in PerpHY created a new EbfC-binding consensus immediately 5′ of site II, which we designate site III (Figure 1). This alternative site is particularly noteworthy, because the different spacing of the two sites in PerpHY could influence EbfC–DNA interactions. To study that possibility, PerpAB was mutated to resemble PerpHY by disruption of site II and creation of site III, both with and without a consensus site I. As anticipated, in the absence of a consensus site I, changing the site II sequence from that of PerpAB to the non-consensus sequence of PerpHY eliminated specific binding (Figure 5A, lane 5 [b-C1/2]). However, in that same context, creation of a consensus sequence at site III was sufficient for EbfC binding (Figure 5A, lane 6 [b-C1/2+3]). Notably, mobilities of EbfC–DNA complexes formed with probes containing a consensus sequence at site III were comparable to those with wild type PerpAB sequences.Figure 5.

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