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A novel bifunctional histone protein in Streptomyces: a candidate for structural coupling between DNA conformation and transcription during development and stress?

Aldridge M, Facey P, Francis L, Bayliss S, Del Sol R, Dyson P - Nucleic Acids Res. (2013)

Bottom Line: Here, we describe a novel developmentally regulated nucleoid-associated protein, DdbA, of the genus that consists of an N-terminal DNA-binding histone H1-like domain and a C-terminal DksA-like domain that can potentially modulate RNA polymerase activity in conjunction with ppGpp.The mutant is also sensitive to oxidative stress owing to impaired upregulation of transcription of sigR, encoding an alternative sigma factor.Consequently, we propose this bifunctional histone-like protein as a candidate that could structurally couple changes in DNA conformation and transcription during the streptomycete life-cycle and in response to stress.

View Article: PubMed Central - PubMed

Affiliation: Institute of Life Science, College of Medicine, Swansea University, Singleton Park, Swansea SA2 8PP, UK.

ABSTRACT
Antibiotic-producing Streptomyces are complex bacteria that remodel global transcription patterns and their nucleoids during development. Here, we describe a novel developmentally regulated nucleoid-associated protein, DdbA, of the genus that consists of an N-terminal DNA-binding histone H1-like domain and a C-terminal DksA-like domain that can potentially modulate RNA polymerase activity in conjunction with ppGpp. Owing to its N-terminal domain, the protein can efficiently bind and condense DNA in vitro. Loss of function of this DNA-binding protein results in changes in both DNA condensation during development and the ability to adjust DNA supercoiling in response to osmotic stress. Initial analysis of the DksA-like activity of DdbA indicates that overexpression of the protein suppresses a conditional deficiency in antibiotic production of relA mutants that are unable to synthesise ppGpp, just as DksA overexpression in Escherichia coli can suppress ppGpp(0) phenotypes. The mutant is also sensitive to oxidative stress owing to impaired upregulation of transcription of sigR, encoding an alternative sigma factor. Consequently, we propose this bifunctional histone-like protein as a candidate that could structurally couple changes in DNA conformation and transcription during the streptomycete life-cycle and in response to stress.

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Related in: MedlinePlus

DdbA is a DNA-binding protein as revealed by EMSA. Panel A: Lane 1 contain 300 ng pUC18 supercoiled DNA substrate in the absence of protein, lane 2: 1 μg BSA, lane 3: 1 μg BSA and 300 ng DNA, lane 4: 1 μg DdbA. Lanes 5 and 6 contain pUC18 substrate DNA with DdbA in a range of 0.1, 0.25, 0.5, 0.75 and 1 μM of protein. The open triangle on the top of the gel image indicates DdbA protein concentration range used. Panel B: Reactions were performed with 300 ng of pUC18 as substrate DNA in the absence of protein (lane 1) or in the presence of 0.25, 0.5 0.75 or 1 μM of DdbA-NTD protein (lanes 2–5). The open triangle on the top of the gel image denotes increasing concentrations of the protein. Panel C: All lanes contain 300 ng supercoiled plasmid pUC18 substrate DNA and respectively no protein (lane1), 1 μM DdbA-CTD protein (lane 2), 1 μM full-length DdbA protein (lane 3) and 1.8 μM DdbA-CTD protein (lane 4).
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gkt180-F1: DdbA is a DNA-binding protein as revealed by EMSA. Panel A: Lane 1 contain 300 ng pUC18 supercoiled DNA substrate in the absence of protein, lane 2: 1 μg BSA, lane 3: 1 μg BSA and 300 ng DNA, lane 4: 1 μg DdbA. Lanes 5 and 6 contain pUC18 substrate DNA with DdbA in a range of 0.1, 0.25, 0.5, 0.75 and 1 μM of protein. The open triangle on the top of the gel image indicates DdbA protein concentration range used. Panel B: Reactions were performed with 300 ng of pUC18 as substrate DNA in the absence of protein (lane 1) or in the presence of 0.25, 0.5 0.75 or 1 μM of DdbA-NTD protein (lanes 2–5). The open triangle on the top of the gel image denotes increasing concentrations of the protein. Panel C: All lanes contain 300 ng supercoiled plasmid pUC18 substrate DNA and respectively no protein (lane1), 1 μM DdbA-CTD protein (lane 2), 1 μM full-length DdbA protein (lane 3) and 1.8 μM DdbA-CTD protein (lane 4).

Mentions: To analyse the DNA-binding ability of SCO2075, three different His-tagged recombinant proteins were produced: full-length SCO2075, a 138 amino acid protein consisting of the predicted N-terminal DNA-binding domain and a 124 amino acid protein containing the complete C-terminal DksA-like domain (Supplementary Figure S1). EMSAs using supercoiled plasmid DNA revealed gel retardation by both the full-length and the N-terminal domain proteins, but not by the C-terminal DksA-like domain (Figure 1). Similar results were obtained with linearized plasmid DNA (results not shown). Hereafter, the product of SCO2075 is referred to as DdbA (Dks-like DNA-binding protein).Figure 1.


A novel bifunctional histone protein in Streptomyces: a candidate for structural coupling between DNA conformation and transcription during development and stress?

Aldridge M, Facey P, Francis L, Bayliss S, Del Sol R, Dyson P - Nucleic Acids Res. (2013)

DdbA is a DNA-binding protein as revealed by EMSA. Panel A: Lane 1 contain 300 ng pUC18 supercoiled DNA substrate in the absence of protein, lane 2: 1 μg BSA, lane 3: 1 μg BSA and 300 ng DNA, lane 4: 1 μg DdbA. Lanes 5 and 6 contain pUC18 substrate DNA with DdbA in a range of 0.1, 0.25, 0.5, 0.75 and 1 μM of protein. The open triangle on the top of the gel image indicates DdbA protein concentration range used. Panel B: Reactions were performed with 300 ng of pUC18 as substrate DNA in the absence of protein (lane 1) or in the presence of 0.25, 0.5 0.75 or 1 μM of DdbA-NTD protein (lanes 2–5). The open triangle on the top of the gel image denotes increasing concentrations of the protein. Panel C: All lanes contain 300 ng supercoiled plasmid pUC18 substrate DNA and respectively no protein (lane1), 1 μM DdbA-CTD protein (lane 2), 1 μM full-length DdbA protein (lane 3) and 1.8 μM DdbA-CTD protein (lane 4).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3643593&req=5

gkt180-F1: DdbA is a DNA-binding protein as revealed by EMSA. Panel A: Lane 1 contain 300 ng pUC18 supercoiled DNA substrate in the absence of protein, lane 2: 1 μg BSA, lane 3: 1 μg BSA and 300 ng DNA, lane 4: 1 μg DdbA. Lanes 5 and 6 contain pUC18 substrate DNA with DdbA in a range of 0.1, 0.25, 0.5, 0.75 and 1 μM of protein. The open triangle on the top of the gel image indicates DdbA protein concentration range used. Panel B: Reactions were performed with 300 ng of pUC18 as substrate DNA in the absence of protein (lane 1) or in the presence of 0.25, 0.5 0.75 or 1 μM of DdbA-NTD protein (lanes 2–5). The open triangle on the top of the gel image denotes increasing concentrations of the protein. Panel C: All lanes contain 300 ng supercoiled plasmid pUC18 substrate DNA and respectively no protein (lane1), 1 μM DdbA-CTD protein (lane 2), 1 μM full-length DdbA protein (lane 3) and 1.8 μM DdbA-CTD protein (lane 4).
Mentions: To analyse the DNA-binding ability of SCO2075, three different His-tagged recombinant proteins were produced: full-length SCO2075, a 138 amino acid protein consisting of the predicted N-terminal DNA-binding domain and a 124 amino acid protein containing the complete C-terminal DksA-like domain (Supplementary Figure S1). EMSAs using supercoiled plasmid DNA revealed gel retardation by both the full-length and the N-terminal domain proteins, but not by the C-terminal DksA-like domain (Figure 1). Similar results were obtained with linearized plasmid DNA (results not shown). Hereafter, the product of SCO2075 is referred to as DdbA (Dks-like DNA-binding protein).Figure 1.

Bottom Line: Here, we describe a novel developmentally regulated nucleoid-associated protein, DdbA, of the genus that consists of an N-terminal DNA-binding histone H1-like domain and a C-terminal DksA-like domain that can potentially modulate RNA polymerase activity in conjunction with ppGpp.The mutant is also sensitive to oxidative stress owing to impaired upregulation of transcription of sigR, encoding an alternative sigma factor.Consequently, we propose this bifunctional histone-like protein as a candidate that could structurally couple changes in DNA conformation and transcription during the streptomycete life-cycle and in response to stress.

View Article: PubMed Central - PubMed

Affiliation: Institute of Life Science, College of Medicine, Swansea University, Singleton Park, Swansea SA2 8PP, UK.

ABSTRACT
Antibiotic-producing Streptomyces are complex bacteria that remodel global transcription patterns and their nucleoids during development. Here, we describe a novel developmentally regulated nucleoid-associated protein, DdbA, of the genus that consists of an N-terminal DNA-binding histone H1-like domain and a C-terminal DksA-like domain that can potentially modulate RNA polymerase activity in conjunction with ppGpp. Owing to its N-terminal domain, the protein can efficiently bind and condense DNA in vitro. Loss of function of this DNA-binding protein results in changes in both DNA condensation during development and the ability to adjust DNA supercoiling in response to osmotic stress. Initial analysis of the DksA-like activity of DdbA indicates that overexpression of the protein suppresses a conditional deficiency in antibiotic production of relA mutants that are unable to synthesise ppGpp, just as DksA overexpression in Escherichia coli can suppress ppGpp(0) phenotypes. The mutant is also sensitive to oxidative stress owing to impaired upregulation of transcription of sigR, encoding an alternative sigma factor. Consequently, we propose this bifunctional histone-like protein as a candidate that could structurally couple changes in DNA conformation and transcription during the streptomycete life-cycle and in response to stress.

Show MeSH
Related in: MedlinePlus