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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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Reduced induction of sigR expression in a ddbA mutant. The qRT-PCR analysis of sigR transcript abundance at 15 and 30 min after induction of thiol stress in M145 and the ddbA mutant. The data were normalized using hrdB as a control. Strains were grown over night on NMMP and transferred to minimal liquid medium (NMMP) + 0.5 mM diamide. The data represent averages obtained from three biological replicates, with three experimental replicate performed on each sample.
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gkt180-F8: Reduced induction of sigR expression in a ddbA mutant. The qRT-PCR analysis of sigR transcript abundance at 15 and 30 min after induction of thiol stress in M145 and the ddbA mutant. The data were normalized using hrdB as a control. Strains were grown over night on NMMP and transferred to minimal liquid medium (NMMP) + 0.5 mM diamide. The data represent averages obtained from three biological replicates, with three experimental replicate performed on each sample.

Mentions: In E. coli, ppGpp/DksA-dependent remodelling of transcription is in part mediated by alternative sigma factors changing the promoter specificity of RNAP (5). For alternative sigma factors such as SigS and SigN, a sigma competition model has been proposed whereby ppGpp/DksA alters the specificity of interaction of the housekeeping Sig70 to core RNAP, allowing other sigma factors to bind (31). We discovered that the ddbA mutant is sensitive to diamide, a thiol-specific oxidising agent, and this sensitivity is restored by genetic complementation with ddbA (Table 3). In streptomycetes, thiol oxidation is sensed by a complex of the alternative sigma factor, SigR, bound to its antisigma factor RsrA (32). Disulphide bond formation in RsrA causes conformational changes, releasing SigR that directs RNAP to transcribe genes belonging to the SigR regulon that include sigR itself, the trxAB operon encoding thioredoxin reductase and thioredoxin, and the relA gene (33), the latter encoding an enzyme that is the principal ppGpp synthase. To investigate the sensitivity of the ddbA mutant to thiol stress and its relation to sigR expression, the induction of the gene after exposure to diamide was quantified in the wild-type and mutant, indicating impaired upregulation of sigR in the mutant (Figure 8). No significant differences in very low, uninduced, levels of expression between wild-type and mutant were detected. Hence, the sensitivity of the mutant to thiol stress is likely due to reduced expression of the SigR regulon.Figure 8.


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)

Reduced induction of sigR expression in a ddbA mutant. The qRT-PCR analysis of sigR transcript abundance at 15 and 30 min after induction of thiol stress in M145 and the ddbA mutant. The data were normalized using hrdB as a control. Strains were grown over night on NMMP and transferred to minimal liquid medium (NMMP) + 0.5 mM diamide. The data represent averages obtained from three biological replicates, with three experimental replicate performed on each sample.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt180-F8: Reduced induction of sigR expression in a ddbA mutant. The qRT-PCR analysis of sigR transcript abundance at 15 and 30 min after induction of thiol stress in M145 and the ddbA mutant. The data were normalized using hrdB as a control. Strains were grown over night on NMMP and transferred to minimal liquid medium (NMMP) + 0.5 mM diamide. The data represent averages obtained from three biological replicates, with three experimental replicate performed on each sample.
Mentions: In E. coli, ppGpp/DksA-dependent remodelling of transcription is in part mediated by alternative sigma factors changing the promoter specificity of RNAP (5). For alternative sigma factors such as SigS and SigN, a sigma competition model has been proposed whereby ppGpp/DksA alters the specificity of interaction of the housekeeping Sig70 to core RNAP, allowing other sigma factors to bind (31). We discovered that the ddbA mutant is sensitive to diamide, a thiol-specific oxidising agent, and this sensitivity is restored by genetic complementation with ddbA (Table 3). In streptomycetes, thiol oxidation is sensed by a complex of the alternative sigma factor, SigR, bound to its antisigma factor RsrA (32). Disulphide bond formation in RsrA causes conformational changes, releasing SigR that directs RNAP to transcribe genes belonging to the SigR regulon that include sigR itself, the trxAB operon encoding thioredoxin reductase and thioredoxin, and the relA gene (33), the latter encoding an enzyme that is the principal ppGpp synthase. To investigate the sensitivity of the ddbA mutant to thiol stress and its relation to sigR expression, the induction of the gene after exposure to diamide was quantified in the wild-type and mutant, indicating impaired upregulation of sigR in the mutant (Figure 8). No significant differences in very low, uninduced, levels of expression between wild-type and mutant were detected. Hence, the sensitivity of the mutant to thiol stress is likely due to reduced expression of the SigR regulon.Figure 8.

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