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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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The ddbA is involved in the osmotic stress response. Panel A: increased production of blue actinorhodin as a result of osmotic stress was evident with surface grown cultures of the ddbA mutant (1) and the mutant with an empty vector, pSH152 (2), compared with the genetically complemented mutant containing plasmid pSH2075 (3). The wild-type strain, M145 is also shown for comparison. M145 containing pSH152, not shown, has a similar phenotype to the plasmid-free wild-type strain. The strains were grown on NMMP medium containing 250 mM KCl. Panel B: qRT-PCR quantifying ddbA transcript abundance before and after of osmotic up-shock with 250 mM KCl. S.coelicolor M145 was grown for 16 h on cellophane discs on the surface of MS agar and then transferred to MS/250 mM KCl plates and incubated for 1 h before total RNA extraction. A non-stressed control sample was processed in parallel by transfer of a cellophane disc to an MS plate, followed by 1 h incubation. The data represent averages obtained from three biological replicates, with three experimental replicates performed on each sample.
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gkt180-F5: The ddbA is involved in the osmotic stress response. Panel A: increased production of blue actinorhodin as a result of osmotic stress was evident with surface grown cultures of the ddbA mutant (1) and the mutant with an empty vector, pSH152 (2), compared with the genetically complemented mutant containing plasmid pSH2075 (3). The wild-type strain, M145 is also shown for comparison. M145 containing pSH152, not shown, has a similar phenotype to the plasmid-free wild-type strain. The strains were grown on NMMP medium containing 250 mM KCl. Panel B: qRT-PCR quantifying ddbA transcript abundance before and after of osmotic up-shock with 250 mM KCl. S.coelicolor M145 was grown for 16 h on cellophane discs on the surface of MS agar and then transferred to MS/250 mM KCl plates and incubated for 1 h before total RNA extraction. A non-stressed control sample was processed in parallel by transfer of a cellophane disc to an MS plate, followed by 1 h incubation. The data represent averages obtained from three biological replicates, with three experimental replicates performed on each sample.

Mentions: The only discernable macroscopic phenotype of the ddbA mutant was both a delay in sporulation and concomitant increase in production of the antibiotic actinorhodin compared with either the wild-type or complemented mutant when these strains were grown on media containing 250 mM potassium chloride (Figure 5). To explore this link further, expression of ddbA was quantified by qRT-PCR, indicating that it is induced during vegetative growth slightly >2-fold (significantly different, one-way ANOVA, P < 0.05) 60 min after osmotic stress (Figure 5). This induction implies another aspect of the gene’s regulation, in addition to its developmental control.Figure 5.


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)

The ddbA is involved in the osmotic stress response. Panel A: increased production of blue actinorhodin as a result of osmotic stress was evident with surface grown cultures of the ddbA mutant (1) and the mutant with an empty vector, pSH152 (2), compared with the genetically complemented mutant containing plasmid pSH2075 (3). The wild-type strain, M145 is also shown for comparison. M145 containing pSH152, not shown, has a similar phenotype to the plasmid-free wild-type strain. The strains were grown on NMMP medium containing 250 mM KCl. Panel B: qRT-PCR quantifying ddbA transcript abundance before and after of osmotic up-shock with 250 mM KCl. S.coelicolor M145 was grown for 16 h on cellophane discs on the surface of MS agar and then transferred to MS/250 mM KCl plates and incubated for 1 h before total RNA extraction. A non-stressed control sample was processed in parallel by transfer of a cellophane disc to an MS plate, followed by 1 h incubation. The data represent averages obtained from three biological replicates, with three experimental replicates performed on each sample.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt180-F5: The ddbA is involved in the osmotic stress response. Panel A: increased production of blue actinorhodin as a result of osmotic stress was evident with surface grown cultures of the ddbA mutant (1) and the mutant with an empty vector, pSH152 (2), compared with the genetically complemented mutant containing plasmid pSH2075 (3). The wild-type strain, M145 is also shown for comparison. M145 containing pSH152, not shown, has a similar phenotype to the plasmid-free wild-type strain. The strains were grown on NMMP medium containing 250 mM KCl. Panel B: qRT-PCR quantifying ddbA transcript abundance before and after of osmotic up-shock with 250 mM KCl. S.coelicolor M145 was grown for 16 h on cellophane discs on the surface of MS agar and then transferred to MS/250 mM KCl plates and incubated for 1 h before total RNA extraction. A non-stressed control sample was processed in parallel by transfer of a cellophane disc to an MS plate, followed by 1 h incubation. The data represent averages obtained from three biological replicates, with three experimental replicates performed on each sample.
Mentions: The only discernable macroscopic phenotype of the ddbA mutant was both a delay in sporulation and concomitant increase in production of the antibiotic actinorhodin compared with either the wild-type or complemented mutant when these strains were grown on media containing 250 mM potassium chloride (Figure 5). To explore this link further, expression of ddbA was quantified by qRT-PCR, indicating that it is induced during vegetative growth slightly >2-fold (significantly different, one-way ANOVA, P < 0.05) 60 min after osmotic stress (Figure 5). This induction implies another aspect of the gene’s regulation, in addition to its developmental control.Figure 5.

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