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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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DdbA modulates DNA supercoiling. For panels A and B, plasmid pROT219 DNA was extracted and supercoils separated on chloroquine gels together with HindIII restricted λ DNA (lane M). Quantitation of the intensity of bands corresponding to individual topoisomers from each lane is shown next to each gel. Lkm, the most abundant topoisomer is indicated by the vertical arrow and the change in linking number, ΔLk is shown for each lane using plasmid isolated from M145 with no osmolyte (Panel A, lane 1) as a reference point. The horizontal arrow indicates direction of migration in gel. Panel A: Lanes 1 and 3—plasmid from M145 grown without and with 1 M sucrose; lanes 2 and 4—plasmid from the ddbA mutant grown without and with 1 M sucrose. Panel B: lane 1—plasmid from the M145 parental strain grown with 1 M sucrose; lane 2—plasmid from the genetically complemented ddbA mutant with integrated pSH2075 grown with 1 M sucrose. Panel C: qRT-PCR quantifying gyrB transcript abundance in strains M145 and the ddbA mutant 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 between 5 and 60 min before total RNA extraction. The data represent averages obtained from three biological replicates, with three experimental replicate performed on each sample.
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gkt180-F6: DdbA modulates DNA supercoiling. For panels A and B, plasmid pROT219 DNA was extracted and supercoils separated on chloroquine gels together with HindIII restricted λ DNA (lane M). Quantitation of the intensity of bands corresponding to individual topoisomers from each lane is shown next to each gel. Lkm, the most abundant topoisomer is indicated by the vertical arrow and the change in linking number, ΔLk is shown for each lane using plasmid isolated from M145 with no osmolyte (Panel A, lane 1) as a reference point. The horizontal arrow indicates direction of migration in gel. Panel A: Lanes 1 and 3—plasmid from M145 grown without and with 1 M sucrose; lanes 2 and 4—plasmid from the ddbA mutant grown without and with 1 M sucrose. Panel B: lane 1—plasmid from the M145 parental strain grown with 1 M sucrose; lane 2—plasmid from the genetically complemented ddbA mutant with integrated pSH2075 grown with 1 M sucrose. Panel C: qRT-PCR quantifying gyrB transcript abundance in strains M145 and the ddbA mutant 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 between 5 and 60 min before total RNA extraction. The data represent averages obtained from three biological replicates, with three experimental replicate performed on each sample.

Mentions: Certain nucleoid-associated proteins in other bacteria have the ability to affect DNA topology and thereby influence gene expression in response to changes in osmotic conditions (8). Moreover, using a reporter plasmid to monitor DNA supercoiling, an increase in negative supercoiling was observed in S. lividans grown in the presence of increased osmolyte (23). We investigated the influence of DdbA on changes to DNA supercoiling in response to varying osmolyte concentration using a similar approach. As observed in S. lividans, 2 day growth of the M145 S. coelicolor wild-type strain in the presence of 1M sucrose results in a net increase in the mean linking number (∼ΔLk) of plasmid supercoils of +6 (Figure 6). In the ddbA mutant, however, no detectable osmolyte-induced increase in negative supercoiling was observed; the difference in mean linking number between the mutant and M145 under osmotic stress was ∼ΔLk −4, −7 and −6 for 0.72 M sucrose, 1 M sucrose and 250 mM KCl, respectively. To confirm the apparent role of DdbA in altering DNA supercoiling, plasmid DNA from the complemented mutant grown in medium supplemented with osmolyte was analysed. In this strain, we observed a similar increase in negative supercoiling as seen for the wild-type (Figure 6).Figure 6.


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 modulates DNA supercoiling. For panels A and B, plasmid pROT219 DNA was extracted and supercoils separated on chloroquine gels together with HindIII restricted λ DNA (lane M). Quantitation of the intensity of bands corresponding to individual topoisomers from each lane is shown next to each gel. Lkm, the most abundant topoisomer is indicated by the vertical arrow and the change in linking number, ΔLk is shown for each lane using plasmid isolated from M145 with no osmolyte (Panel A, lane 1) as a reference point. The horizontal arrow indicates direction of migration in gel. Panel A: Lanes 1 and 3—plasmid from M145 grown without and with 1 M sucrose; lanes 2 and 4—plasmid from the ddbA mutant grown without and with 1 M sucrose. Panel B: lane 1—plasmid from the M145 parental strain grown with 1 M sucrose; lane 2—plasmid from the genetically complemented ddbA mutant with integrated pSH2075 grown with 1 M sucrose. Panel C: qRT-PCR quantifying gyrB transcript abundance in strains M145 and the ddbA mutant 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 between 5 and 60 min before total RNA extraction. 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-F6: DdbA modulates DNA supercoiling. For panels A and B, plasmid pROT219 DNA was extracted and supercoils separated on chloroquine gels together with HindIII restricted λ DNA (lane M). Quantitation of the intensity of bands corresponding to individual topoisomers from each lane is shown next to each gel. Lkm, the most abundant topoisomer is indicated by the vertical arrow and the change in linking number, ΔLk is shown for each lane using plasmid isolated from M145 with no osmolyte (Panel A, lane 1) as a reference point. The horizontal arrow indicates direction of migration in gel. Panel A: Lanes 1 and 3—plasmid from M145 grown without and with 1 M sucrose; lanes 2 and 4—plasmid from the ddbA mutant grown without and with 1 M sucrose. Panel B: lane 1—plasmid from the M145 parental strain grown with 1 M sucrose; lane 2—plasmid from the genetically complemented ddbA mutant with integrated pSH2075 grown with 1 M sucrose. Panel C: qRT-PCR quantifying gyrB transcript abundance in strains M145 and the ddbA mutant 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 between 5 and 60 min before total RNA extraction. The data represent averages obtained from three biological replicates, with three experimental replicate performed on each sample.
Mentions: Certain nucleoid-associated proteins in other bacteria have the ability to affect DNA topology and thereby influence gene expression in response to changes in osmotic conditions (8). Moreover, using a reporter plasmid to monitor DNA supercoiling, an increase in negative supercoiling was observed in S. lividans grown in the presence of increased osmolyte (23). We investigated the influence of DdbA on changes to DNA supercoiling in response to varying osmolyte concentration using a similar approach. As observed in S. lividans, 2 day growth of the M145 S. coelicolor wild-type strain in the presence of 1M sucrose results in a net increase in the mean linking number (∼ΔLk) of plasmid supercoils of +6 (Figure 6). In the ddbA mutant, however, no detectable osmolyte-induced increase in negative supercoiling was observed; the difference in mean linking number between the mutant and M145 under osmotic stress was ∼ΔLk −4, −7 and −6 for 0.72 M sucrose, 1 M sucrose and 250 mM KCl, respectively. To confirm the apparent role of DdbA in altering DNA supercoiling, plasmid DNA from the complemented mutant grown in medium supplemented with osmolyte was analysed. In this strain, we observed a similar increase in negative supercoiling as seen for the wild-type (Figure 6).Figure 6.

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