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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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Over-expression of DdbA restores antibiotic production in relA mutants. Strains were grown for 5 days on SMMS medium supplemented with indicated concentrations of the inducer, thiostrepton. Panel A: clockwise from top: M145 with the empty vector pIJ8600H; two independent relA mutants of M145 with the empty vector pIJ8600H; the same two relA mutants with plasmid pDksA3H. Panel B: the top two strains are two independent clones of M570relA with plasmid pDksA3; the bottom strain is M570relA with the empty vector pIJ8600.
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gkt180-F7: Over-expression of DdbA restores antibiotic production in relA mutants. Strains were grown for 5 days on SMMS medium supplemented with indicated concentrations of the inducer, thiostrepton. Panel A: clockwise from top: M145 with the empty vector pIJ8600H; two independent relA mutants of M145 with the empty vector pIJ8600H; the same two relA mutants with plasmid pDksA3H. Panel B: the top two strains are two independent clones of M570relA with plasmid pDksA3; the bottom strain is M570relA with the empty vector pIJ8600.

Mentions: In E. coli, DksA augments the role of ppGpp in the stringent response and consequently has been termed a ppGpp cofactor (29). In addition, overexpression of DksA can suppress the phenotypes of ppGpp0 mutants (9). In S. coelicolor, ppGpp is synthesised towards the end of exponential growth and during the so-called transition phase immediately before antibiotic production; a relA mutant is conditionally deficient in antibiotic production (14). We investigated overexpression of ddbA in S. coelicolor relA mutants constructed in two different genetic backgrounds. DdbA was overexpressed by fusing the gene to the thiostrepton-inducible tipA promoter in pIJ8600 or pIJ8600H, and consequently all strains tested contained either the empty vector or the ddbA overexpression plasmid. As our previous experiments were performed in M145, we constructed a mutant containing a Tn5062 insertion in relA and tested two independent isolates of this mutant. Although M145 produced red prodigines on nitrogen-limiting supplemented minimal medium, solid (SMMS) medium, the independently isolated relA mutants did not (Figure 7a). The second genetic background we tested was S. coelicolor M570, using a previously published relA mutant (30). The parental M570 produced actinorhodin on SMMS, whereas the mutant did not (Figure 7b). In both mutants containing a ddbA overexpression plasmid, antibiotic production was restored in response to addition of inducer to the growth medium; overexpression of ddbA triggered production of red prodiginines in the M145 genetic background and blue actinorhodin in the M570 background (Figure 7). To test whether this apparent suppression of the phenotype of the M570 relA mutant could be attributed to the DksA-like domain of DdbA, we also overexpressed just the 138 amino acid N-terminal DNA-binding domain of the protein. Antibiotic production was not restored in the mutant, indicating that the full-length protein is required for relA suppression (results not shown). Overexpression of either full-length DdbA or the N-terminal DNA-binding domain in M145 did not affect antibiotic production.Figure 7.


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)

Over-expression of DdbA restores antibiotic production in relA mutants. Strains were grown for 5 days on SMMS medium supplemented with indicated concentrations of the inducer, thiostrepton. Panel A: clockwise from top: M145 with the empty vector pIJ8600H; two independent relA mutants of M145 with the empty vector pIJ8600H; the same two relA mutants with plasmid pDksA3H. Panel B: the top two strains are two independent clones of M570relA with plasmid pDksA3; the bottom strain is M570relA with the empty vector pIJ8600.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt180-F7: Over-expression of DdbA restores antibiotic production in relA mutants. Strains were grown for 5 days on SMMS medium supplemented with indicated concentrations of the inducer, thiostrepton. Panel A: clockwise from top: M145 with the empty vector pIJ8600H; two independent relA mutants of M145 with the empty vector pIJ8600H; the same two relA mutants with plasmid pDksA3H. Panel B: the top two strains are two independent clones of M570relA with plasmid pDksA3; the bottom strain is M570relA with the empty vector pIJ8600.
Mentions: In E. coli, DksA augments the role of ppGpp in the stringent response and consequently has been termed a ppGpp cofactor (29). In addition, overexpression of DksA can suppress the phenotypes of ppGpp0 mutants (9). In S. coelicolor, ppGpp is synthesised towards the end of exponential growth and during the so-called transition phase immediately before antibiotic production; a relA mutant is conditionally deficient in antibiotic production (14). We investigated overexpression of ddbA in S. coelicolor relA mutants constructed in two different genetic backgrounds. DdbA was overexpressed by fusing the gene to the thiostrepton-inducible tipA promoter in pIJ8600 or pIJ8600H, and consequently all strains tested contained either the empty vector or the ddbA overexpression plasmid. As our previous experiments were performed in M145, we constructed a mutant containing a Tn5062 insertion in relA and tested two independent isolates of this mutant. Although M145 produced red prodigines on nitrogen-limiting supplemented minimal medium, solid (SMMS) medium, the independently isolated relA mutants did not (Figure 7a). The second genetic background we tested was S. coelicolor M570, using a previously published relA mutant (30). The parental M570 produced actinorhodin on SMMS, whereas the mutant did not (Figure 7b). In both mutants containing a ddbA overexpression plasmid, antibiotic production was restored in response to addition of inducer to the growth medium; overexpression of ddbA triggered production of red prodiginines in the M145 genetic background and blue actinorhodin in the M570 background (Figure 7). To test whether this apparent suppression of the phenotype of the M570 relA mutant could be attributed to the DksA-like domain of DdbA, we also overexpressed just the 138 amino acid N-terminal DNA-binding domain of the protein. Antibiotic production was not restored in the mutant, indicating that the full-length protein is required for relA suppression (results not shown). Overexpression of either full-length DdbA or the N-terminal DNA-binding domain in M145 did not affect antibiotic production.Figure 7.

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