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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 is a developmentally regulated NAP. Panel A: DdbA::mCherry is present in a germinating spore (arrowed) and in a small region of the adjacent young aerial hyphae. Panel B: DdbA::mCherry is subsequently expressed in non-branching aerial hyphae. Panel C: DdbA::mCherry co-localizes with nucleoids in spore compartments. Panel D: little or no fusion protein was detected in nucleoid-free spore compartments of an smc mutant. Bars indicate 10 µm. Panel E: total protein extracts from S.coelicolor M145/ p2075His grown on the surface of SFM plates were collected and immunobloted. Spores were collected after 4 days, filtered through cotton and a total protein extract prepared. In all, 10 mg of total protein were loaded in each of the first four lanes. Adjacent panel (R) shows purified recombinant DbdAHis expressed in E.coli BL21 (DE3), and detected as above.
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gkt180-F2: DdbA is a developmentally regulated NAP. Panel A: DdbA::mCherry is present in a germinating spore (arrowed) and in a small region of the adjacent young aerial hyphae. Panel B: DdbA::mCherry is subsequently expressed in non-branching aerial hyphae. Panel C: DdbA::mCherry co-localizes with nucleoids in spore compartments. Panel D: little or no fusion protein was detected in nucleoid-free spore compartments of an smc mutant. Bars indicate 10 µm. Panel E: total protein extracts from S.coelicolor M145/ p2075His grown on the surface of SFM plates were collected and immunobloted. Spores were collected after 4 days, filtered through cotton and a total protein extract prepared. In all, 10 mg of total protein were loaded in each of the first four lanes. Adjacent panel (R) shows purified recombinant DbdAHis expressed in E.coli BL21 (DE3), and detected as above.

Mentions: To examine ddbA function in vivo, a mutant was constructed with a Tn5062 insertion 77 bp after the start of the gene. With respect to its macroscopic phenotype, the mutant did not differ from the wild-type in terms of sporulation. In addition, a mCherry translational fusion was constructed to determine time of expression and localization of the protein by fluorescence microscopy. The gene encoding the fusion protein restored normal growth of the ddbA mutant grown under osmotic stress (see later in the text), indicating functionality. Under control of the native promoter, the mCherry-tagged protein is abundant in spores before and immediately after germination, with the protein apparently diffusing along the young hyphae (Figure 2). However, beyond a short distance from the germinated spore and in older vegetative hyphae, there was very little detectable fluorescent signal, indicating low levels of expression and/or rapid turnover of the fusion protein. Only during growth and development of aerial hyphae was the mCherry fusion protein detectable once again, with the intensity of the signal increasing as the aerial hyphae matured (Figure 2). The protein appears to be nucleoid-associated within pre-spores as the diffuse nature of the protein evident in early aerial hyphae changes to co-localization with condensing nucleoids during early sporulation. During late sporulation, when the unigenomic spores are mature, the localization of the DdbA::mCherry fusion protein is consistent with the shape of the nucleoid (Figure 2). This association was confirmed by examining localization of the fusion protein in a smc mutant that generates nucleoid-free spore compartments at high frequency (13). Little or no fusion protein could be detected in anucleate cells (Figure 2). In addition to the cytological analysis, the developmental regulation of DdbA was confirmed by western blot, indicating abundant expression of the His-tagged protein (that retained function from evidence of complementation of the osmotic stress-induced phenotype of the ddbA mutant—see later in the text) under the control of the native promoter after 72 h growth and in spores (Figure 2). The 25 kDa DdbA protein from both S. coelicolor and purified from E. coli migrates slower than predicted in SDS–PAGE gels.Figure 2.


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 developmentally regulated NAP. Panel A: DdbA::mCherry is present in a germinating spore (arrowed) and in a small region of the adjacent young aerial hyphae. Panel B: DdbA::mCherry is subsequently expressed in non-branching aerial hyphae. Panel C: DdbA::mCherry co-localizes with nucleoids in spore compartments. Panel D: little or no fusion protein was detected in nucleoid-free spore compartments of an smc mutant. Bars indicate 10 µm. Panel E: total protein extracts from S.coelicolor M145/ p2075His grown on the surface of SFM plates were collected and immunobloted. Spores were collected after 4 days, filtered through cotton and a total protein extract prepared. In all, 10 mg of total protein were loaded in each of the first four lanes. Adjacent panel (R) shows purified recombinant DbdAHis expressed in E.coli BL21 (DE3), and detected as above.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC3643593&req=5

gkt180-F2: DdbA is a developmentally regulated NAP. Panel A: DdbA::mCherry is present in a germinating spore (arrowed) and in a small region of the adjacent young aerial hyphae. Panel B: DdbA::mCherry is subsequently expressed in non-branching aerial hyphae. Panel C: DdbA::mCherry co-localizes with nucleoids in spore compartments. Panel D: little or no fusion protein was detected in nucleoid-free spore compartments of an smc mutant. Bars indicate 10 µm. Panel E: total protein extracts from S.coelicolor M145/ p2075His grown on the surface of SFM plates were collected and immunobloted. Spores were collected after 4 days, filtered through cotton and a total protein extract prepared. In all, 10 mg of total protein were loaded in each of the first four lanes. Adjacent panel (R) shows purified recombinant DbdAHis expressed in E.coli BL21 (DE3), and detected as above.
Mentions: To examine ddbA function in vivo, a mutant was constructed with a Tn5062 insertion 77 bp after the start of the gene. With respect to its macroscopic phenotype, the mutant did not differ from the wild-type in terms of sporulation. In addition, a mCherry translational fusion was constructed to determine time of expression and localization of the protein by fluorescence microscopy. The gene encoding the fusion protein restored normal growth of the ddbA mutant grown under osmotic stress (see later in the text), indicating functionality. Under control of the native promoter, the mCherry-tagged protein is abundant in spores before and immediately after germination, with the protein apparently diffusing along the young hyphae (Figure 2). However, beyond a short distance from the germinated spore and in older vegetative hyphae, there was very little detectable fluorescent signal, indicating low levels of expression and/or rapid turnover of the fusion protein. Only during growth and development of aerial hyphae was the mCherry fusion protein detectable once again, with the intensity of the signal increasing as the aerial hyphae matured (Figure 2). The protein appears to be nucleoid-associated within pre-spores as the diffuse nature of the protein evident in early aerial hyphae changes to co-localization with condensing nucleoids during early sporulation. During late sporulation, when the unigenomic spores are mature, the localization of the DdbA::mCherry fusion protein is consistent with the shape of the nucleoid (Figure 2). This association was confirmed by examining localization of the fusion protein in a smc mutant that generates nucleoid-free spore compartments at high frequency (13). Little or no fusion protein could be detected in anucleate cells (Figure 2). In addition to the cytological analysis, the developmental regulation of DdbA was confirmed by western blot, indicating abundant expression of the His-tagged protein (that retained function from evidence of complementation of the osmotic stress-induced phenotype of the ddbA mutant—see later in the text) under the control of the native promoter after 72 h growth and in spores (Figure 2). The 25 kDa DdbA protein from both S. coelicolor and purified from E. coli migrates slower than predicted in SDS–PAGE gels.Figure 2.

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