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Phosphoproteome dynamics mediate revival of bacterial spores.

Rosenberg A, Soufi B, Ravikumar V, Soares NC, Krug K, Smith Y, Macek B, Ben-Yehuda S - BMC Biol. (2015)

Bottom Line: The phosphoproteome was found to chiefly comprise newly identified phosphorylation sites located within proteins involved in basic biological functions, such as transcription, translation, carbon metabolism, and spore-specific determinants.Herein, we provide, for the first time, a phosphoproteomic view of a germinating bacterial spore.We further show that the spore phosphoproteome is dynamic and present evidence that phosphorylation events play an integral role in facilitating spore revival.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, Institute for Medical Research Israel-Canada (IMRIC), The Hebrew University-Hadassah Medical School, The Hebrew University of Jerusalem, POB 12272, 91120, Jerusalem, Israel.

ABSTRACT

Background: Bacterial spores can remain dormant for decades, yet harbor the exceptional capacity to rapidly resume metabolic activity and recommence life. Although germinants and their corresponding receptors have been known for more than 30 years, the molecular events underlying this remarkable cellular transition from dormancy to full metabolic activity are only partially defined.

Results: Here, we examined whether protein phospho-modifications occur during germination, the first step of exiting dormancy, thereby facilitating spore revival. Utilizing Bacillus subtilis as a model organism, we performed phosphoproteomic analysis to define the Ser/Thr/Tyr phosphoproteome of a reviving spore. The phosphoproteome was found to chiefly comprise newly identified phosphorylation sites located within proteins involved in basic biological functions, such as transcription, translation, carbon metabolism, and spore-specific determinants. Quantitative comparison of dormant and germinating spore phosphoproteomes revealed phosphorylation dynamics, indicating that phospho-modifications could modulate protein activity during this cellular transition. Furthermore, by mutating select phosphorylation sites located within proteins representative of key biological processes, we established a functional connection between phosphorylation and the progression of spore revival.

Conclusions: Herein, we provide, for the first time, a phosphoproteomic view of a germinating bacterial spore. We further show that the spore phosphoproteome is dynamic and present evidence that phosphorylation events play an integral role in facilitating spore revival.

No MeSH data available.


Related in: MedlinePlus

Characterization of SspA-S47 phosphorylation-site mutants. a Spores of PY79 (wild type, WT), AR209 (sspA-S47A), AR210 (sspA-S47D), and AR179 (∆sspA) strains were incubated at 37 °C in S7-defined medium supplemented with L-Ala (10 mM), and optical density (OD600) was measured at the indicated time points. Data are presented as a fraction of the initial OD600 of the phase-bright spores. Decreasing OD600 signifies spore germination while increasing OD600 indicates spore outgrowth. The data points are averages of results obtained from four independent biological repeats. Error bars designate SD. b Spores of PY79 (wild type, WT), AR209 (sspA-S47A), AR210 (sspA-S47D), and AR179 (∆sspA) strains were exposed to increasing UV (254 nm) doses (mj/cm2) and plated on LB for viable count. Survival was calculated by dividing the viable spore titer at any given UV dose (mj/cm2) with the spore titer obtained from the non-irradiated spores. The data points are averages of results obtained from three independent biological repeats. Error bars designate SD. c Spores of PY79 (wild type, WT), AR209 (sspA-S47A), AR210 (sspA-S47D), and AR179 (∆sspA) strains were germinated with L-Ala (10 mM). Samples were taken at the indicated time points, irradiated with 500 mj/cm2 UV (254 nm), and plated on LB. Survival was calculated by dividing the viable spore titer at any given time point with the spore titer obtained from spores irradiated at the 0 time point. The data points are averages of results obtained from three independent biological repeats. Error bars designate SD. d UV resistance of AR186 (∆sspB), AR195 (∆sspA ∆sspB), AR187 (sspA-S47A ∆sspB), and AR188 (sspA-S47D ∆sspB) spores was determined as described in (b). e UV resistance of AR186 (∆sspB), AR187 (sspA-S47A, ∆sspB), and AR188 (sspA-S47D, ∆sspB) germinating spores was determined as described in (c)
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Fig3: Characterization of SspA-S47 phosphorylation-site mutants. a Spores of PY79 (wild type, WT), AR209 (sspA-S47A), AR210 (sspA-S47D), and AR179 (∆sspA) strains were incubated at 37 °C in S7-defined medium supplemented with L-Ala (10 mM), and optical density (OD600) was measured at the indicated time points. Data are presented as a fraction of the initial OD600 of the phase-bright spores. Decreasing OD600 signifies spore germination while increasing OD600 indicates spore outgrowth. The data points are averages of results obtained from four independent biological repeats. Error bars designate SD. b Spores of PY79 (wild type, WT), AR209 (sspA-S47A), AR210 (sspA-S47D), and AR179 (∆sspA) strains were exposed to increasing UV (254 nm) doses (mj/cm2) and plated on LB for viable count. Survival was calculated by dividing the viable spore titer at any given UV dose (mj/cm2) with the spore titer obtained from the non-irradiated spores. The data points are averages of results obtained from three independent biological repeats. Error bars designate SD. c Spores of PY79 (wild type, WT), AR209 (sspA-S47A), AR210 (sspA-S47D), and AR179 (∆sspA) strains were germinated with L-Ala (10 mM). Samples were taken at the indicated time points, irradiated with 500 mj/cm2 UV (254 nm), and plated on LB. Survival was calculated by dividing the viable spore titer at any given time point with the spore titer obtained from spores irradiated at the 0 time point. The data points are averages of results obtained from three independent biological repeats. Error bars designate SD. d UV resistance of AR186 (∆sspB), AR195 (∆sspA ∆sspB), AR187 (sspA-S47A ∆sspB), and AR188 (sspA-S47D ∆sspB) spores was determined as described in (b). e UV resistance of AR186 (∆sspB), AR187 (sspA-S47A, ∆sspB), and AR188 (sspA-S47D, ∆sspB) germinating spores was determined as described in (c)

Mentions: Strains producing SspA-S47A or SspA-S47D were constructed and their ability to sporulate and revive was assessed. The mutant strains sporulated with efficiency comparable to wild type, and their revival capability, as measured by monitoring changes in optical density (OD600), was not affected, while ∆sspA spores exhibited an extended ripening period (Fig. 3a; Additional file 10: Table S6). Since a major function of SspA is protecting the spore DNA from UV damage, we assayed the UV resistance of the mutant strains. Irradiating spores harboring the mutant alleles revealed slightly modified survival kinetics in comparison to wild type spores, while ∆sspA spores were highly sensitive to UV under the tested conditions (Fig. 3b). Additionally, an altered profile of resistance was observed when mutant spores were irradiated during germination (Fig. 3c). These results suggest that the phosphorylation state of Ser47 affects the protein functionality.Fig. 3


Phosphoproteome dynamics mediate revival of bacterial spores.

Rosenberg A, Soufi B, Ravikumar V, Soares NC, Krug K, Smith Y, Macek B, Ben-Yehuda S - BMC Biol. (2015)

Characterization of SspA-S47 phosphorylation-site mutants. a Spores of PY79 (wild type, WT), AR209 (sspA-S47A), AR210 (sspA-S47D), and AR179 (∆sspA) strains were incubated at 37 °C in S7-defined medium supplemented with L-Ala (10 mM), and optical density (OD600) was measured at the indicated time points. Data are presented as a fraction of the initial OD600 of the phase-bright spores. Decreasing OD600 signifies spore germination while increasing OD600 indicates spore outgrowth. The data points are averages of results obtained from four independent biological repeats. Error bars designate SD. b Spores of PY79 (wild type, WT), AR209 (sspA-S47A), AR210 (sspA-S47D), and AR179 (∆sspA) strains were exposed to increasing UV (254 nm) doses (mj/cm2) and plated on LB for viable count. Survival was calculated by dividing the viable spore titer at any given UV dose (mj/cm2) with the spore titer obtained from the non-irradiated spores. The data points are averages of results obtained from three independent biological repeats. Error bars designate SD. c Spores of PY79 (wild type, WT), AR209 (sspA-S47A), AR210 (sspA-S47D), and AR179 (∆sspA) strains were germinated with L-Ala (10 mM). Samples were taken at the indicated time points, irradiated with 500 mj/cm2 UV (254 nm), and plated on LB. Survival was calculated by dividing the viable spore titer at any given time point with the spore titer obtained from spores irradiated at the 0 time point. The data points are averages of results obtained from three independent biological repeats. Error bars designate SD. d UV resistance of AR186 (∆sspB), AR195 (∆sspA ∆sspB), AR187 (sspA-S47A ∆sspB), and AR188 (sspA-S47D ∆sspB) spores was determined as described in (b). e UV resistance of AR186 (∆sspB), AR187 (sspA-S47A, ∆sspB), and AR188 (sspA-S47D, ∆sspB) germinating spores was determined as described in (c)
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Fig3: Characterization of SspA-S47 phosphorylation-site mutants. a Spores of PY79 (wild type, WT), AR209 (sspA-S47A), AR210 (sspA-S47D), and AR179 (∆sspA) strains were incubated at 37 °C in S7-defined medium supplemented with L-Ala (10 mM), and optical density (OD600) was measured at the indicated time points. Data are presented as a fraction of the initial OD600 of the phase-bright spores. Decreasing OD600 signifies spore germination while increasing OD600 indicates spore outgrowth. The data points are averages of results obtained from four independent biological repeats. Error bars designate SD. b Spores of PY79 (wild type, WT), AR209 (sspA-S47A), AR210 (sspA-S47D), and AR179 (∆sspA) strains were exposed to increasing UV (254 nm) doses (mj/cm2) and plated on LB for viable count. Survival was calculated by dividing the viable spore titer at any given UV dose (mj/cm2) with the spore titer obtained from the non-irradiated spores. The data points are averages of results obtained from three independent biological repeats. Error bars designate SD. c Spores of PY79 (wild type, WT), AR209 (sspA-S47A), AR210 (sspA-S47D), and AR179 (∆sspA) strains were germinated with L-Ala (10 mM). Samples were taken at the indicated time points, irradiated with 500 mj/cm2 UV (254 nm), and plated on LB. Survival was calculated by dividing the viable spore titer at any given time point with the spore titer obtained from spores irradiated at the 0 time point. The data points are averages of results obtained from three independent biological repeats. Error bars designate SD. d UV resistance of AR186 (∆sspB), AR195 (∆sspA ∆sspB), AR187 (sspA-S47A ∆sspB), and AR188 (sspA-S47D ∆sspB) spores was determined as described in (b). e UV resistance of AR186 (∆sspB), AR187 (sspA-S47A, ∆sspB), and AR188 (sspA-S47D, ∆sspB) germinating spores was determined as described in (c)
Mentions: Strains producing SspA-S47A or SspA-S47D were constructed and their ability to sporulate and revive was assessed. The mutant strains sporulated with efficiency comparable to wild type, and their revival capability, as measured by monitoring changes in optical density (OD600), was not affected, while ∆sspA spores exhibited an extended ripening period (Fig. 3a; Additional file 10: Table S6). Since a major function of SspA is protecting the spore DNA from UV damage, we assayed the UV resistance of the mutant strains. Irradiating spores harboring the mutant alleles revealed slightly modified survival kinetics in comparison to wild type spores, while ∆sspA spores were highly sensitive to UV under the tested conditions (Fig. 3b). Additionally, an altered profile of resistance was observed when mutant spores were irradiated during germination (Fig. 3c). These results suggest that the phosphorylation state of Ser47 affects the protein functionality.Fig. 3

Bottom Line: The phosphoproteome was found to chiefly comprise newly identified phosphorylation sites located within proteins involved in basic biological functions, such as transcription, translation, carbon metabolism, and spore-specific determinants.Herein, we provide, for the first time, a phosphoproteomic view of a germinating bacterial spore.We further show that the spore phosphoproteome is dynamic and present evidence that phosphorylation events play an integral role in facilitating spore revival.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Genetics, Institute for Medical Research Israel-Canada (IMRIC), The Hebrew University-Hadassah Medical School, The Hebrew University of Jerusalem, POB 12272, 91120, Jerusalem, Israel.

ABSTRACT

Background: Bacterial spores can remain dormant for decades, yet harbor the exceptional capacity to rapidly resume metabolic activity and recommence life. Although germinants and their corresponding receptors have been known for more than 30 years, the molecular events underlying this remarkable cellular transition from dormancy to full metabolic activity are only partially defined.

Results: Here, we examined whether protein phospho-modifications occur during germination, the first step of exiting dormancy, thereby facilitating spore revival. Utilizing Bacillus subtilis as a model organism, we performed phosphoproteomic analysis to define the Ser/Thr/Tyr phosphoproteome of a reviving spore. The phosphoproteome was found to chiefly comprise newly identified phosphorylation sites located within proteins involved in basic biological functions, such as transcription, translation, carbon metabolism, and spore-specific determinants. Quantitative comparison of dormant and germinating spore phosphoproteomes revealed phosphorylation dynamics, indicating that phospho-modifications could modulate protein activity during this cellular transition. Furthermore, by mutating select phosphorylation sites located within proteins representative of key biological processes, we established a functional connection between phosphorylation and the progression of spore revival.

Conclusions: Herein, we provide, for the first time, a phosphoproteomic view of a germinating bacterial spore. We further show that the spore phosphoproteome is dynamic and present evidence that phosphorylation events play an integral role in facilitating spore revival.

No MeSH data available.


Related in: MedlinePlus