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Systems-wide temporal proteomic profiling in glucose-starved Bacillus subtilis.

Otto A, Bernhardt J, Meyer H, Schaffer M, Herbst FA, Siebourg J, Mäder U, Lalk M, Hecker M, Becher D - Nat Commun (2010)

Bottom Line: In this study, we monitor temporal changes in the proteome, transcriptome and extracellular metabolome of B. subtilis caused by glucose starvation.Quantitative proteomic and corresponding transcriptomic data were analysed with Voronoi treemaps linking functional classification and relative expression changes of gene products according to their fate in the stationary phase.The obtained data comprise the first comprehensive profiling of changes in the membrane subfraction and allow in-depth analysis of major physiological processes, including monitoring of protein degradation.

View Article: PubMed Central - PubMed

Affiliation: Ernst-Moritz-Arndt-Universität Greifswald, Institute for Microbiology, Greifswald 17487, Germany.

ABSTRACT
Functional genomics of the Gram-positive model organism Bacillus subtilis reveals valuable insights into basic concepts of cell physiology. In this study, we monitor temporal changes in the proteome, transcriptome and extracellular metabolome of B. subtilis caused by glucose starvation. For proteomic profiling, a combination of in vivo metabolic labelling and shotgun mass spectrometric analysis was carried out for five different proteomic subfractions (cytosolic, integral membrane, membrane, surface and extracellular proteome fraction), leading to the identification of ~52% of the predicted proteome of B. subtilis. Quantitative proteomic and corresponding transcriptomic data were analysed with Voronoi treemaps linking functional classification and relative expression changes of gene products according to their fate in the stationary phase. The obtained data comprise the first comprehensive profiling of changes in the membrane subfraction and allow in-depth analysis of major physiological processes, including monitoring of protein degradation.

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Distribution of proteins identified according to the subfractionation workflow.(a) Workflow of the proteomic experiment. The preparation of the five different subcellular fractions is exemplified with the most crucial steps. The starting point for all proteomic samples is the bacterial batch cell culture. The first crossroads is the separation between complete cells for the BEF (biotinylation-enriched fraction) aiming at the surface-exposed proteins, the cell culture medium for the extracellular proteins and cells for the cytosolic/membrane fractions. 14N/15N mixing depicts the stage in sample preparation, where mixing of the labelled pool and the unlabelled samples occurred. The earliest time point in experimental setup is chosen to minimize introduced effects by the labelling procedure to the quantitative data. Proteomic analyses (GeLC-MS and LC-MS) were performed with high-resolution and high mass accuracy mass spectrometry (2,142 total identified proteins with an FPR (false-positive rate) of 0.11% and 1.86 p.p.m. mass accuracy). (b) Distribution of proteins identified with a predicted subcellular localization in the different subfractions analysed. The area of the circles is proportional to number of identified proteins. (c) VENN plot of protein identifications in the different designated subcellular fractions. Numbers for the membrane fraction are summarized from EMF and MSF. Prediction of subcellular localization is according to Zhou et al.52 Loci of localization assigned were as follows: intracellular; integral membrane proteins (multitransmembrane, multitransmembrane (lipid-modified N termini)); membrane-associated proteins (lipid anchored, LPxTG cell wall anchored, N-terminally anchored (no cleavage site), N-terminally anchored (with cleavage site), C-terminally anchored (with cleavage site), intracellular/TMH start after 60); secreted (secreted via minor pathways (bacteriocin) (no cleavage site), secretory (released) (with cleavage site)).
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f1: Distribution of proteins identified according to the subfractionation workflow.(a) Workflow of the proteomic experiment. The preparation of the five different subcellular fractions is exemplified with the most crucial steps. The starting point for all proteomic samples is the bacterial batch cell culture. The first crossroads is the separation between complete cells for the BEF (biotinylation-enriched fraction) aiming at the surface-exposed proteins, the cell culture medium for the extracellular proteins and cells for the cytosolic/membrane fractions. 14N/15N mixing depicts the stage in sample preparation, where mixing of the labelled pool and the unlabelled samples occurred. The earliest time point in experimental setup is chosen to minimize introduced effects by the labelling procedure to the quantitative data. Proteomic analyses (GeLC-MS and LC-MS) were performed with high-resolution and high mass accuracy mass spectrometry (2,142 total identified proteins with an FPR (false-positive rate) of 0.11% and 1.86 p.p.m. mass accuracy). (b) Distribution of proteins identified with a predicted subcellular localization in the different subfractions analysed. The area of the circles is proportional to number of identified proteins. (c) VENN plot of protein identifications in the different designated subcellular fractions. Numbers for the membrane fraction are summarized from EMF and MSF. Prediction of subcellular localization is according to Zhou et al.52 Loci of localization assigned were as follows: intracellular; integral membrane proteins (multitransmembrane, multitransmembrane (lipid-modified N termini)); membrane-associated proteins (lipid anchored, LPxTG cell wall anchored, N-terminally anchored (no cleavage site), N-terminally anchored (with cleavage site), C-terminally anchored (with cleavage site), intracellular/TMH start after 60); secreted (secreted via minor pathways (bacteriocin) (no cleavage site), secretory (released) (with cleavage site)).

Mentions: Metabolically labelled B. subtilis proteins from growing and non-growing cells were fractionated to obtain five different subproteomes for a comprehensive view on regulatory and physiological changes (Fig. 1). Entry into stationary phase was provoked by glucose deprivation at an optical density (500 nm) of 1 (Supplementary Fig. S1). Based on our workflow, allowing highly accurate (average mass deviation of <2 p.p.m. in our analysis) and reliable data (0.11% false positives on average on protein level), we were able to considerably cover the proteome of B. subtilis. In total, 2,142 proteins were identified with at least two peptides, which represents 52.2% of the entire predicted proteome (Table 1; Supplementary Data 1). We were able to assign 552 proteins solely found in the cytosolic fraction (Fig. 1), while 344 proteins were exclusively found to be associated with or completely inserted into the bacterial membrane. Furthermore, 81 proteins were identified only in the extracellular space. A substantial number of proteins were found in multiple subcellular fractions: 509 proteins were found in two, 389 in three and 266 proteins in all subproteomes.


Systems-wide temporal proteomic profiling in glucose-starved Bacillus subtilis.

Otto A, Bernhardt J, Meyer H, Schaffer M, Herbst FA, Siebourg J, Mäder U, Lalk M, Hecker M, Becher D - Nat Commun (2010)

Distribution of proteins identified according to the subfractionation workflow.(a) Workflow of the proteomic experiment. The preparation of the five different subcellular fractions is exemplified with the most crucial steps. The starting point for all proteomic samples is the bacterial batch cell culture. The first crossroads is the separation between complete cells for the BEF (biotinylation-enriched fraction) aiming at the surface-exposed proteins, the cell culture medium for the extracellular proteins and cells for the cytosolic/membrane fractions. 14N/15N mixing depicts the stage in sample preparation, where mixing of the labelled pool and the unlabelled samples occurred. The earliest time point in experimental setup is chosen to minimize introduced effects by the labelling procedure to the quantitative data. Proteomic analyses (GeLC-MS and LC-MS) were performed with high-resolution and high mass accuracy mass spectrometry (2,142 total identified proteins with an FPR (false-positive rate) of 0.11% and 1.86 p.p.m. mass accuracy). (b) Distribution of proteins identified with a predicted subcellular localization in the different subfractions analysed. The area of the circles is proportional to number of identified proteins. (c) VENN plot of protein identifications in the different designated subcellular fractions. Numbers for the membrane fraction are summarized from EMF and MSF. Prediction of subcellular localization is according to Zhou et al.52 Loci of localization assigned were as follows: intracellular; integral membrane proteins (multitransmembrane, multitransmembrane (lipid-modified N termini)); membrane-associated proteins (lipid anchored, LPxTG cell wall anchored, N-terminally anchored (no cleavage site), N-terminally anchored (with cleavage site), C-terminally anchored (with cleavage site), intracellular/TMH start after 60); secreted (secreted via minor pathways (bacteriocin) (no cleavage site), secretory (released) (with cleavage site)).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3105300&req=5

f1: Distribution of proteins identified according to the subfractionation workflow.(a) Workflow of the proteomic experiment. The preparation of the five different subcellular fractions is exemplified with the most crucial steps. The starting point for all proteomic samples is the bacterial batch cell culture. The first crossroads is the separation between complete cells for the BEF (biotinylation-enriched fraction) aiming at the surface-exposed proteins, the cell culture medium for the extracellular proteins and cells for the cytosolic/membrane fractions. 14N/15N mixing depicts the stage in sample preparation, where mixing of the labelled pool and the unlabelled samples occurred. The earliest time point in experimental setup is chosen to minimize introduced effects by the labelling procedure to the quantitative data. Proteomic analyses (GeLC-MS and LC-MS) were performed with high-resolution and high mass accuracy mass spectrometry (2,142 total identified proteins with an FPR (false-positive rate) of 0.11% and 1.86 p.p.m. mass accuracy). (b) Distribution of proteins identified with a predicted subcellular localization in the different subfractions analysed. The area of the circles is proportional to number of identified proteins. (c) VENN plot of protein identifications in the different designated subcellular fractions. Numbers for the membrane fraction are summarized from EMF and MSF. Prediction of subcellular localization is according to Zhou et al.52 Loci of localization assigned were as follows: intracellular; integral membrane proteins (multitransmembrane, multitransmembrane (lipid-modified N termini)); membrane-associated proteins (lipid anchored, LPxTG cell wall anchored, N-terminally anchored (no cleavage site), N-terminally anchored (with cleavage site), C-terminally anchored (with cleavage site), intracellular/TMH start after 60); secreted (secreted via minor pathways (bacteriocin) (no cleavage site), secretory (released) (with cleavage site)).
Mentions: Metabolically labelled B. subtilis proteins from growing and non-growing cells were fractionated to obtain five different subproteomes for a comprehensive view on regulatory and physiological changes (Fig. 1). Entry into stationary phase was provoked by glucose deprivation at an optical density (500 nm) of 1 (Supplementary Fig. S1). Based on our workflow, allowing highly accurate (average mass deviation of <2 p.p.m. in our analysis) and reliable data (0.11% false positives on average on protein level), we were able to considerably cover the proteome of B. subtilis. In total, 2,142 proteins were identified with at least two peptides, which represents 52.2% of the entire predicted proteome (Table 1; Supplementary Data 1). We were able to assign 552 proteins solely found in the cytosolic fraction (Fig. 1), while 344 proteins were exclusively found to be associated with or completely inserted into the bacterial membrane. Furthermore, 81 proteins were identified only in the extracellular space. A substantial number of proteins were found in multiple subcellular fractions: 509 proteins were found in two, 389 in three and 266 proteins in all subproteomes.

Bottom Line: In this study, we monitor temporal changes in the proteome, transcriptome and extracellular metabolome of B. subtilis caused by glucose starvation.Quantitative proteomic and corresponding transcriptomic data were analysed with Voronoi treemaps linking functional classification and relative expression changes of gene products according to their fate in the stationary phase.The obtained data comprise the first comprehensive profiling of changes in the membrane subfraction and allow in-depth analysis of major physiological processes, including monitoring of protein degradation.

View Article: PubMed Central - PubMed

Affiliation: Ernst-Moritz-Arndt-Universität Greifswald, Institute for Microbiology, Greifswald 17487, Germany.

ABSTRACT
Functional genomics of the Gram-positive model organism Bacillus subtilis reveals valuable insights into basic concepts of cell physiology. In this study, we monitor temporal changes in the proteome, transcriptome and extracellular metabolome of B. subtilis caused by glucose starvation. For proteomic profiling, a combination of in vivo metabolic labelling and shotgun mass spectrometric analysis was carried out for five different proteomic subfractions (cytosolic, integral membrane, membrane, surface and extracellular proteome fraction), leading to the identification of ~52% of the predicted proteome of B. subtilis. Quantitative proteomic and corresponding transcriptomic data were analysed with Voronoi treemaps linking functional classification and relative expression changes of gene products according to their fate in the stationary phase. The obtained data comprise the first comprehensive profiling of changes in the membrane subfraction and allow in-depth analysis of major physiological processes, including monitoring of protein degradation.

Show MeSH
Related in: MedlinePlus