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

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          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.

          Abstract

          Identifying the transcripts and proteins that fluctuate in response to stimuli provides important information for understanding cell physiology. In this study, 52% of the Bacillus subtilis predicted proteome is identified following glucose starvation, revealing further insight into protein dynamics at a global scale.

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          Most cited references45

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          Comprehensive mass-spectrometry-based proteome quantification of haploid versus diploid yeast.

          Mass spectrometry is a powerful technology for the analysis of large numbers of endogenous proteins. However, the analytical challenges associated with comprehensive identification and relative quantification of cellular proteomes have so far appeared to be insurmountable. Here, using advances in computational proteomics, instrument performance and sample preparation strategies, we compare protein levels of essentially all endogenous proteins in haploid yeast cells to their diploid counterparts. Our analysis spans more than four orders of magnitude in protein abundance with no discrimination against membrane or low level regulatory proteins. Stable-isotope labelling by amino acids in cell culture (SILAC) quantification was very accurate across the proteome, as demonstrated by one-to-one ratios of most yeast proteins. Key members of the pheromone pathway were specific to haploid yeast but others were unaltered, suggesting an efficient control mechanism of the mating response. Several retrotransposon-associated proteins were specific to haploid yeast. Gene ontology analysis pinpointed a significant change for cell wall components in agreement with geometrical considerations: diploid cells have twice the volume but not twice the surface area of haploid cells. Transcriptome levels agreed poorly with proteome changes overall. However, after filtering out low confidence microarray measurements, messenger RNA changes and SILAC ratios correlated very well for pheromone pathway components. Systems-wide, precise quantification directly at the protein level opens up new perspectives in post-genomics and systems biology.
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            Evaluation of multidimensional chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS) for large-scale protein analysis: the yeast proteome.

            Highly complex protein mixtures can be directly analyzed after proteolysis by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). In this paper, we have utilized the combination of strong cation exchange (SCX) and reversed-phase (RP) chromatography to achieve two-dimensional separation prior to MS/MS. One milligram of whole yeast protein was proteolyzed and separated by SCX chromatography (2.1 mm i.d.) with fraction collection every minute during an 80-min elution. Eighty fractions were reduced in volume and then re-injected via an autosampler in an automated fashion using a vented-column (100 microm i.d.) approach for RP-LC-MS/MS analysis. More than 162,000 MS/MS spectra were collected with 26,815 matched to yeast peptides (7,537 unique peptides). A total of 1,504 yeast proteins were unambiguously identified in this single analysis. We present a comparison of this experiment with a previously published yeast proteome analysis by Yates and colleagues (Washburn, M. P.; Wolters, D.; Yates, J. R., III. Nat. Biotechnol. 2001, 19, 242-7). In addition, we report an in-depth analysis of the false-positive rates associated with peptide identification using the Sequest algorithm and a reversed yeast protein database. New criteria are proposed to decrease false-positives to less than 1% and to greatly reduce the need for manual interpretation while permitting more proteins to be identified.
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              Cannibalism by sporulating bacteria.

              Spore formation by the bacterium Bacillus subtilis is an elaborate developmental process that is triggered by nutrient limitation. Here we report that cells that have entered the pathway to sporulate produce and export a killing factor and a signaling protein that act cooperatively to block sister cells from sporulating and to cause them to lyse. The sporulating cells feed on the nutrients thereby released, which allows them to keep growing rather than to complete morphogenesis. We propose that sporulation is a stress-response pathway of last resort and that B. subtilis delays a commitment to spore formation by cannibalizing its siblings.
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                Author and article information

                Journal
                Nat Commun
                Nature Communications
                Nature Publishing Group
                2041-1723
                14 December 2010
                : 1
                : 137
                Affiliations
                [1 ]simpleErnst-Moritz-Arndt-Universität Greifswald, Institute for Microbiology , Greifswald 17487, Germany
                [2 ]simpleErnst-Moritz-Arndt-Universität Greifswald, Institute of Pharmacy , Greifswald 17487, Germany
                [3 ]simpleETH Zürich, Department of Biosystems Science and Engeneering, Mattenstrasse 26 , Basel CH-4058, Switzerland
                [4 ]simpleErnst-Moritz-Arndt-Universität Greifswald, Interfaculty Institute for Genetics and Functional Genomics , Greifswald 17487, Germany
                Author notes
                Article
                ncomms1137
                10.1038/ncomms1137
                3105300
                21266987
                2add4f33-1c1b-4e05-9917-53f3b9b558f8
                Copyright © 2010, Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.

                This work is licensed under a Creative Commons Attribution-NonCommercial-No Derivative Works 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/3.0/

                History
                : 16 March 2010
                : 15 November 2010
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