12 research outputs found
Analyses of competent and non-competent subpopulations of Bacillus subtilis reveal yhfW, yhxC and ncRNAs as novel players in competence
Upon competence-inducing nutrient-limited conditions, only part of the Bacillus subtilis population becomes competent. Here, we separated the two subpopulations by fluorescence-assisted cell sorting (FACS). Using RNA-seq, we confirmed the previously described ComK regulon. We also found for the first time significantly downregulated genes in the competent subpopulation. The downregulated genes are not under direct control by ComK but have higher levels of corresponding antisense RNAs in the competent subpopulation. During competence, cell division and replication are halted. By investigating the proteome during competence, we found higher levels of the regulators of cell division, MinD and Noc. The exonucleases SbcC and SbcD were also primarily regulated at the post-transcriptional level. In the competent subpopulation, yhfW was newly identified as being highly upregulated. Its absence reduces the expression of comG, and has a modest, but statistically significant effect on the expression of comK. Although expression of yhfW is higher in the competent subpopulation, no ComK-binding site is present in its promoter region. Mutants of yhfW have a small but significant defect in transformation. Metabolomic analyses revealed significant reductions in tricarboxylic acid (TCA) cycle metabolites and several amino acids in a ÎyhfW mutant. RNA-seq analysis of ÎyhfW revealed higher expression of the NAD synthesis genes nadA, nadB and nadC
Multi-omics based characterization of various stress responses in Bacillus subtilis
The soil bacterium Bacillus subtilis is capable of surviving most of the ensuing environmental stress conditions. The dynamic nature of the soil habitat is manifested with varying amounts of nutrients, frequent flooding, drying and variation of other growth parameters like temperature, acidity, aeration etc. In order to survive in these conditions, B. subtilis has evolved to employ very complex adaptational responses. These adaptational responses are often multi-faceted; hence comprehensive understanding of the adaptational responses requires generation and integration of data on multi-omics level. Hence, multi-omics based detailed analysis was performed for the molecules involved in the central carbon metabolism (CCM) and proline biosynthesis pathway. In the current study two major stress conditions were extensively investigated: 1) energy limitation/starvation which is achieved by limiting glucose in the growth medium, 2) osmostress resulting from frequent drying out of soil which is simulated by adding 1.2 M NaCl to the growth medium. In addition to osmostress, the naturally available osmoprotectant glycine betaine (GB) was supplemented to understand the simultaneous influence of osmostress and osmoprotection on cellular physiology. To measure absolute protein abundances by mass spectrometry, a targeted approach (SRM âsingle reaction monitoring) using stable heavy isotope labeled artificial standard proteins known as QconCATs was optimized and implemented in the current study. The SRM technique in combination with QconCAT provided absolute quantitative data with high dynamic range for the 45 targeted CCM proteins. Transcriptome data was obtained from microarray analysis. The resulting data were integrated with the other omics data sets obtained by metabolome and flux analysis. As part of a joint study conducted by the BaCell-SysMO and BaSysBio consortia which aimed for the genome wide mapping of transcription units and previously unannotated RNAs of B. subtilis by means of tiling array hybridizations, mRNA samples from growth at high and low temperatures (51°C and 16°C) and in the presence of 1.2 M NaCl, shake flask experiments during transition from exponential growth to the stationary phase, and high density batch fermentation. Time course analysis of B. subtilis transitioning from exponential to stationary phase was investigated by high cell density fed-batch fermentation (glucose limitation) and batch fermentation (glucose exhaustion) with glucose as a limiting factor. A multi-omics analysis of the CCM for the batch fermentation was performed and the time course data was integrated and visualized. In conclusion, pathway based multi-omics data were generated, integrated and visualized as a prerequisite for systems biology approaches and for a better understanding of the complex adaptational responses of B. subtilis.Der Gram-positive Bodenorganismus Bacillus subtilis muss in seinem natĂŒrlichen Habitat, dem Boden, eine Vielzahl von Stressbedingungen ĂŒberleben. Um diese Anpassungen zu verstehen wurde in dieser Arbeit eine multi-omics Analyse mit Fokus auf den zentralen Kohlenstoff Metabolismus durchgefĂŒhrt. Hierbei wurde insbesondere die Anpassung an hohe OsmolaritĂ€ten unter natĂŒrlichen, Glukose limitierten, und Laborbedingungen, mit ausreichend vorhandener Glukose, betrachtet. Weiterhin wurde die Auswirkung des kompatiblen Solutes Glycin-Betain betrachtet. Neben der Transkiptomanalyse mitteln Microarrays und der absolute Quantifizierung der Proteine mittels LC-ESI-MS/MS, wurden stöchiometrische Daten der Proteinmengen der Proteine des zentralen Kohlenstoff Metabolismus und der Enzyme der Prolin Biosynthese erhoben. Hierzu wurden vier QconCAT Proteine (Quantification Concatemer) mit Peptiden der Zielproteine entworfen und als interne Standardproteine in einer SRM (Single Reaction Monitoring) Analyse eingesetzt. Zusammenfassend wurden die DatensĂ€tze aus Transkriptom, Proteom and Metabolom integriert und mit den ebenfalls erhobenen und Flux-Analysen verglichen
Brain Derived Neurotrophic Factor Contributes to the Cardiogenic Potential of Adult Resident Progenitor Cells in Failing Murine Heart
<div><p>Aims</p><p>Resident cardiac progenitor cells show homing properties when injected into the injured but not to the healthy myocardium. The molecular background behind this difference in behavior needs to be studied to elucidate how adult progenitor cells can restore cardiac function of the damaged myocardium. Since the brain derived neurotrophic factor (BDNF) moderates cardioprotection in injured hearts, we focused on delineating its regulatory role in the damaged myocardium.</p><p>Methods and Results</p><p>Comparative gene expression profiling of freshly isolated undifferentiated Sca-1 progenitor cells derived either from heart failure transgenic αMHC-CyclinT1/Gαq overexpressing mice or wildtype littermates revealed transcriptional variations. Bdnf expression was up regulated 5-fold during heart failure which was verified by qRT-PCR and confirmed at protein level. The migratory capacity of Sca-1 cells from transgenic hearts was improved by 15% in the presence of 25ng/ml BDNF. Furthermore, BDNF-mediated effects on Sca-1 cells were studied via pulsed Stable Isotope Labeling of Amino acids in Cell Culture (pSILAC) proteomics approach. After BDNF treatment significant differences between newly synthesized proteins in Sca-1 cells from control and transgenic hearts were observed for CDK1, SRRT, HDGF, and MAP2K3 which are known to regulate cell cycle, survival and differentiation. Moreover BDNF repressed the proliferation of Sca-1 cells from transgenic hearts.</p><p>Conclusion</p><p>Comparative profiling of resident Sca-1 cells revealed elevated BDNF levels in the failing heart. Exogenous BDNF (i) stimulated migration, which might improve the homing ability of Sca-1 cells derived from the failing heart and (ii) repressed the cell cycle progression suggesting its potency to ameliorate heart failure.</p></div
pSILAC analysis of BDNF induced changes in protein synthesis.
<p>(A) Schematic representation of pulsed SILAC analysis. Sca-1 cells were cultured for 8days in light medium (red) followed by exchange to medium with heavy lysine and arginine (blue) containing 25ng/ml BDNF or 400nM K252a or a combination of both and allowed to grow for 24 hours. Cell lysates were digested and then subjected to mass spectrometric analysis. Newly synthesized proteins were determined based on H/L ratios. (B) Comparison of BDNF induced changes in the protein synthesis of Cyc and Wt cells (n = 3). Majority of proteins showed moderate change in protein synthesis depicted as grey spots; those identified with statistical significance (<i>p</i> < 0.05) are highlighted in black while only differentially regulated proteins in Cyc and Wt cells (<i>p</i> < 0.05, log2 ratio ℠±0.3) are highlighted in red and white spots respectively. (C) Venn diagram illustrates the overlap of displays protein numbers with altered synthesis rate as a result of BDNF treatment (BDNF vs Co, <i>p</i> < 0.05) in both groups of cells.</p
Expression of BDNF and receptor TrkB on progenitor cells.
<p>(A) Immunoblot data depicted higher expression of BDNF in freshly isolated Cyc cells in comparison to Wt. Bar graph indicates relative protein abundance of BDNF normalized to Ă-actin control quantified by densitometry (n = 3; mean ± SD; *<i>p</i><0.05; t-test). (B) mRNA intensity signals of TrkB receptor determined by microarray analysis (n = 2; mean ± SD). (C-D) Immunofluorescence micrographs display the expression of TrkB receptor on Sca-1 cells. Nuclei were stained with DAPI. (Scale bar: 50ÎŒm, image magnification: 40x). (E) Protein abundance of TrkB receptor on Sca-1 cells quantified against Ă-actin control by densitometry (n = 3, mean ± SD). Representative immunoblot of both full length and truncated forms of TrkB receptor on Wt and Cyc cells is shown below.</p
BDNF enhances migration of progenitor cells.
<p>Migration of Sca-1 cells (4 x 10<sup>4</sup>) was evaluated using a modified Boyden chamber assay. Freshly isolated cells were exposed to varying concentrations of BDNF (10, 25, 50ng/ml) and allowed to migrate for 2 hours (n = 3; mean ± SD; ANOVA; ***<i>p</i> < 0.0001, **<i>p</i> < 0.001, *<i>p</i> < 0.05 vs Control; §§<i>p</i> < 0.001, §<i>p</i> < 0.05 vs BDNF).</p
Gene expression alterations in Sca-1 cells in response to heart failure.
<p>(A) Relative fold change in expression of genes associated with heart failure in Cyc compared to Wt cells. (B) Top canonical pathways overrepresented by differentially expressed genes in Cyc vs Wt cells.âlog <i>p-</i>value displays the significance of association dependent of the number of genes in the class calculated by Fisherâs exact test in IPA. Numbers of up-regulated/down-regulated genes are indicated above each bar. (C) Biological functions predicted to be differentially affected based on the differentially expressed genes in Cyc vs Wt cells based on activation z-score calculated in IPA. Black dots denote functional activation (z-score â„ 1.5) and grey dots functional inhibition (z-score †â1.5). (D) Validation of microarray results by quantitative RT-PCR (n = 3; <i>p</i><0.05; ANOVA).</p
K-means clustering of newly synthesized proteins.
<p>K-means clustering of differentially regulated proteins under different treatment conditions compared to their respective controls. (A) represents BDNF induced up-regulation in protein synthesis, while (B) represents down regulation. Top regulated proteins in response to BDNF treatment are specified in each cluster (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120360#pone.0120360.s010" target="_blank">S5 Table</a><i>)</i>.</p
Global secretome analysis of resident cardiac progenitor cells from wildâtype and transgenic heart failure mice: Why ambience matters
Large-scale reduction of the <em>Bacillus subtilis</em> genome: consequences for the transcriptional network, resource allocation, and metabolism
International audienceUnderstanding cellular life requires a comprehensive knowledge of the essential cellular functions, the components involved, and their interactions. Minimized genomes are an important tool to gain this knowledge. We have constructed strains of the model bacterium, Bacillus subtilis, whose genomes have been reduced by similar to ~ 36%. These strains are fully viable, and their growth rates in complex medium are comparable to those of wild type strains. An in-depth multi-omics analysis of the genome reduced strains revealed how the deletions affect the transcription regulatory network of the cell, translation resource allocation, and metabolism. A comparison of gene counts and resource allocation demonstrates drastic differences in the two parameters, with 50% of the genes using as little as 10% of translation capacity, whereas the 6% essential genes require 57% of the translation resources. Taken together, the results are a valuable resource on gene dispensability in B. subtilis, and they suggest the roads to further genome reduction to approach the final aim of a minimal cell in which all functions are understood