E 10 mL of fresh LB medium. To study the effect of fusaricidin on B. subtilis 168 cells and the corresponding transcriptomic profiles, fusaricidin (1.713 mg/mL) was added at an OD600 of approximately 1.30 at the exponential growth phase (7-h culture period). Two independently cultured replicates were performed, respectively. Samples were taken to measure the OD600 at designated time points (5, 20, and 170 min) and to extract RNA for the following experiments.MISP AnalysisThe differentially expressed genes chosen with a coefficient of variation .0.1 were distributed over the MIPS functional categories for their classification (http://mips.gsf.de/projects/).Results and Discussion B. subtilis 168 Cell Growth was Inhibited by FusaricidinChanges in cell growth (measured as change in cell concentration) were studied for 3.5 h after the addition of fusaricidin (,1 minimal inhibitory concentration [MIC], 1.713 mg/mL). As shown in Figure 1, the replicates of the cells MedChemExpress AKT inhibitor 2 treated with fusaricidin grew more slowly; by contrast, the replication of the control was continuous. This indicates that fusaricidin is toxic to B. subtilis 168. The influence of fusaricidin on the transcriptome of logarithmically growing B. subtilis cells was quantified using fluorescent DNA microarray technology. Changes in gene Pleuromutilin site expression were studied by the addition of fusaricidin. Samples were taken at 5, 20, and 170 min after the addition of fusaricidin and compared with an untreated control sample taken at 5 min. When a 3-fold change (p value log ratio ,0.05) relative to the control was used as a cutoff value, 18, 415, and 415 genes (approximately 0.44 , 10.11 , and 10.11 of all B. subtilis genes, respectively) were identified as significantly induced by fusaricidin at the respective time points.RNA Preparation and Microarray AnalysesThe cultures were grown to mid-log phase (OD600 of 1.30) and split into 2 flasks. One culture was treated with fusaricidin (1.6 mg/ mL), and the other was untreated as a control. In parallel, 2 independent array experiments from separate cultures with fusaricidin treatment were performed with biological duplicates. The B. subtilis cells were harvested at 5, 20, and 170 min after fusaricidin addition. RNA isolation and microarray analysis were performed as previously described for amino acid addition [9]. 23727046 All the microarray data reported in the manuscript are described in accordance 1326631 with the MIAME (Minimum Information About a Microarray Experiment) guidelines. A 3-fold change was used as the threshold for selection of fusaricidin-induced genes.Fusaricidin Rapidly Induced sW Regulon in B. subtilisIn this study, DNA microarrays were used to investigate the global transcriptional 
response to fusaricidin. ApproximatelyMechanisms of Fusaricidins to Bacillus subtilisFigure 4. The metabolic changes of carbon and nitrogen. The expression of genes related to the central carbon and nitrogen pathways are schematically presented. The 3 bars from left to right represent the fold changes of the gene expressions in response to the 3 time points (5, 20, and 170 min). The red bars represent an upregulation; the green bars, a downregulation; and the gray bars, the messages that did not significantly change relative to our cutoff (3-fold increase in expression). doi:10.1371/journal.pone.0050003.ggenes were found to be induced by 3-fold during the first 5 min of the fusaricidin treatment, including yqfB (3.1-fold), which codes for a hypothetical protein; sporulat.E 10 mL of fresh LB medium. To study the effect of fusaricidin on B. subtilis 168 cells and the corresponding transcriptomic profiles, fusaricidin (1.713 mg/mL) was added at an OD600 of approximately 1.30 at the exponential growth phase (7-h culture period). Two independently cultured replicates were performed, respectively. Samples were taken to measure the OD600 at designated time points (5, 20, and 170 min) and to extract RNA for the following experiments.MISP AnalysisThe differentially expressed genes chosen with a coefficient of variation .0.1 were distributed over the MIPS functional categories for their classification (http://mips.gsf.de/projects/).Results and Discussion B. subtilis 168 Cell Growth was Inhibited by FusaricidinChanges in cell growth (measured as change in cell concentration) were studied for 3.5 h after the addition of fusaricidin (,1 minimal inhibitory concentration [MIC], 1.713 mg/mL). As shown in Figure 1, the replicates of the cells treated with fusaricidin grew more slowly; by contrast, the replication of the control was continuous. This indicates that fusaricidin is toxic to B. subtilis 168. The influence of fusaricidin on the transcriptome of logarithmically growing B. subtilis cells was quantified using fluorescent DNA microarray technology. Changes in gene expression were studied by the addition of fusaricidin. Samples were taken at 5, 20, and 170 min after the addition of fusaricidin and compared with an untreated control sample taken at 5 min. When a 3-fold change (p value log ratio ,0.05) relative to the control was used as a cutoff value, 18, 415, and 415 genes (approximately 0.44 , 10.11 , and 10.11 of all B. subtilis genes, respectively) were identified as significantly induced by fusaricidin at the respective time points.RNA Preparation and Microarray AnalysesThe cultures were grown to mid-log phase (OD600 of 1.30) and split into 2 flasks. One culture was treated with fusaricidin (1.6 mg/ mL), and the other was untreated as a control. In parallel, 2 independent array experiments from separate cultures with 
fusaricidin treatment were performed with biological duplicates. The B. subtilis cells were harvested at 5, 20, and 170 min after fusaricidin addition. RNA isolation and microarray analysis were performed as previously described for amino acid addition [9]. 23727046 All the microarray data reported in the manuscript are described in accordance 1326631 with the MIAME (Minimum Information About a Microarray Experiment) guidelines. A 3-fold change was used as the threshold for selection of fusaricidin-induced genes.Fusaricidin Rapidly Induced sW Regulon in B. subtilisIn this study, DNA microarrays were used to investigate the global transcriptional response to fusaricidin. ApproximatelyMechanisms of Fusaricidins to Bacillus subtilisFigure 4. The metabolic changes of carbon and nitrogen. The expression of genes related to the central carbon and nitrogen pathways are schematically presented. The 3 bars from left to right represent the fold changes of the gene expressions in response to the 3 time points (5, 20, and 170 min). The red bars represent an upregulation; the green bars, a downregulation; and the gray bars, the messages that did not significantly change relative to our cutoff (3-fold increase in expression). doi:10.1371/journal.pone.0050003.ggenes were found to be induced by 3-fold during the first 5 min of the fusaricidin treatment, including yqfB (3.1-fold), which codes for a hypothetical protein; sporulat.