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The Role of α-CTD in the Genome-Wide Transcriptional Regulation of the Bacillus subtilis Cells.

Murayama S, Ishikawa S, Chumsakul O, Ogasawara N, Oshima T - PLoS ONE (2015)

Bottom Line: Transcriptomic and ChAP-chip analyses revealed that α-CTD deficiency reduced the transcription and RNAP binding of genes related to the utilization of secondary carbon sources, transition state responses, and ribosome synthesis.In E. coli, it is known that α-CTD also contributes to the expression of genes related to the utilization of secondary carbon sources and ribosome synthesis.Our results suggest that the biological importance of α-CTD is conserved in B. subtilis and E. coli, but that its specific roles have diversified between these two bacteria.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5, Takayama, Ikoma, Nara 630-0192, Japan.

ABSTRACT
The amino acid sequence of the RNA polymerase (RNAP) α-subunit is well conserved throughout the Eubacteria. Its C-terminal domain (α-CTD) is important for the transcriptional regulation of specific promoters in both Escherichia coli and Bacillus subtilis, through interactions with transcription factors and/or a DNA element called the "UP element". However, there is only limited information regarding the α-CTD regulated genes in B. subtilis and the importance of this subunit in the transcriptional regulation of B. subtilis. Here, we established strains and the growth conditions in which the α-subunit of RNAP was replaced with a C-terminally truncated version. Transcriptomic and ChAP-chip analyses revealed that α-CTD deficiency reduced the transcription and RNAP binding of genes related to the utilization of secondary carbon sources, transition state responses, and ribosome synthesis. In E. coli, it is known that α-CTD also contributes to the expression of genes related to the utilization of secondary carbon sources and ribosome synthesis. Our results suggest that the biological importance of α-CTD is conserved in B. subtilis and E. coli, but that its specific roles have diversified between these two bacteria.

No MeSH data available.


Related in: MedlinePlus

Transcriptome analysis of rpoAint-expressing cells (SMS08) and rpoAdel-expressing cells (SMS09).A scatter plot (log2 scale) of the transcriptional signal intensity (averaged from duplicate experiments) of each gene in rpoAdel-expressing cells (vertical axis) versus rpoAint-expressing cells (horizontal axis) at 0 hour (A) and at 1 hour (B), 2 hours (C) and 3 hours (D) after the beginning of the RpoAdel induction. The correlation coefficients between the transcriptomes of rpoAint and rpoAdel-expressing cells are indicated as (r) in each panel. The average signal intensities from two independent experiments are plotted. For each gene plotted, the sum of the signal intensities for all experiments performed at the same time point (two experiments each for rpoAint- and rpoAdel-expressing cells) was > 400; this avoided the inclusion of minimally expressed genes in our analysis. We analyzed a total of 2755 (0 hour), 2855 (1 hour), 2921 (2 hour) and 2956 (3 hour) genes. The genes found to be down-regulated in rpoAdel-expressing cells compared to rpoAint-expressing cells at 3 hours after the beginning of the RpoAdel induction are shown as blue dots, and the up-regulated genes are shown by red dots.
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pone.0131588.g003: Transcriptome analysis of rpoAint-expressing cells (SMS08) and rpoAdel-expressing cells (SMS09).A scatter plot (log2 scale) of the transcriptional signal intensity (averaged from duplicate experiments) of each gene in rpoAdel-expressing cells (vertical axis) versus rpoAint-expressing cells (horizontal axis) at 0 hour (A) and at 1 hour (B), 2 hours (C) and 3 hours (D) after the beginning of the RpoAdel induction. The correlation coefficients between the transcriptomes of rpoAint and rpoAdel-expressing cells are indicated as (r) in each panel. The average signal intensities from two independent experiments are plotted. For each gene plotted, the sum of the signal intensities for all experiments performed at the same time point (two experiments each for rpoAint- and rpoAdel-expressing cells) was > 400; this avoided the inclusion of minimally expressed genes in our analysis. We analyzed a total of 2755 (0 hour), 2855 (1 hour), 2921 (2 hour) and 2956 (3 hour) genes. The genes found to be down-regulated in rpoAdel-expressing cells compared to rpoAint-expressing cells at 3 hours after the beginning of the RpoAdel induction are shown as blue dots, and the up-regulated genes are shown by red dots.

Mentions: The differences between the transcriptomes of rpoAint-expressing cells and rpoAdel-expressing cells gradually increased during their cultivation in LBxyl. The correlation coefficient, therefore, gradually decreased over the same period (Fig 3). To identify the genes whose expression levels were affected by α-CTD deficiency, we extracted data for genes whose expression levels differed by > 4- or < -0.25 fold (> 2 or < -2 in log2 scale) in the rpoAdel-expressing cells compared to the rpoAint-expressing cells and which had a false discovery rate (q-value) of < 0.2. After 3 hours cultivation, we identified 53 down-regulated genes and 27 up-regulated genes differentially expressed between the rpoAint-expressing cells (SMS08) and the rpoAdel-expressing cells (SMS09) (S5 Table). Amongst them, there were 20 down- and 5 up-regulated genes that were also differentially expressed after 2 hours of cultivation (S2 Table). At 2 hours, we identified four additional down-regulated genes (yhfH, yhzC, ykbA and yokG) and one additional up-regulated gene (ybcP) in the rpoAdel-expressing cells. At 3 hours, these genes were excluded from the list of differentially expressed genes for the following reasons: yhzC showed a low expression level; yhfH had a high q-value (log2 fold = -4.13, q = 0.201); ykbA and yokG had moderate fold changes or a high q-value (ykbA, log2 fold = -1.95, q = 0.14; yokG, log2 fold = -2.47, q = 0.24); and ybcP had a high q-value (log2 fold = 2.64, q = 0.24). In general, however, the up- and down-regulated genes overlapped at 2 and 3 hours. At 0 and 1 hour, no up- or down-regulation was identified by the criteria used in this study. We detected the known down-regulation of the srf operon that was previously associated with α-CTD deficiency [20], but not the known down-regulation of flagellar genes, hag, fliD and motA [25,26]. These results might indicate that the transcriptional regulation of hag, fliD and motA have more factors affecting them than simple UP element dependent regulation.


The Role of α-CTD in the Genome-Wide Transcriptional Regulation of the Bacillus subtilis Cells.

Murayama S, Ishikawa S, Chumsakul O, Ogasawara N, Oshima T - PLoS ONE (2015)

Transcriptome analysis of rpoAint-expressing cells (SMS08) and rpoAdel-expressing cells (SMS09).A scatter plot (log2 scale) of the transcriptional signal intensity (averaged from duplicate experiments) of each gene in rpoAdel-expressing cells (vertical axis) versus rpoAint-expressing cells (horizontal axis) at 0 hour (A) and at 1 hour (B), 2 hours (C) and 3 hours (D) after the beginning of the RpoAdel induction. The correlation coefficients between the transcriptomes of rpoAint and rpoAdel-expressing cells are indicated as (r) in each panel. The average signal intensities from two independent experiments are plotted. For each gene plotted, the sum of the signal intensities for all experiments performed at the same time point (two experiments each for rpoAint- and rpoAdel-expressing cells) was > 400; this avoided the inclusion of minimally expressed genes in our analysis. We analyzed a total of 2755 (0 hour), 2855 (1 hour), 2921 (2 hour) and 2956 (3 hour) genes. The genes found to be down-regulated in rpoAdel-expressing cells compared to rpoAint-expressing cells at 3 hours after the beginning of the RpoAdel induction are shown as blue dots, and the up-regulated genes are shown by red dots.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4495994&req=5

pone.0131588.g003: Transcriptome analysis of rpoAint-expressing cells (SMS08) and rpoAdel-expressing cells (SMS09).A scatter plot (log2 scale) of the transcriptional signal intensity (averaged from duplicate experiments) of each gene in rpoAdel-expressing cells (vertical axis) versus rpoAint-expressing cells (horizontal axis) at 0 hour (A) and at 1 hour (B), 2 hours (C) and 3 hours (D) after the beginning of the RpoAdel induction. The correlation coefficients between the transcriptomes of rpoAint and rpoAdel-expressing cells are indicated as (r) in each panel. The average signal intensities from two independent experiments are plotted. For each gene plotted, the sum of the signal intensities for all experiments performed at the same time point (two experiments each for rpoAint- and rpoAdel-expressing cells) was > 400; this avoided the inclusion of minimally expressed genes in our analysis. We analyzed a total of 2755 (0 hour), 2855 (1 hour), 2921 (2 hour) and 2956 (3 hour) genes. The genes found to be down-regulated in rpoAdel-expressing cells compared to rpoAint-expressing cells at 3 hours after the beginning of the RpoAdel induction are shown as blue dots, and the up-regulated genes are shown by red dots.
Mentions: The differences between the transcriptomes of rpoAint-expressing cells and rpoAdel-expressing cells gradually increased during their cultivation in LBxyl. The correlation coefficient, therefore, gradually decreased over the same period (Fig 3). To identify the genes whose expression levels were affected by α-CTD deficiency, we extracted data for genes whose expression levels differed by > 4- or < -0.25 fold (> 2 or < -2 in log2 scale) in the rpoAdel-expressing cells compared to the rpoAint-expressing cells and which had a false discovery rate (q-value) of < 0.2. After 3 hours cultivation, we identified 53 down-regulated genes and 27 up-regulated genes differentially expressed between the rpoAint-expressing cells (SMS08) and the rpoAdel-expressing cells (SMS09) (S5 Table). Amongst them, there were 20 down- and 5 up-regulated genes that were also differentially expressed after 2 hours of cultivation (S2 Table). At 2 hours, we identified four additional down-regulated genes (yhfH, yhzC, ykbA and yokG) and one additional up-regulated gene (ybcP) in the rpoAdel-expressing cells. At 3 hours, these genes were excluded from the list of differentially expressed genes for the following reasons: yhzC showed a low expression level; yhfH had a high q-value (log2 fold = -4.13, q = 0.201); ykbA and yokG had moderate fold changes or a high q-value (ykbA, log2 fold = -1.95, q = 0.14; yokG, log2 fold = -2.47, q = 0.24); and ybcP had a high q-value (log2 fold = 2.64, q = 0.24). In general, however, the up- and down-regulated genes overlapped at 2 and 3 hours. At 0 and 1 hour, no up- or down-regulation was identified by the criteria used in this study. We detected the known down-regulation of the srf operon that was previously associated with α-CTD deficiency [20], but not the known down-regulation of flagellar genes, hag, fliD and motA [25,26]. These results might indicate that the transcriptional regulation of hag, fliD and motA have more factors affecting them than simple UP element dependent regulation.

Bottom Line: Transcriptomic and ChAP-chip analyses revealed that α-CTD deficiency reduced the transcription and RNAP binding of genes related to the utilization of secondary carbon sources, transition state responses, and ribosome synthesis.In E. coli, it is known that α-CTD also contributes to the expression of genes related to the utilization of secondary carbon sources and ribosome synthesis.Our results suggest that the biological importance of α-CTD is conserved in B. subtilis and E. coli, but that its specific roles have diversified between these two bacteria.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5, Takayama, Ikoma, Nara 630-0192, Japan.

ABSTRACT
The amino acid sequence of the RNA polymerase (RNAP) α-subunit is well conserved throughout the Eubacteria. Its C-terminal domain (α-CTD) is important for the transcriptional regulation of specific promoters in both Escherichia coli and Bacillus subtilis, through interactions with transcription factors and/or a DNA element called the "UP element". However, there is only limited information regarding the α-CTD regulated genes in B. subtilis and the importance of this subunit in the transcriptional regulation of B. subtilis. Here, we established strains and the growth conditions in which the α-subunit of RNAP was replaced with a C-terminally truncated version. Transcriptomic and ChAP-chip analyses revealed that α-CTD deficiency reduced the transcription and RNAP binding of genes related to the utilization of secondary carbon sources, transition state responses, and ribosome synthesis. In E. coli, it is known that α-CTD also contributes to the expression of genes related to the utilization of secondary carbon sources and ribosome synthesis. Our results suggest that the biological importance of α-CTD is conserved in B. subtilis and E. coli, but that its specific roles have diversified between these two bacteria.

No MeSH data available.


Related in: MedlinePlus