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Regulation of 6S RNA by pRNA synthesis is required for efficient recovery from stationary phase in E. coli and B. subtilis.

Cavanagh AT, Sperger JM, Wassarman KM - Nucleic Acids Res. (2011)

Bottom Line: Intriguingly, 6S-2 RNA does not direct pRNA synthesis under physiological conditions and its non-release from Eσ(A) prevents efficient outgrowth in cells lacking 6S-1 RNA.The behavioral differences in the two B. subtilis RNAs clearly demonstrate that they act independently, revealing a higher than anticipated diversity in 6S RNA function globally.Overexpression of a pRNA-synthesis-defective 6S RNA in E. coli leads to decreased cell viability, suggesting pRNA synthesis-mediated regulation of 6S RNA function is important at other times of growth as well.

View Article: PubMed Central - PubMed

Affiliation: Department of Bacteriology, University of Wisconsin-Madison, Madison, WI 53706, USA.

ABSTRACT
6S RNAs function through interaction with housekeeping forms of RNA polymerase holoenzyme (Eσ(70) in Escherichia coli, Eσ(A) in Bacillus subtilis). Escherichia coli 6S RNA accumulates to high levels during stationary phase, and has been shown to be released from Eσ(70) during exit from stationary phase by a process in which 6S RNA serves as a template for Eσ(70) to generate product RNAs (pRNAs). Here, we demonstrate that not only does pRNA synthesis occur, but it is an important mechanism for regulation of 6S RNA function that is required for cells to exit stationary phase efficiently in both E. coli and B. subtilis. Bacillus subtilis has two 6S RNAs, 6S-1 and 6S-2. Intriguingly, 6S-2 RNA does not direct pRNA synthesis under physiological conditions and its non-release from Eσ(A) prevents efficient outgrowth in cells lacking 6S-1 RNA. The behavioral differences in the two B. subtilis RNAs clearly demonstrate that they act independently, revealing a higher than anticipated diversity in 6S RNA function globally. Overexpression of a pRNA-synthesis-defective 6S RNA in E. coli leads to decreased cell viability, suggesting pRNA synthesis-mediated regulation of 6S RNA function is important at other times of growth as well.

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Related in: MedlinePlus

Escherichia coli cells overexpressing 6S(M68) RNA have decreased viability in stationary phase compared to cells overexpressing wild-type 6S RNA. Viability of E. coli ssrS1 cells containing pKK-6S+Y (left panel) or pKK-6S+Y(M68) was monitored by spot titration from 0 to 24 h after growth in culture was initiated from a suspension of cells scraped off agar plates. Cultures were serially diluted 1:10 and 10 µl of each dilution was spotted onto LB plates. The red boxes surrounding 10-4 dilutions are to facilitate comparison. Experiments were done with at least three cultures per cell type. Results from matching cell types were very similar; a representative set is shown here.
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gkr1003-F8: Escherichia coli cells overexpressing 6S(M68) RNA have decreased viability in stationary phase compared to cells overexpressing wild-type 6S RNA. Viability of E. coli ssrS1 cells containing pKK-6S+Y (left panel) or pKK-6S+Y(M68) was monitored by spot titration from 0 to 24 h after growth in culture was initiated from a suspension of cells scraped off agar plates. Cultures were serially diluted 1:10 and 10 µl of each dilution was spotted onto LB plates. The red boxes surrounding 10-4 dilutions are to facilitate comparison. Experiments were done with at least three cultures per cell type. Results from matching cell types were very similar; a representative set is shown here.

Mentions: To check that the OD600 readings were representative of viable cell numbers in the starting cultures, the diluted stationary phase cells (the initial 0.3 OD dilutions) were serially diluted in 1× M9 salts and 10 µl of each dilution was spotted onto LB agar plates. The extent of growth was assessed in each spot after overnight incubation at 30°C. With the exception of E. coli cells containing pKK-6S+Y(M68) (Figure 8), 18 h overnight cultures gave similar OD600 readings (within 15% of wild-type) and normalized dilutions used for outgrowth assays had viable cell counts within ~2-fold for each experiment. For cells containing plasmids, growth was compared on plates with and without antibiotic to test for plasmid loss. No significant plasmid loss was observed in most experiments; results from experiments showing any detectable plasmid loss were discarded.


Regulation of 6S RNA by pRNA synthesis is required for efficient recovery from stationary phase in E. coli and B. subtilis.

Cavanagh AT, Sperger JM, Wassarman KM - Nucleic Acids Res. (2011)

Escherichia coli cells overexpressing 6S(M68) RNA have decreased viability in stationary phase compared to cells overexpressing wild-type 6S RNA. Viability of E. coli ssrS1 cells containing pKK-6S+Y (left panel) or pKK-6S+Y(M68) was monitored by spot titration from 0 to 24 h after growth in culture was initiated from a suspension of cells scraped off agar plates. Cultures were serially diluted 1:10 and 10 µl of each dilution was spotted onto LB plates. The red boxes surrounding 10-4 dilutions are to facilitate comparison. Experiments were done with at least three cultures per cell type. Results from matching cell types were very similar; a representative set is shown here.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3299989&req=5

gkr1003-F8: Escherichia coli cells overexpressing 6S(M68) RNA have decreased viability in stationary phase compared to cells overexpressing wild-type 6S RNA. Viability of E. coli ssrS1 cells containing pKK-6S+Y (left panel) or pKK-6S+Y(M68) was monitored by spot titration from 0 to 24 h after growth in culture was initiated from a suspension of cells scraped off agar plates. Cultures were serially diluted 1:10 and 10 µl of each dilution was spotted onto LB plates. The red boxes surrounding 10-4 dilutions are to facilitate comparison. Experiments were done with at least three cultures per cell type. Results from matching cell types were very similar; a representative set is shown here.
Mentions: To check that the OD600 readings were representative of viable cell numbers in the starting cultures, the diluted stationary phase cells (the initial 0.3 OD dilutions) were serially diluted in 1× M9 salts and 10 µl of each dilution was spotted onto LB agar plates. The extent of growth was assessed in each spot after overnight incubation at 30°C. With the exception of E. coli cells containing pKK-6S+Y(M68) (Figure 8), 18 h overnight cultures gave similar OD600 readings (within 15% of wild-type) and normalized dilutions used for outgrowth assays had viable cell counts within ~2-fold for each experiment. For cells containing plasmids, growth was compared on plates with and without antibiotic to test for plasmid loss. No significant plasmid loss was observed in most experiments; results from experiments showing any detectable plasmid loss were discarded.

Bottom Line: Intriguingly, 6S-2 RNA does not direct pRNA synthesis under physiological conditions and its non-release from Eσ(A) prevents efficient outgrowth in cells lacking 6S-1 RNA.The behavioral differences in the two B. subtilis RNAs clearly demonstrate that they act independently, revealing a higher than anticipated diversity in 6S RNA function globally.Overexpression of a pRNA-synthesis-defective 6S RNA in E. coli leads to decreased cell viability, suggesting pRNA synthesis-mediated regulation of 6S RNA function is important at other times of growth as well.

View Article: PubMed Central - PubMed

Affiliation: Department of Bacteriology, University of Wisconsin-Madison, Madison, WI 53706, USA.

ABSTRACT
6S RNAs function through interaction with housekeeping forms of RNA polymerase holoenzyme (Eσ(70) in Escherichia coli, Eσ(A) in Bacillus subtilis). Escherichia coli 6S RNA accumulates to high levels during stationary phase, and has been shown to be released from Eσ(70) during exit from stationary phase by a process in which 6S RNA serves as a template for Eσ(70) to generate product RNAs (pRNAs). Here, we demonstrate that not only does pRNA synthesis occur, but it is an important mechanism for regulation of 6S RNA function that is required for cells to exit stationary phase efficiently in both E. coli and B. subtilis. Bacillus subtilis has two 6S RNAs, 6S-1 and 6S-2. Intriguingly, 6S-2 RNA does not direct pRNA synthesis under physiological conditions and its non-release from Eσ(A) prevents efficient outgrowth in cells lacking 6S-1 RNA. The behavioral differences in the two B. subtilis RNAs clearly demonstrate that they act independently, revealing a higher than anticipated diversity in 6S RNA function globally. Overexpression of a pRNA-synthesis-defective 6S RNA in E. coli leads to decreased cell viability, suggesting pRNA synthesis-mediated regulation of 6S RNA function is important at other times of growth as well.

Show MeSH
Related in: MedlinePlus