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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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Bacillus subtilis cells expressing 6S-2 RNA but lacking 6S-1 RNA are delayed in outgrowth from stationary phase compared to wild-type cells. Growth of B. subtilis cells as monitored by optical density at 595 nm (OD595) in an absorbance plate reader after stationary phase cells were diluted ~1:500 into 2× YT medium. Data shown are averages of three independent experiments with three biological replicates per experiment. Error bars correspond to ± standard deviations from the averages. (A) Comparing growth of B. subtilis wild-type 168 (red; KW586), ΔbsrA (green; KW587), ΔbsrB (orange; KW589) or ΔbsrAΔbsrB (blue; KW590) cells. (B) Comparing growth of B. subtilis ΔbsrA cells (KW587) containing plasmids pSP-Bs6S-1 (red), pSP-Bs6S-1(M6) (blue), pSP-Bs6S-2 (green) or pSP-Bs6S-2(M6) (purple). (C) Comparing growth of B. subtilis ΔbsrAΔbsrB cells (KW590) containing plasmids pSP-Bs6S-1 (red), pSP-Bs6S-1(M6) (blue), pSP-Bs6S-2 (green) or pSP-Bs6S-2(M6) (purple).
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gkr1003-F2: Bacillus subtilis cells expressing 6S-2 RNA but lacking 6S-1 RNA are delayed in outgrowth from stationary phase compared to wild-type cells. Growth of B. subtilis cells as monitored by optical density at 595 nm (OD595) in an absorbance plate reader after stationary phase cells were diluted ~1:500 into 2× YT medium. Data shown are averages of three independent experiments with three biological replicates per experiment. Error bars correspond to ± standard deviations from the averages. (A) Comparing growth of B. subtilis wild-type 168 (red; KW586), ΔbsrA (green; KW587), ΔbsrB (orange; KW589) or ΔbsrAΔbsrB (blue; KW590) cells. (B) Comparing growth of B. subtilis ΔbsrA cells (KW587) containing plasmids pSP-Bs6S-1 (red), pSP-Bs6S-1(M6) (blue), pSP-Bs6S-2 (green) or pSP-Bs6S-2(M6) (purple). (C) Comparing growth of B. subtilis ΔbsrAΔbsrB cells (KW590) containing plasmids pSP-Bs6S-1 (red), pSP-Bs6S-1(M6) (blue), pSP-Bs6S-2 (green) or pSP-Bs6S-2(M6) (purple).

Mentions: Growth experiments using B. subtilis strains lacking 6S-1 RNA (ΔbsrA; KW587), 6S-2 RNA (ΔbsrB; KW589) or both (ΔbsrAΔbsrB; KW590) revealed that cells lacking 6S-1 RNA were delayed in outgrowth from stationary phase. For example, when stationary phase cells were diluted ~1:500 into fresh medium, cells lacking 6S-1 RNA were delayed for entry into exponential phase by 50 min compared to wild-type cells or cells lacking 6S-2 RNA (Figure 2A). The delay appeared to result primarily from an increase in lag time; once the ΔbsrA cells entered exponential phase, they grew with a similar doubling time to wild-type cells and obtained a similar maximal cell density in stationary phase. Intriguingly, cells lacking both 6S-1 and 6S-2 RNAs (ΔbsrAΔbsrB) were not delayed in outgrowth, suggesting it is the presence of 6S-2 RNA in the absence of 6S-1 RNA that is required for the outgrowth delay.Figure 2.


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)

Bacillus subtilis cells expressing 6S-2 RNA but lacking 6S-1 RNA are delayed in outgrowth from stationary phase compared to wild-type cells. Growth of B. subtilis cells as monitored by optical density at 595 nm (OD595) in an absorbance plate reader after stationary phase cells were diluted ~1:500 into 2× YT medium. Data shown are averages of three independent experiments with three biological replicates per experiment. Error bars correspond to ± standard deviations from the averages. (A) Comparing growth of B. subtilis wild-type 168 (red; KW586), ΔbsrA (green; KW587), ΔbsrB (orange; KW589) or ΔbsrAΔbsrB (blue; KW590) cells. (B) Comparing growth of B. subtilis ΔbsrA cells (KW587) containing plasmids pSP-Bs6S-1 (red), pSP-Bs6S-1(M6) (blue), pSP-Bs6S-2 (green) or pSP-Bs6S-2(M6) (purple). (C) Comparing growth of B. subtilis ΔbsrAΔbsrB cells (KW590) containing plasmids pSP-Bs6S-1 (red), pSP-Bs6S-1(M6) (blue), pSP-Bs6S-2 (green) or pSP-Bs6S-2(M6) (purple).
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gkr1003-F2: Bacillus subtilis cells expressing 6S-2 RNA but lacking 6S-1 RNA are delayed in outgrowth from stationary phase compared to wild-type cells. Growth of B. subtilis cells as monitored by optical density at 595 nm (OD595) in an absorbance plate reader after stationary phase cells were diluted ~1:500 into 2× YT medium. Data shown are averages of three independent experiments with three biological replicates per experiment. Error bars correspond to ± standard deviations from the averages. (A) Comparing growth of B. subtilis wild-type 168 (red; KW586), ΔbsrA (green; KW587), ΔbsrB (orange; KW589) or ΔbsrAΔbsrB (blue; KW590) cells. (B) Comparing growth of B. subtilis ΔbsrA cells (KW587) containing plasmids pSP-Bs6S-1 (red), pSP-Bs6S-1(M6) (blue), pSP-Bs6S-2 (green) or pSP-Bs6S-2(M6) (purple). (C) Comparing growth of B. subtilis ΔbsrAΔbsrB cells (KW590) containing plasmids pSP-Bs6S-1 (red), pSP-Bs6S-1(M6) (blue), pSP-Bs6S-2 (green) or pSP-Bs6S-2(M6) (purple).
Mentions: Growth experiments using B. subtilis strains lacking 6S-1 RNA (ΔbsrA; KW587), 6S-2 RNA (ΔbsrB; KW589) or both (ΔbsrAΔbsrB; KW590) revealed that cells lacking 6S-1 RNA were delayed in outgrowth from stationary phase. For example, when stationary phase cells were diluted ~1:500 into fresh medium, cells lacking 6S-1 RNA were delayed for entry into exponential phase by 50 min compared to wild-type cells or cells lacking 6S-2 RNA (Figure 2A). The delay appeared to result primarily from an increase in lag time; once the ΔbsrA cells entered exponential phase, they grew with a similar doubling time to wild-type cells and obtained a similar maximal cell density in stationary phase. Intriguingly, cells lacking both 6S-1 and 6S-2 RNAs (ΔbsrAΔbsrB) were not delayed in outgrowth, suggesting it is the presence of 6S-2 RNA in the absence of 6S-1 RNA that is required for the outgrowth delay.Figure 2.

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