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Sigma E controls biogenesis of the antisense RNA MicA.

Udekwu KI, Wagner EG - Nucleic Acids Res. (2007)

Bottom Line: This regulation is Hfq-dependent and occurs by MicA-dependent translational inhibition which facilitates mRNA decay.Such treatments are sensed as envelope stress, upon which the extracytoplasmic sigma factor sigma(E) is activated.The strict dependence of micA transcription on sigma(E) is supported by three observations.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell & Molecular Biology, Uppsala university, Biomedical Center, Box 596, S-75124 Uppsala, Sweden. klas.udekwu@icm.uu.se

ABSTRACT
Adaptation stress responses in the Gram-negative bacterium Escherichia coli and its relatives involve a growing list of small regulatory RNAs (sRNAs). Previous work by us and others showed that the antisense RNA MicA downregulates the synthesis of the outer membrane protein OmpA upon entry into stationary phase. This regulation is Hfq-dependent and occurs by MicA-dependent translational inhibition which facilitates mRNA decay. In this article, we investigate the transcriptional regulation of the micA gene. Induction of MicA is dependent on the alarmone ppGpp, suggestive of alternative sigma factor involvement, yet MicA accumulates in the absence of the general stress/stationary phase sigma(S). We identified stress conditions that induce high MicA levels even during exponential growth-a phase in which MicA levels are low (ethanol, hyperosmolarity and heat shock). Such treatments are sensed as envelope stress, upon which the extracytoplasmic sigma factor sigma(E) is activated. The strict dependence of micA transcription on sigma(E) is supported by three observations. Induced overexpression of sigma(E) increases micA transcription, an DeltarpoE mutant displays undetectable MicA levels and the micA promoter has the consensus sigma(E) signature. Thus, MicA is part of the sigma(E) regulon and downregulates its target gene, ompA, probably to alleviate membrane stress.

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MicA accumulation and transcription rate in different growth phases. (A) Exponentially growing E. coli cells either carrying the micA promoter-luc fusion plasmid pluc-30, or the promoterless control plasmid, were assayed for specific luciferase activity at the OD600 values indicated (squares). Values depicted show values relative to that obtained at OD600 = 0.2. In parallel, MicA accumulation was quantified by Northern analysis, and normalized to 5S rRNA signals. These values are likewise shown as relative induction, compared to that at OD600 = 0.2 (triangles). (B) The kinetics of σE-dependent induction of MicA transcription was analyzed using strain LMG194::pAC-rpoE4, carrying in addition the reporter plasmid pluc-30. At time 0, the culture was either induced (squares) or mock-treated (triangles). Samples were taken for determination of luciferase activity at the indicated time points. Values show relative increases over that obtained at t = 0 min.
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Figure 5: MicA accumulation and transcription rate in different growth phases. (A) Exponentially growing E. coli cells either carrying the micA promoter-luc fusion plasmid pluc-30, or the promoterless control plasmid, were assayed for specific luciferase activity at the OD600 values indicated (squares). Values depicted show values relative to that obtained at OD600 = 0.2. In parallel, MicA accumulation was quantified by Northern analysis, and normalized to 5S rRNA signals. These values are likewise shown as relative induction, compared to that at OD600 = 0.2 (triangles). (B) The kinetics of σE-dependent induction of MicA transcription was analyzed using strain LMG194::pAC-rpoE4, carrying in addition the reporter plasmid pluc-30. At time 0, the culture was either induced (squares) or mock-treated (triangles). Samples were taken for determination of luciferase activity at the indicated time points. Values show relative increases over that obtained at t = 0 min.

Mentions: The experiment in Figure 4 suggests that σE is responsible for initiation at the micA promoter. If true, a micA promoter fused to a reporter gene should show growth-phase-dependent upregulation. Plasmid p30-luc carries the micA promoter in front of a luc (luciferase) reporter gene. The control plasmid pZE12b-luc is promoterless. E. coli cells harboring either of the two plasmids were diluted after overnight growth and allowed to grow. Samples were withdrawn for determination of luciferase activity at the OD600 values indicated in Figure 5A. Background luciferase activity from the control strain (promoterless plasmid) was subtracted from that obtained from the corresponding lysates of the strain carrying p30-luc, and induction values were calculated setting the luciferase activity at OD600 = 0.2 to unity. In parallel, MicA accumulation was measured by quantitation of Northern blots and, at the same time, OD-values. Here, RNA samples were taken from the control-plasmid-carrying strain since the high-copy plasmid p30-luc might out-titrate σE, resulting in lower-than-normal MicA levels (data not shown). Figure 5A shows both the induction of luciferase activity driven by the micA promoter (values normalized to OD600) and the MicA RNA levels from the control strain (normalized to 5S rRNA signals). Induction kinetics were similar for micA promoter activity (luc-values) and MicA accumulation. At the latest time point, MicA levels had dropped somewhat, whereas transcription activity stayed higher. Additional confirmation of direct σE involvement emerges from the kinetics of micA transcription (assayed by luc expression from pluc-30) upon arabinose induction of plasmid pAC-rpoE4 (Figure 5B). An overnight culture of strain LMG194, harboring the two compatible plasmids, was diluted into fresh medium, split in two, and luc expression was measured ±σE induction at 5-min intervals. Figure 5B shows a marked increase in transcription already at 5 min post-induction compared to the non-induced strain. No significant increase in luminescence was measured in a strain carrying the promoterless plasmid pZE12b-luc (data not shown). Taken together, these results confirm that the effect of the rpoE gene product on MicA levels can be accounted for, primarily or exclusively, by an increase in transcription rate.Figure 5.


Sigma E controls biogenesis of the antisense RNA MicA.

Udekwu KI, Wagner EG - Nucleic Acids Res. (2007)

MicA accumulation and transcription rate in different growth phases. (A) Exponentially growing E. coli cells either carrying the micA promoter-luc fusion plasmid pluc-30, or the promoterless control plasmid, were assayed for specific luciferase activity at the OD600 values indicated (squares). Values depicted show values relative to that obtained at OD600 = 0.2. In parallel, MicA accumulation was quantified by Northern analysis, and normalized to 5S rRNA signals. These values are likewise shown as relative induction, compared to that at OD600 = 0.2 (triangles). (B) The kinetics of σE-dependent induction of MicA transcription was analyzed using strain LMG194::pAC-rpoE4, carrying in addition the reporter plasmid pluc-30. At time 0, the culture was either induced (squares) or mock-treated (triangles). Samples were taken for determination of luciferase activity at the indicated time points. Values show relative increases over that obtained at t = 0 min.
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Related In: Results  -  Collection

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Figure 5: MicA accumulation and transcription rate in different growth phases. (A) Exponentially growing E. coli cells either carrying the micA promoter-luc fusion plasmid pluc-30, or the promoterless control plasmid, were assayed for specific luciferase activity at the OD600 values indicated (squares). Values depicted show values relative to that obtained at OD600 = 0.2. In parallel, MicA accumulation was quantified by Northern analysis, and normalized to 5S rRNA signals. These values are likewise shown as relative induction, compared to that at OD600 = 0.2 (triangles). (B) The kinetics of σE-dependent induction of MicA transcription was analyzed using strain LMG194::pAC-rpoE4, carrying in addition the reporter plasmid pluc-30. At time 0, the culture was either induced (squares) or mock-treated (triangles). Samples were taken for determination of luciferase activity at the indicated time points. Values show relative increases over that obtained at t = 0 min.
Mentions: The experiment in Figure 4 suggests that σE is responsible for initiation at the micA promoter. If true, a micA promoter fused to a reporter gene should show growth-phase-dependent upregulation. Plasmid p30-luc carries the micA promoter in front of a luc (luciferase) reporter gene. The control plasmid pZE12b-luc is promoterless. E. coli cells harboring either of the two plasmids were diluted after overnight growth and allowed to grow. Samples were withdrawn for determination of luciferase activity at the OD600 values indicated in Figure 5A. Background luciferase activity from the control strain (promoterless plasmid) was subtracted from that obtained from the corresponding lysates of the strain carrying p30-luc, and induction values were calculated setting the luciferase activity at OD600 = 0.2 to unity. In parallel, MicA accumulation was measured by quantitation of Northern blots and, at the same time, OD-values. Here, RNA samples were taken from the control-plasmid-carrying strain since the high-copy plasmid p30-luc might out-titrate σE, resulting in lower-than-normal MicA levels (data not shown). Figure 5A shows both the induction of luciferase activity driven by the micA promoter (values normalized to OD600) and the MicA RNA levels from the control strain (normalized to 5S rRNA signals). Induction kinetics were similar for micA promoter activity (luc-values) and MicA accumulation. At the latest time point, MicA levels had dropped somewhat, whereas transcription activity stayed higher. Additional confirmation of direct σE involvement emerges from the kinetics of micA transcription (assayed by luc expression from pluc-30) upon arabinose induction of plasmid pAC-rpoE4 (Figure 5B). An overnight culture of strain LMG194, harboring the two compatible plasmids, was diluted into fresh medium, split in two, and luc expression was measured ±σE induction at 5-min intervals. Figure 5B shows a marked increase in transcription already at 5 min post-induction compared to the non-induced strain. No significant increase in luminescence was measured in a strain carrying the promoterless plasmid pZE12b-luc (data not shown). Taken together, these results confirm that the effect of the rpoE gene product on MicA levels can be accounted for, primarily or exclusively, by an increase in transcription rate.Figure 5.

Bottom Line: This regulation is Hfq-dependent and occurs by MicA-dependent translational inhibition which facilitates mRNA decay.Such treatments are sensed as envelope stress, upon which the extracytoplasmic sigma factor sigma(E) is activated.The strict dependence of micA transcription on sigma(E) is supported by three observations.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell & Molecular Biology, Uppsala university, Biomedical Center, Box 596, S-75124 Uppsala, Sweden. klas.udekwu@icm.uu.se

ABSTRACT
Adaptation stress responses in the Gram-negative bacterium Escherichia coli and its relatives involve a growing list of small regulatory RNAs (sRNAs). Previous work by us and others showed that the antisense RNA MicA downregulates the synthesis of the outer membrane protein OmpA upon entry into stationary phase. This regulation is Hfq-dependent and occurs by MicA-dependent translational inhibition which facilitates mRNA decay. In this article, we investigate the transcriptional regulation of the micA gene. Induction of MicA is dependent on the alarmone ppGpp, suggestive of alternative sigma factor involvement, yet MicA accumulates in the absence of the general stress/stationary phase sigma(S). We identified stress conditions that induce high MicA levels even during exponential growth-a phase in which MicA levels are low (ethanol, hyperosmolarity and heat shock). Such treatments are sensed as envelope stress, upon which the extracytoplasmic sigma factor sigma(E) is activated. The strict dependence of micA transcription on sigma(E) is supported by three observations. Induced overexpression of sigma(E) increases micA transcription, an DeltarpoE mutant displays undetectable MicA levels and the micA promoter has the consensus sigma(E) signature. Thus, MicA is part of the sigma(E) regulon and downregulates its target gene, ompA, probably to alleviate membrane stress.

Show MeSH
Related in: MedlinePlus