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The extracytoplasmic stress factor, sigmaE, is required to maintain cell envelope integrity in Escherichia coli.

Hayden JD, Ades SE - PLoS ONE (2008)

Bottom Line: Many cells lyse and some develop blebs containing cytoplasmic material along their sides.To better understand the connection between transcription by sigma(E) and cell envelope integrity, we identified two multicopy suppressors of the essentiality of sigma(E), ptsN and yhbW. yhbW is a gene of unknown function, while ptsN is a member of the sigma(E) regulon.Overexpression of ptsN lowers the basal level of multiple envelope stress responses, but not that of a cytoplasmic stress response.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania, USA.

ABSTRACT
Extracytoplasmic function or ECF sigma factors are the most abundant class of alternative sigma factors in bacteria. Members of the rpoE subclass of ECF sigma factors are implicated in sensing stress in the cell envelope of Gram-negative bacteria and are required for virulence in many pathogens. The best-studied member of this family is rpoE from Escherichia coli, encoding the sigma(E) protein. sigma(E) has been well studied for its role in combating extracytoplasmic stress, and the members of its regulon have been largely defined. sigma(E) is required for viability of E. coli, yet none of the studies to date explain why sigma(E) is essential in seemingly unstressed cells. In this work we investigate the essential role of sigma(E) in E. coli by analyzing the phenotypes associated with loss of sigma(E) activity and isolating suppressors that allow cells to live in the absence of sigma(E). We demonstrate that when sigma(E) is inhibited, cell envelope stress increases and envelope integrity is lost. Many cells lyse and some develop blebs containing cytoplasmic material along their sides. To better understand the connection between transcription by sigma(E) and cell envelope integrity, we identified two multicopy suppressors of the essentiality of sigma(E), ptsN and yhbW. yhbW is a gene of unknown function, while ptsN is a member of the sigma(E) regulon. Overexpression of ptsN lowers the basal level of multiple envelope stress responses, but not that of a cytoplasmic stress response. Our results are consistent with a model in which overexpression of ptsN reduces stress in the cell envelope, thereby promoting survival in the absence of sigma(E).

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ptsN requires known regulators to reduce stress sensed by the σE, Rcs, and Cpx envelope stress pathways.In each panel the β-galactosidase activity was measured with and without overexpression of ptsN in a WT strain and in a strain lacking known regulators of the respective stress responses. Measurements are expressed as the percentage of reporter gene activity in the WT strain without ptsN overexpression and this value is noted above the columns. Relative reporter gene activities are shown for (A) the rpoHP3-lacZ fusion in WT and ΔrseA strains (SEA4254 and SEA4228), (B) the rprA-lacZ fusion in WT and ΔrcsB strains (SEA4199 and SEA4203), and (C) the cpxP-lacZ fusion in WT and cpxR::Ωspec strains (SEA 4179 and SEA4251). The inset in C shows the cpxR::Ωspec strain with and without ptsN overexpression. The basal level of expression is >250-fold lower in cpxR::Ωspec cells than in WT cells and is not easily seen on the main plot.
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pone-0001573-g008: ptsN requires known regulators to reduce stress sensed by the σE, Rcs, and Cpx envelope stress pathways.In each panel the β-galactosidase activity was measured with and without overexpression of ptsN in a WT strain and in a strain lacking known regulators of the respective stress responses. Measurements are expressed as the percentage of reporter gene activity in the WT strain without ptsN overexpression and this value is noted above the columns. Relative reporter gene activities are shown for (A) the rpoHP3-lacZ fusion in WT and ΔrseA strains (SEA4254 and SEA4228), (B) the rprA-lacZ fusion in WT and ΔrcsB strains (SEA4199 and SEA4203), and (C) the cpxP-lacZ fusion in WT and cpxR::Ωspec strains (SEA 4179 and SEA4251). The inset in C shows the cpxR::Ωspec strain with and without ptsN overexpression. The basal level of expression is >250-fold lower in cpxR::Ωspec cells than in WT cells and is not easily seen on the main plot.

Mentions: The basal levels of several envelope stress responses are lower in a ΔydcQ strain, the other characterized suppressor ΔrpoE lethality [10]. In addition, σE-dependent rpoHP3-lacZ activity is low in a strain containing an unmapped ΔrpoE suppressor [46]. Consistent with these results, overexpression of ptsN lowered activity of the σE-dependent rpoHP3-lacZ reporter in a wild-type rpoE+ strain (Fig. 7 and 8A). To determine whether this effect was specific to σE, or if ptsN overexpression affected other envelope stress responses, we measured the effects of ptsN overexpression on reporters for the following envelope stress responses (Fig. 3): Cpx (cpxP-lacZ reporter), σE and Cpx (degP-lacZ reporter), Bae and Cpx (spy-lacZ reporter), and Rcs (rprA-lacZ reporter). A strain carrying a reporter for the σ32-dependent cytoplasmic heat shock response (htpG-lacZ) was included to assay whether ptsN also lowered cytoplasmic stress (Fig. 3). Overexpression of ptsN significantly lowered expression of the lacZ fusions that are regulated by envelope stress response factors, but had no effect on the σ32-dependent cytoplasmic stress reporter (Fig. 7). The latter result indicates that overexpression of ptsN does not lower β-galactosidase activity per se, nor does it have a dampening effect on overall gene expression.


The extracytoplasmic stress factor, sigmaE, is required to maintain cell envelope integrity in Escherichia coli.

Hayden JD, Ades SE - PLoS ONE (2008)

ptsN requires known regulators to reduce stress sensed by the σE, Rcs, and Cpx envelope stress pathways.In each panel the β-galactosidase activity was measured with and without overexpression of ptsN in a WT strain and in a strain lacking known regulators of the respective stress responses. Measurements are expressed as the percentage of reporter gene activity in the WT strain without ptsN overexpression and this value is noted above the columns. Relative reporter gene activities are shown for (A) the rpoHP3-lacZ fusion in WT and ΔrseA strains (SEA4254 and SEA4228), (B) the rprA-lacZ fusion in WT and ΔrcsB strains (SEA4199 and SEA4203), and (C) the cpxP-lacZ fusion in WT and cpxR::Ωspec strains (SEA 4179 and SEA4251). The inset in C shows the cpxR::Ωspec strain with and without ptsN overexpression. The basal level of expression is >250-fold lower in cpxR::Ωspec cells than in WT cells and is not easily seen on the main plot.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2215328&req=5

pone-0001573-g008: ptsN requires known regulators to reduce stress sensed by the σE, Rcs, and Cpx envelope stress pathways.In each panel the β-galactosidase activity was measured with and without overexpression of ptsN in a WT strain and in a strain lacking known regulators of the respective stress responses. Measurements are expressed as the percentage of reporter gene activity in the WT strain without ptsN overexpression and this value is noted above the columns. Relative reporter gene activities are shown for (A) the rpoHP3-lacZ fusion in WT and ΔrseA strains (SEA4254 and SEA4228), (B) the rprA-lacZ fusion in WT and ΔrcsB strains (SEA4199 and SEA4203), and (C) the cpxP-lacZ fusion in WT and cpxR::Ωspec strains (SEA 4179 and SEA4251). The inset in C shows the cpxR::Ωspec strain with and without ptsN overexpression. The basal level of expression is >250-fold lower in cpxR::Ωspec cells than in WT cells and is not easily seen on the main plot.
Mentions: The basal levels of several envelope stress responses are lower in a ΔydcQ strain, the other characterized suppressor ΔrpoE lethality [10]. In addition, σE-dependent rpoHP3-lacZ activity is low in a strain containing an unmapped ΔrpoE suppressor [46]. Consistent with these results, overexpression of ptsN lowered activity of the σE-dependent rpoHP3-lacZ reporter in a wild-type rpoE+ strain (Fig. 7 and 8A). To determine whether this effect was specific to σE, or if ptsN overexpression affected other envelope stress responses, we measured the effects of ptsN overexpression on reporters for the following envelope stress responses (Fig. 3): Cpx (cpxP-lacZ reporter), σE and Cpx (degP-lacZ reporter), Bae and Cpx (spy-lacZ reporter), and Rcs (rprA-lacZ reporter). A strain carrying a reporter for the σ32-dependent cytoplasmic heat shock response (htpG-lacZ) was included to assay whether ptsN also lowered cytoplasmic stress (Fig. 3). Overexpression of ptsN significantly lowered expression of the lacZ fusions that are regulated by envelope stress response factors, but had no effect on the σ32-dependent cytoplasmic stress reporter (Fig. 7). The latter result indicates that overexpression of ptsN does not lower β-galactosidase activity per se, nor does it have a dampening effect on overall gene expression.

Bottom Line: Many cells lyse and some develop blebs containing cytoplasmic material along their sides.To better understand the connection between transcription by sigma(E) and cell envelope integrity, we identified two multicopy suppressors of the essentiality of sigma(E), ptsN and yhbW. yhbW is a gene of unknown function, while ptsN is a member of the sigma(E) regulon.Overexpression of ptsN lowers the basal level of multiple envelope stress responses, but not that of a cytoplasmic stress response.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park, Pennsylvania, USA.

ABSTRACT
Extracytoplasmic function or ECF sigma factors are the most abundant class of alternative sigma factors in bacteria. Members of the rpoE subclass of ECF sigma factors are implicated in sensing stress in the cell envelope of Gram-negative bacteria and are required for virulence in many pathogens. The best-studied member of this family is rpoE from Escherichia coli, encoding the sigma(E) protein. sigma(E) has been well studied for its role in combating extracytoplasmic stress, and the members of its regulon have been largely defined. sigma(E) is required for viability of E. coli, yet none of the studies to date explain why sigma(E) is essential in seemingly unstressed cells. In this work we investigate the essential role of sigma(E) in E. coli by analyzing the phenotypes associated with loss of sigma(E) activity and isolating suppressors that allow cells to live in the absence of sigma(E). We demonstrate that when sigma(E) is inhibited, cell envelope stress increases and envelope integrity is lost. Many cells lyse and some develop blebs containing cytoplasmic material along their sides. To better understand the connection between transcription by sigma(E) and cell envelope integrity, we identified two multicopy suppressors of the essentiality of sigma(E), ptsN and yhbW. yhbW is a gene of unknown function, while ptsN is a member of the sigma(E) regulon. Overexpression of ptsN lowers the basal level of multiple envelope stress responses, but not that of a cytoplasmic stress response. Our results are consistent with a model in which overexpression of ptsN reduces stress in the cell envelope, thereby promoting survival in the absence of sigma(E).

Show MeSH
Related in: MedlinePlus