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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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Overexpression of ptsN reduces cell envelope stress.β-galactosidase activity from the rpoHP3-lacZ (σE, SEA4254), degP-lacZ (σE and Cpx, SEA4181), cpxP-lacZ (Cpx, SEA4179), rprA-lacZ (Rcs, SEA4199), spy-lacZ (Cpx and Bae, SEA4189), and htpG-lacZ (σ32, SEA4185) fusions was measured in overnight cultures, with and without overexpression of ptsN. Activity of each lacZ fusion was normalized to that of cultures with no ptsN overexpression and set to 100%. Average values and standard deviations from a minimum of 3 experiments are shown.
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pone-0001573-g007: Overexpression of ptsN reduces cell envelope stress.β-galactosidase activity from the rpoHP3-lacZ (σE, SEA4254), degP-lacZ (σE and Cpx, SEA4181), cpxP-lacZ (Cpx, SEA4179), rprA-lacZ (Rcs, SEA4199), spy-lacZ (Cpx and Bae, SEA4189), and htpG-lacZ (σ32, SEA4185) fusions was measured in overnight cultures, with and without overexpression of ptsN. Activity of each lacZ fusion was normalized to that of cultures with no ptsN overexpression and set to 100%. Average values and standard deviations from a minimum of 3 experiments are shown.

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)

Overexpression of ptsN reduces cell envelope stress.β-galactosidase activity from the rpoHP3-lacZ (σE, SEA4254), degP-lacZ (σE and Cpx, SEA4181), cpxP-lacZ (Cpx, SEA4179), rprA-lacZ (Rcs, SEA4199), spy-lacZ (Cpx and Bae, SEA4189), and htpG-lacZ (σ32, SEA4185) fusions was measured in overnight cultures, with and without overexpression of ptsN. Activity of each lacZ fusion was normalized to that of cultures with no ptsN overexpression and set to 100%. Average values and standard deviations from a minimum of 3 experiments are shown.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2215328&req=5

pone-0001573-g007: Overexpression of ptsN reduces cell envelope stress.β-galactosidase activity from the rpoHP3-lacZ (σE, SEA4254), degP-lacZ (σE and Cpx, SEA4181), cpxP-lacZ (Cpx, SEA4179), rprA-lacZ (Rcs, SEA4199), spy-lacZ (Cpx and Bae, SEA4189), and htpG-lacZ (σ32, SEA4185) fusions was measured in overnight cultures, with and without overexpression of ptsN. Activity of each lacZ fusion was normalized to that of cultures with no ptsN overexpression and set to 100%. Average values and standard deviations from a minimum of 3 experiments are shown.
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