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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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Inhibition of σE activity caused cell death.Strains SEA007 (squares, pRseAB), SEA008 (circles, pTrc99a vector control), and SEA4044 (triangles, pRseAD11H) were grown in LB at 30°C with shaking in a gyrotary waterbath. IPTG was added at OD600 ∼0.1 to induce overexpression of rseA and rseB, indicated by the arrow. (A) σE was inhibited by overexpression of rseA and rseB, but not rseAD11H and rseB, as shown by β-galactosidase activity measured from the σE-dependent rpoHP3-lacZ reporter. Miller units (open symbols, right axis) and OD600 (closed symbols, left axis) are plotted at each time point. (B) Overexpression of rseA and rseB, but not rseAD11H and rseB, caused a reduction in the colony forming units and optical density. OD600 (closed symbols, left axis) and cfu/ml (open symbols, right axis) were measured throughout the growth curve. A representative experiment is shown in both panels.
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pone-0001573-g002: Inhibition of σE activity caused cell death.Strains SEA007 (squares, pRseAB), SEA008 (circles, pTrc99a vector control), and SEA4044 (triangles, pRseAD11H) were grown in LB at 30°C with shaking in a gyrotary waterbath. IPTG was added at OD600 ∼0.1 to induce overexpression of rseA and rseB, indicated by the arrow. (A) σE was inhibited by overexpression of rseA and rseB, but not rseAD11H and rseB, as shown by β-galactosidase activity measured from the σE-dependent rpoHP3-lacZ reporter. Miller units (open symbols, right axis) and OD600 (closed symbols, left axis) are plotted at each time point. (B) Overexpression of rseA and rseB, but not rseAD11H and rseB, caused a reduction in the colony forming units and optical density. OD600 (closed symbols, left axis) and cfu/ml (open symbols, right axis) were measured throughout the growth curve. A representative experiment is shown in both panels.

Mentions: To gain a better understanding of the role of a particular gene in cellular physiology, it is often informative to examine the phenotypes of a deletion mutant lacking the gene of interest. Because σE is encoded by an essential gene, it is not possible to examine the phenotype of a ΔrpoE strain. Therefore we examined the effects of loss of σE activity by inducing the overexpression of its inhibitors, rseA and rseB, encoded under the IPTG-inducible trc promoter on the pTrc99a plasmid, pRseAB. This method efficiently inhibits σE (Fig. 2A) by preventing its association with RNA polymerase [15]–[17]. Overexpression of rseA and rseB and the consequent sequestration of σE were lethal for the bacterium. In liquid culture, the cfu/ml began to decrease after 2 hours (∼2.5 generations) following the addition of IPTG and the optical density stopped increasing and began to decrease slightly within 3 hours (∼3.5 generation) (Fig. 2B and [15]). On solid media, the plating efficiency in the presence of IPTG was reduced by three orders of magnitude compared with non-inducing conditions (Table 1). To ensure that the observed phenotypes were due to inhibition of σE and not overproduction of RseA and RseB, we introduced a point mutation into the rseA gene on the plasmid encoding rseA and rseB, changing the aspartate residue at position 11 of RseA to histidine. This mutation abrogates the antisigma factor activity of RseA (Fig. 2A and [16]). When overproduced along with RseB, the RseAD11H protein reached similar steady-state levels as the wild-type protein, was properly localized to the inner membrane, did not inhibit σE activity, did not induce lysis, and did not reduce the plating efficiency (Fig. 2, Table 1, and data not shown).


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

Hayden JD, Ades SE - PLoS ONE (2008)

Inhibition of σE activity caused cell death.Strains SEA007 (squares, pRseAB), SEA008 (circles, pTrc99a vector control), and SEA4044 (triangles, pRseAD11H) were grown in LB at 30°C with shaking in a gyrotary waterbath. IPTG was added at OD600 ∼0.1 to induce overexpression of rseA and rseB, indicated by the arrow. (A) σE was inhibited by overexpression of rseA and rseB, but not rseAD11H and rseB, as shown by β-galactosidase activity measured from the σE-dependent rpoHP3-lacZ reporter. Miller units (open symbols, right axis) and OD600 (closed symbols, left axis) are plotted at each time point. (B) Overexpression of rseA and rseB, but not rseAD11H and rseB, caused a reduction in the colony forming units and optical density. OD600 (closed symbols, left axis) and cfu/ml (open symbols, right axis) were measured throughout the growth curve. A representative experiment is shown in both panels.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2215328&req=5

pone-0001573-g002: Inhibition of σE activity caused cell death.Strains SEA007 (squares, pRseAB), SEA008 (circles, pTrc99a vector control), and SEA4044 (triangles, pRseAD11H) were grown in LB at 30°C with shaking in a gyrotary waterbath. IPTG was added at OD600 ∼0.1 to induce overexpression of rseA and rseB, indicated by the arrow. (A) σE was inhibited by overexpression of rseA and rseB, but not rseAD11H and rseB, as shown by β-galactosidase activity measured from the σE-dependent rpoHP3-lacZ reporter. Miller units (open symbols, right axis) and OD600 (closed symbols, left axis) are plotted at each time point. (B) Overexpression of rseA and rseB, but not rseAD11H and rseB, caused a reduction in the colony forming units and optical density. OD600 (closed symbols, left axis) and cfu/ml (open symbols, right axis) were measured throughout the growth curve. A representative experiment is shown in both panels.
Mentions: To gain a better understanding of the role of a particular gene in cellular physiology, it is often informative to examine the phenotypes of a deletion mutant lacking the gene of interest. Because σE is encoded by an essential gene, it is not possible to examine the phenotype of a ΔrpoE strain. Therefore we examined the effects of loss of σE activity by inducing the overexpression of its inhibitors, rseA and rseB, encoded under the IPTG-inducible trc promoter on the pTrc99a plasmid, pRseAB. This method efficiently inhibits σE (Fig. 2A) by preventing its association with RNA polymerase [15]–[17]. Overexpression of rseA and rseB and the consequent sequestration of σE were lethal for the bacterium. In liquid culture, the cfu/ml began to decrease after 2 hours (∼2.5 generations) following the addition of IPTG and the optical density stopped increasing and began to decrease slightly within 3 hours (∼3.5 generation) (Fig. 2B and [15]). On solid media, the plating efficiency in the presence of IPTG was reduced by three orders of magnitude compared with non-inducing conditions (Table 1). To ensure that the observed phenotypes were due to inhibition of σE and not overproduction of RseA and RseB, we introduced a point mutation into the rseA gene on the plasmid encoding rseA and rseB, changing the aspartate residue at position 11 of RseA to histidine. This mutation abrogates the antisigma factor activity of RseA (Fig. 2A and [16]). When overproduced along with RseB, the RseAD11H protein reached similar steady-state levels as the wild-type protein, was properly localized to the inner membrane, did not inhibit σE activity, did not induce lysis, and did not reduce the plating efficiency (Fig. 2, Table 1, and data not shown).

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