Limits...
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).

Show MeSH

Related in: MedlinePlus

Fractionation of strain SEA007 lysates with and without inhibition of σE on sucrose density gradients.At 2.5 hours following inhibition of σE, cells were harvested and lysates separated by discontinuous sucrose density gradient centrifugation. Lysates from comparable numbers of cells (as determined by OD600) were loaded on the gradients. (A) Coomassie-stained 12% SDS-polyacrylamide gels of gradient fractions from the control culture (top) and culture in which σE activity was inhibited (bottom). M denotes molecular weight markers. (B) β-NADH oxidase activity (closed symbols) and specific gravity (open symbols) of the fractions in (A), inhibition of σE (circles) and control (squares). (C) Western blots using polyclonal antibodies to detect FtsH (inner membrane) and FepA (outer membrane) in fractions from (A), control (top panels) and inhibition of σE (bottom panels). L denotes whole-cell lysates before fractionation.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2215328&req=5

pone-0001573-g005: Fractionation of strain SEA007 lysates with and without inhibition of σE on sucrose density gradients.At 2.5 hours following inhibition of σE, cells were harvested and lysates separated by discontinuous sucrose density gradient centrifugation. Lysates from comparable numbers of cells (as determined by OD600) were loaded on the gradients. (A) Coomassie-stained 12% SDS-polyacrylamide gels of gradient fractions from the control culture (top) and culture in which σE activity was inhibited (bottom). M denotes molecular weight markers. (B) β-NADH oxidase activity (closed symbols) and specific gravity (open symbols) of the fractions in (A), inhibition of σE (circles) and control (squares). (C) Western blots using polyclonal antibodies to detect FtsH (inner membrane) and FepA (outer membrane) in fractions from (A), control (top panels) and inhibition of σE (bottom panels). L denotes whole-cell lysates before fractionation.

Mentions: Since inhibition of σE increases envelope stress and σE can transcribe genes that are localized to the inner and outer membranes, we next asked if the envelope membranes were altered. Samples were taken from cultures 2.5 hours following overexpression of rseA and rseB. This time point was chosen for analysis because the cell envelope stress responses were activated, the cfu/ml had started to decrease, and the morphological defects described below were evident. Cell lysates were fractionated by centrifugation through discontinuous sucrose density gradients and several assays were used to detect fractions containing inner and outer membrane components. In each of these assays the results from cultures in which σE activity was inhibited were similar to those from control cultures. No alterations in the overall protein content of the fractions were detected on Coomassie-stained SDS polyacrylamide gels (Fig. 5A). The inner and outer membrane fractions were well separated, as determined by western blots probed with antibodies to the inner membrane protein, FtsH, and the outer membrane protein, FepA (Fig. 5C). Fractions containing inner membrane were also identified using a β-NADH oxidase activity assay (Fig. 5B). Thus, loss of σE activity does not result in gross defects in the steady-state protein composition or density of the inner and outer membranes.


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

Hayden JD, Ades SE - PLoS ONE (2008)

Fractionation of strain SEA007 lysates with and without inhibition of σE on sucrose density gradients.At 2.5 hours following inhibition of σE, cells were harvested and lysates separated by discontinuous sucrose density gradient centrifugation. Lysates from comparable numbers of cells (as determined by OD600) were loaded on the gradients. (A) Coomassie-stained 12% SDS-polyacrylamide gels of gradient fractions from the control culture (top) and culture in which σE activity was inhibited (bottom). M denotes molecular weight markers. (B) β-NADH oxidase activity (closed symbols) and specific gravity (open symbols) of the fractions in (A), inhibition of σE (circles) and control (squares). (C) Western blots using polyclonal antibodies to detect FtsH (inner membrane) and FepA (outer membrane) in fractions from (A), control (top panels) and inhibition of σE (bottom panels). L denotes whole-cell lysates before fractionation.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2215328&req=5

pone-0001573-g005: Fractionation of strain SEA007 lysates with and without inhibition of σE on sucrose density gradients.At 2.5 hours following inhibition of σE, cells were harvested and lysates separated by discontinuous sucrose density gradient centrifugation. Lysates from comparable numbers of cells (as determined by OD600) were loaded on the gradients. (A) Coomassie-stained 12% SDS-polyacrylamide gels of gradient fractions from the control culture (top) and culture in which σE activity was inhibited (bottom). M denotes molecular weight markers. (B) β-NADH oxidase activity (closed symbols) and specific gravity (open symbols) of the fractions in (A), inhibition of σE (circles) and control (squares). (C) Western blots using polyclonal antibodies to detect FtsH (inner membrane) and FepA (outer membrane) in fractions from (A), control (top panels) and inhibition of σE (bottom panels). L denotes whole-cell lysates before fractionation.
Mentions: Since inhibition of σE increases envelope stress and σE can transcribe genes that are localized to the inner and outer membranes, we next asked if the envelope membranes were altered. Samples were taken from cultures 2.5 hours following overexpression of rseA and rseB. This time point was chosen for analysis because the cell envelope stress responses were activated, the cfu/ml had started to decrease, and the morphological defects described below were evident. Cell lysates were fractionated by centrifugation through discontinuous sucrose density gradients and several assays were used to detect fractions containing inner and outer membrane components. In each of these assays the results from cultures in which σE activity was inhibited were similar to those from control cultures. No alterations in the overall protein content of the fractions were detected on Coomassie-stained SDS polyacrylamide gels (Fig. 5A). The inner and outer membrane fractions were well separated, as determined by western blots probed with antibodies to the inner membrane protein, FtsH, and the outer membrane protein, FepA (Fig. 5C). Fractions containing inner membrane were also identified using a β-NADH oxidase activity assay (Fig. 5B). Thus, loss of σE activity does not result in gross defects in the steady-state protein composition or density of the inner and outer membranes.

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