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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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Cell envelope defects resulting from inhibition of σE.Cultures of SEA007 and SEA008 were grown as in Fig. 2. Samples were taken approximately 2.5 hrs. after induction of rseA and rseB overexpression. (A–D) Images of live cells are shown using differential interference microscopy (DIC, column 1) and fluorescence microscopy following addition of FM4-64 to visualize membranes (red, column 2), DAPI to visualize DNA (blue, column 3), and expression of YFP to visualize the cytoplasm (green, column 4). The three fluorescent micrographs are overlaid in column 5. (A) Images of the SEA008 control strain in which σE was not inhibited are shown. (B–D) Images of SEA007 following σE inhibition, reveal blebs that contain YFP and stain with DAPI (B and C) and that contain YFP but do not stain with DAPI (D). In (D), the arrow marks a bleb lacking DAPI staining and the arrowhead marks a lysed cell that retained DAPI staining, but lost YFP. Scale bars are 2 µm. Over 1,000 cells were examined by fluorescence microscopy and typical micrographs are represented here. Scanning electron micrographs (E) and transmission electron micrographs (F) of SEA007 following σE inhibition. Scale bars are 1 µm. No blebs were seen on cells in control cultures.
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pone-0001573-g006: Cell envelope defects resulting from inhibition of σE.Cultures of SEA007 and SEA008 were grown as in Fig. 2. Samples were taken approximately 2.5 hrs. after induction of rseA and rseB overexpression. (A–D) Images of live cells are shown using differential interference microscopy (DIC, column 1) and fluorescence microscopy following addition of FM4-64 to visualize membranes (red, column 2), DAPI to visualize DNA (blue, column 3), and expression of YFP to visualize the cytoplasm (green, column 4). The three fluorescent micrographs are overlaid in column 5. (A) Images of the SEA008 control strain in which σE was not inhibited are shown. (B–D) Images of SEA007 following σE inhibition, reveal blebs that contain YFP and stain with DAPI (B and C) and that contain YFP but do not stain with DAPI (D). In (D), the arrow marks a bleb lacking DAPI staining and the arrowhead marks a lysed cell that retained DAPI staining, but lost YFP. Scale bars are 2 µm. Over 1,000 cells were examined by fluorescence microscopy and typical micrographs are represented here. Scanning electron micrographs (E) and transmission electron micrographs (F) of SEA007 following σE inhibition. Scale bars are 1 µm. No blebs were seen on cells in control cultures.

Mentions: We next examined the morphology of cells following inhibition of σE. By 2 hours after addition of IPTG to overexpress rseA and rseB (the time that the cfu/ml began to drop), two phenotypes were evident by phase contrast microscopy. Ghosted cells and cells with blebs began to appear in the culture. The blebs formed primarily along the lateral wall of the cells and were found less frequently at either the poles or the septum. Usually only one bleb formed per cell. By 2.5 hours after addition of IPTG, approximately 20% of cells in the culture had blebs (Fig. 6) and the number of ghosted cells increased along with the amount of cellular debris, indicative of lysis. In a typical experiment, approximately 100 cells were viewed at each time point. The location of the blebs and the timing of their appearance with respect to loss of viability were very reproducible, suggesting that the phenotypes were related. By 3–4 hours after addition of IPTG some cells had additional blebs, the number of ghosted cells and the amount of cell debris increased, and many cells clumped together. Similar phenotypes were observed when σE was inhibited by proteolytic stabilization of RseA through depletion of the DegS or RseP proteases (data not shown) and were not found following overexpression of rseAD11H and rseB from pRseAD11HB, providing further evidence that the phenotypes were due to inhibition of σE and not overexpression of rseA and rseB. In addition, the same phenotypes were seen when the cells were grown in glucose or glycerol minimal media supplemented with amino acids indicating that the phenotypes were not a function of growth rate or medium composition.


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

Hayden JD, Ades SE - PLoS ONE (2008)

Cell envelope defects resulting from inhibition of σE.Cultures of SEA007 and SEA008 were grown as in Fig. 2. Samples were taken approximately 2.5 hrs. after induction of rseA and rseB overexpression. (A–D) Images of live cells are shown using differential interference microscopy (DIC, column 1) and fluorescence microscopy following addition of FM4-64 to visualize membranes (red, column 2), DAPI to visualize DNA (blue, column 3), and expression of YFP to visualize the cytoplasm (green, column 4). The three fluorescent micrographs are overlaid in column 5. (A) Images of the SEA008 control strain in which σE was not inhibited are shown. (B–D) Images of SEA007 following σE inhibition, reveal blebs that contain YFP and stain with DAPI (B and C) and that contain YFP but do not stain with DAPI (D). In (D), the arrow marks a bleb lacking DAPI staining and the arrowhead marks a lysed cell that retained DAPI staining, but lost YFP. Scale bars are 2 µm. Over 1,000 cells were examined by fluorescence microscopy and typical micrographs are represented here. Scanning electron micrographs (E) and transmission electron micrographs (F) of SEA007 following σE inhibition. Scale bars are 1 µm. No blebs were seen on cells in control cultures.
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pone-0001573-g006: Cell envelope defects resulting from inhibition of σE.Cultures of SEA007 and SEA008 were grown as in Fig. 2. Samples were taken approximately 2.5 hrs. after induction of rseA and rseB overexpression. (A–D) Images of live cells are shown using differential interference microscopy (DIC, column 1) and fluorescence microscopy following addition of FM4-64 to visualize membranes (red, column 2), DAPI to visualize DNA (blue, column 3), and expression of YFP to visualize the cytoplasm (green, column 4). The three fluorescent micrographs are overlaid in column 5. (A) Images of the SEA008 control strain in which σE was not inhibited are shown. (B–D) Images of SEA007 following σE inhibition, reveal blebs that contain YFP and stain with DAPI (B and C) and that contain YFP but do not stain with DAPI (D). In (D), the arrow marks a bleb lacking DAPI staining and the arrowhead marks a lysed cell that retained DAPI staining, but lost YFP. Scale bars are 2 µm. Over 1,000 cells were examined by fluorescence microscopy and typical micrographs are represented here. Scanning electron micrographs (E) and transmission electron micrographs (F) of SEA007 following σE inhibition. Scale bars are 1 µm. No blebs were seen on cells in control cultures.
Mentions: We next examined the morphology of cells following inhibition of σE. By 2 hours after addition of IPTG to overexpress rseA and rseB (the time that the cfu/ml began to drop), two phenotypes were evident by phase contrast microscopy. Ghosted cells and cells with blebs began to appear in the culture. The blebs formed primarily along the lateral wall of the cells and were found less frequently at either the poles or the septum. Usually only one bleb formed per cell. By 2.5 hours after addition of IPTG, approximately 20% of cells in the culture had blebs (Fig. 6) and the number of ghosted cells increased along with the amount of cellular debris, indicative of lysis. In a typical experiment, approximately 100 cells were viewed at each time point. The location of the blebs and the timing of their appearance with respect to loss of viability were very reproducible, suggesting that the phenotypes were related. By 3–4 hours after addition of IPTG some cells had additional blebs, the number of ghosted cells and the amount of cell debris increased, and many cells clumped together. Similar phenotypes were observed when σE was inhibited by proteolytic stabilization of RseA through depletion of the DegS or RseP proteases (data not shown) and were not found following overexpression of rseAD11H and rseB from pRseAD11HB, providing further evidence that the phenotypes were due to inhibition of σE and not overexpression of rseA and rseB. In addition, the same phenotypes were seen when the cells were grown in glucose or glycerol minimal media supplemented with amino acids indicating that the phenotypes were not a function of growth rate or medium composition.

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