Constitutive expression of the maltoporin LamB in the absence of OmpR damages the cell envelope

Abstract

Cells experience multiple environmental stimuli simultaneously. To survive, they must respond accordingly. Unfortunately, the proper response to one stress easily could make the cell more susceptible to a second coexistent stress. To deal with such a problem, a cell must possess a mechanism that balances the need to respond simultaneously to both stresses. Our recent studies of ompR malT(Con) double mutants show that elevated expression of LamB, the outer membrane porin responsible for maltose uptake, causes cell death when the osmoregulator OmpR is disabled. To obtain insight into the nature of the death experienced by ompR malT(Con) mutants, we described the death process. On the basis of microscopic and biochemical approaches, we conclude that death results from a loss of membrane integrity. On the basis of an unbiased genome-wide search for suppressor mutations, we conclude that this loss of membrane integrity results from a LamB-induced envelope stress that the cells do not sufficiently perceive and thus do not adequately accommodate. Finally, we conclude that this envelope stress involves an imbalance in the lipopolysaccharide/porin composition of the outer membrane and an increased requirement for inorganic phosphate.

FIG. 1.

Death phenotypes of ompR malT(Con) mutants. (A) Colony phenotype of ompR malT(Con) mutants (strain AJW3098) grown on LB plates at 37°C for 48 h. Note the translucent colony appearance and the formation of papillae. (B and C) Colony phenotype of ompR malT(Con) mutants on LB plates supplemented with X-Gal at 37°C. ompR malT(Con) mutants were transformed with the empty mF vector (B) (20) or the vector carrying ompR (C). Note the halo around the colony in panel B. (D and E) FM4-64 fluorescent membrane stain (D) and LIVE/DEAD stain with propidium iodide (E) of ompR malT(Con) mutants during late exponential phase in LB at 37°C.

FIG. 2.

Time course of ompR malT(Con) mutant phenotypes. (A) Growth curve of the ompR malT(Con) mutant (strain AJW3098) in LB at 37°C. Arrows and letters refer to time points depicted in panels B to F. (B to F) TEM images of the ompR malT(Con) mutant harvested after 90 min (B), 216 min (C), 320 min (D), 500 min (E), and 1,440 min (F) of growth. Note the plasmolytic bays in panel F. (G) Enlargement of an ompR malT(Con) mutant cell at 320 min. The arrows point to disruptions of the cytoplasmic membrane.

FIG. 3.

Envelope permeability of ompR malT(Con) mutants. (A) Inner membrane permeability of WT (AJW678) (circles), ompR (AJW2050) (squares), malT(Con) (AJW3499) (triangles), and ompR malT(Con) (AJW3098) (diamonds) mutants. Cells were grown in LB at 37°C. At regular intervals, samples were taken for OD readings and β-galactosidase measurements as described in Materials and Methods. Upper panel, growth curves. Lower panel, percent β-galactosidase activity. Values represent the mean of triplicates. Error bars indicate standard deviations and are shown only when greater than the size of the symbol. (B) Outer membrane permeativity of ompR malT(Con) mutants. Cells were grown in LB at 37°C. After 150 min (left panel) and 400 min (right panel), cells were harvested, stained with Nile red, and observed under a fluorescence microscope. The total cell numbers in the two images are identical.

FIG. 4.

Effect of σ^E activation on ompR malT(Con) survival. (A) Colony phenotypes of the ompR malT(Con) mutant (AJW3098) (left) and the ompR malT(Con) rseA::Tn5 mutant (strain AJW3855) (right). Colonies were grown in LB at 37°C. (B) Growth curves of WT (AJW678) (closed circles), ompR malT(Con) (AJW3098) (closed diamonds), rseA (AJW3815) (open circles), and ompR malT(Con) rseA (AJW3818) (open diamonds) strains. Cells were grown in LB at 37°C. Values represent the means of triplicates, and error bars indicate standard deviations and are shown only when greater than the size of the symbol. (C) β-Galactosidase activities of WT (AJW678), ompR malT(Con) (AJW3098), rseA (AJW3815), and ompR malT(Con) rseA (AJW3818) strains carrying a Φ(rpoHP3-lacZ) promoter fusion grown under nonpermissive conditions. Cells were harvested at an OD₆₀₀ of 1. Values represent the means and standard deviations for triplicates.

FIG. 5.

Effect of σ^E overexpression on ompR malT(Con) survival. (A) Growth curves of the ompR malT(Con) mutant (strain AJW3098) transformed with the empty pTrc99a vector (circles) or the vector carrying rpoE (triangles) under nonpermissive conditions. Expression of rpoE was induced with 50 μM IPTG. Values represent the means of triplicates, and error bars indicate standard deviations and are shown only when greater than the size of the symbol. (B) β-Galactosidase activity of the ompR malT(Con) mutant carrying an Φ(rpoHP3-lacZ) promoter fusion transformed with the empty pTrc99a vector (left bar) or the vector expressing rpoE (right bar). Cells were grown under nonpermissive conditions, and expression of rpoE was induced with 50 μM IPTG. Cells were harvested at an OD₆₀₀ of 1. Values represent the means and standard deviations for triplicates.

FIG. 6.

Effect of PhoB activation on ompR malT(Con) mutant survival. (A) Deletion of the pst-phoU genes rescues lethality. Growth curve of the WT (black circles) (AJW678), ompR malT(Con) (black diamonds) (AJW3098), ompR malT(Con) pstS (gray diamonds) (AJW3785), ompR malT(Con) pstC (gray triangles) (AJW3783), ompR malT(Con) pstA (gray circles) (AJW3781), ompR malT(Con) pstB (gray squares) (strain AJW3782), and ompR malT(Con) phoU (white triangles) (AJW3780) strains under nonpermissive conditions are shown. Values represent the mean of triplicates, and error bars indicate standard deviations and are shown only when greater than the size of the symbol. (B) Expression of constitutive PhoB^CA(E11K) delays death. Growth curves of the ompR malT(Con) mutant (black diamonds) (AJW3098) and the ompR malT(Con) mutant expressing phoB^CA(E11K) (white triangles) are shown. For comparative purposes, the growth curve of the suppressed ompR malT(Con) rseA strain (black circles) is included. Values represent the means of triplicates, and error bars indicate standard deviations and are shown only when greater than the size of the symbol. (C) Activation of the PhoB regulon increases alkaline phosphatase activity in the ompR malT(Con) mutant. Cells were grown under nonpermissive conditions and harvested at an OD of approximately 0.3. Alkaline phosphatase activity was measured using p-NPP as a substrate and is expressed as arbitrary units as described in Materials and Methods. Error bars indicate the standard deviations for triplicates.

FIG. 7.

Effect of phoE on survival of ompR malT(Con) pstB and ompR malT(Con) pstC mutants. (A) Colony phenotype of the ompR malT(Con) pstC (AJW3946) (left) and the ompR malT(Con) pstC phoE (AJW4197) (right) mutants. Colonies were grown in LB at 37°C. (B) Growth curves of the ompR malT(Con) (AJW3098) (gray diamonds), ompR malT(Con) pstB (AJW3945) (white squares), ompR malT(Con) pstB phoE (AJW4196) (black squares), ompR malT(Con) pstC (AJW3946) (white triangles), and ompR malT(Con) pstC phoE (AJW4197) (black triangles) mutants. Cells were grown in LB at 37°C, and samples were taken at regular intervals. Values represent the means of triplicates, and error bars indicate standard deviations and are shown only when greater than the size of the symbol. (C) Deletion of phoE was confirmed using outer membrane preparations. Cells were grown in LB at 37°C and harvested during late exponential phase. Gels were stained with Coomassie brilliant blue. Lane 1, ompR malT(Con) mutant (AJW3098); lane 2, ompR malT(Con) pstB mutant (AJW3945); lane 3, ompR malT(Con) pstB phoE mutant (AJW4196); lane 4, ompR malT(Con) pstC mutant (AJW3946); lane 5, ompR malT(Con) pstC phoE mutant (AJW4197).

FIG. 8.

Effect of inorganic phosphate on ompR malT(Con) survival. Growth curves of the ompR malT(Con) mutant (AJW3098) in LB medium containing 80 mM NaCl (gray diamonds) and in LB containing 100 mM sodium phosphate (pH 7) instead of NaCl (white squares) are shown. Cells were grown at 37°C, and samples were taken at regular intervals. Values represent the means of triplicates, and error bars indicate standard deviations and are shown only when greater than the size of the symbol.

FIG. 9.

The multifactorial role of OmpR in the prevention of cell death caused by increased LamB levels in malT(Con) mutants. The main cause of ompR malT(Con) synthetic lethality is increased LamB expression in the absence of OmpR. Cell death can be prevented by several avenues, as follows. (i) Reduced expression of LamB eliminates the main cause of lethality. (ii) The activation of σ^E by OmpR leads to increased abundance of LPS in the outer membrane, restoring the porin/LPS balance. (iii) The increased abundance of the OMP PhoE restores the porin/LPS balance. (iv) The uptake of inorganic phosphate by PhoE enables phosphate or a derivative to prevent cell death.