Expression of outer membrane proteins in Escherichia coli growing at acid pH

Abstract

It is generally accepted for Escherichia coli that (i) the level of OmpC increases with increased osmolarity when cells are growing in neutral and alkaline media, whereas the level of OmpF decreases at high osmolarity, and that (ii) the two-component system composed of OmpR (regulator) and EnvZ (sensor) regulates porin expression. In this study, we found that OmpC was expressed at low osmolarity in medium of pH below 6 and that the expression was repressed when medium osmolarity was increased. In contrast, the expression of ompF at acidic pH was essentially the same as that at alkaline pH. Neither OmpC nor OmpF was detectable in an ompR mutant at both acid and alkaline pH values. However, OmpC and OmpF were well expressed at acid pH in a mutant envZ strain, and their expression was regulated by medium osmolarity. Thus, it appears that E. coli has a different mechanism for porin expression at acid pH. A mutant deficient in ompR grew slower than its parent strain in low-osmolarity medium at acid pH (below 5.5). The same growth diminution was observed when ompC and ompF were deleted, suggesting that both OmpF and OmpC are required for optimal growth under hypoosmosis at acid pH.

FIG. 1

Urea-SDS–polyacrylamide gel electrophoresis of outer membrane proteins. W3110 (wild type) was grown in medium A of initial pH 5.5 (lanes 1 to 3) and pH 8.5 (lanes 4 to 6) and harvested at an absorbance of 0.3 to 0.4 as described in Materials and Methods. When cells were harvested, the medium pH values were 5.1 to 5.3 and 7.9 to 8.1, respectively. Densities of lane 1 were taken as 100, and relative densities are represented. Lanes: 1 and 4, cells grown without the addition of NaCl; 2 and 5, cells grown in the presence of 100 mM NaCl; 3 and 6, cells grown in the presence of 200 mM NaCl.

FIG. 2

Expression of OmpC and OmpF at low pH. Cells were grown in medium A of initial pHs 5.5 and 8.5 with and without the addition of 200 mM NaCl. Cells were harvested as described in the legend of Fig. 1. One Miller unit of β-galactosidase activity was defined as described previously (13). Each bar represents the mean ± standard deviation of four independent experiments. (A) MF8.1 (ompF-lacZ; open bars), MSR6 (ompF-lacZ ompR::Tn10; solid bars), and MSZ6 (ompF-lacZ envZ::kan; hatched bars). (B) MKC5.4 (ompC-lacZ; gray bars), MKCR5.4 (ompC-lacZ ompR::Tn10; dotted bars), and MKCZ5.4 (ompC-lacZ envZ::kan; horizontally striped bars).

FIG. 3

Urea-SDS–polyacrylamide gel electrophoresis of outer membrane proteins. Cells grown in medium A of initial pH 5.5 (lanes 1, 2, 5, and 6) and pH 8.5 (lanes 3, 4, 7, and 8) were harvested as described in the legend of Fig. 1. Densities of lanes 1 and 5 were taken as 100, and relative densities are represented. Lanes: 1 and 3, W3110 (wild type); 2 and 4, MH1160 (ompR101); 5 and 7, MSZ31 (W3110 envZ) grown without the addition of NaCl; 6 and 8, MSZ31 (W3110 envZ) grown in the presence of 200 mM NaCl.

FIG. 4

Growth of W3110 and MSR31. W3110 (wild type; solid symbols) and MSR31 (W3110 ompR::Tn10; open symbols) were grown in media A (A) and B (B) of initial pH 5.5. Growth was monitored by measuring the absorbance of the medium.

FIG. 5

Growth of W3110 and MSR in low-osmolarity medium. W3110 (wild type; solid symbols) and MSR31 (W3110 ompR::Tn10; open symbols) were grown in medium C of initial pHs 8.5 (A) and 5.5 (B). Growth was monitored by determining the viable cell count. Symbols: circles, no addition of NaCl; triangles, 200 mM NaCl added.

FIG. 6

Growth of mutants deficient in OmpC and OmpF in medium of initial pH 5.0. Cells were grown in medium B of initial pH 5.0 (open symbols) and the same medium containing 200 mM NaCl (solid symbols). Growth was monitored by measuring the absorbance of the medium. (A) ● and ○, W3110 (ompR⁺); ■ and □, MSR31 (ompR). (B) ● and ○, MKW505 (ompC⁺ ompF⁺); ■ and □, MKCF36 (ompC ompF); Δ, MKC505 (ompC ompF⁺); ▿, MH621 (ompC⁺ ompF).