An early response to environmental stress involves regulation of OmpX and OmpF, two enterobacterial outer membrane pore-forming proteins

Abstract

Bacterial adaptation to external stresses and toxic compounds is a key step in the emergence of multidrug-resistant strains that are a serious threat to human health. Although some of the proteins and regulators involved in antibiotic resistance mechanisms have been described, no information is available to date concerning the early bacterial response to external stresses. Here we report that the expression of ompX, encoding an outer membrane protein, is increased during early exposure to drugs or environmental stresses. At the same time, the level of ompF porin expression is noticeably affected. Because of the role of these proteins in membrane permeability, these data suggest that OmpF and OmpX are involved in the control of the penetration of antibiotics such as beta-lactams and fluoroquinolones through the enterobacterial outer membrane. Consequently, the early control of ompX and ompF induced by external stresses may represent a preliminary response to antibiotics, thus triggering the initial bacterial line of defense against antibiotherapy.

FIG. 1.

Activation of the ompX-lacZ fusion in response to several compounds and external factors in E. coli ΔompX. The growth rate was controlled in each used condition. Miller units were calculated as described in Materials and Methods. Osmolarity corresponded to osmotic shock obtained in the presence of NaCl. Values are means from five independent experiments, and standard deviations were calculated. The top part presents the Miller units after 60 min of incubation with various conditions (standard deviations were included); the bottom part presents the complete curves (standard deviations were calculated but were omitted for figure clarity).

FIG. 2.

Effect of several compounds and the mar background on the ompX-lacZ fusion in E. coli after 60 min of incubation. Values are means from five independent determinations, and standard deviation are represented.

FIG. 3.

Semiquantitative analysis of ompX expression using RT-PCR. (A and B) RT-PCR of 16S rRNA (A) or ompX (B) on total RNA extracted from ATCC strain 15038 with no treatment (lane 1), novobiocin added (lane 2); osmotic shock (in the presence of NaCl) (lane 3), or salicylate added (lane 4). The concentrations of total RNA were evaluated as described in Materials and Methods. Serial dilutions of total RNA template were performed; lanes a and b show cDNA products amplified from dilutions 1/10⁴ and 1/10⁵, respectively. Only the relevant parts of the gel are shown.

FIG. 4.

Effect of hns on ompX expression in E. coli. (A) Histograms representiing the β-galactosidase activity in the presence or absence of functional hns. Values are means from five independent experiments, and standard deviation are represented. (B) Immunoblots showing analysis of porin production (OmpF and OmpC) in response to the overproduction of OmpX and the presence or absence of hns expression. Lane 1, PS2209; lane 2, PS2652; lane 3, PS2209+pMD05 (OmpX-overproducing strain); lane 4, PS2652+pMD05 (OmpX-overproducing strain). Control strains were presented for comparison: lane 5, BZB1107 (porin less strain); lane 6, BZB1107+pLG361 (OmpF-producing strain); and lane 7, BZB1107+pMY150 (OmpC-producing strain).