Functional heterogeneity of RpoS in stress tolerance of enterohemorrhagic Escherichia coli strains

Abstract

The stationary-phase sigma factor (RpoS) regulates many cellular responses to environmental stress conditions such as heat, acid, and alkali shocks. On the other hand, mutations at the rpoS locus have frequently been detected among pathogenic as well as commensal strains of Escherichia coli. The objective of this study was to perform a functional analysis of the RpoS-mediated stress responses of enterohemorrhagic E. coli strains from food-borne outbreaks. E. coli strains belonging to serotypes O157:H7, O111:H11, and O26:H11 exhibited polymorphisms for two phenotypes widely used to monitor rpoS mutations, heat tolerance and glycogen synthesis, as well as for two others, alkali tolerance and adherence to Caco-2 cells. However, these strains synthesized the oxidative acid resistance system through an rpoS-dependent pathway. During the transition from mildly acidic growth conditions (pH 5.5) to alkaline stress (pH 10.2), cell survival was dependent on rpoS functionality. Some strains were able to overcome negative regulation by RpoS and induced higher beta-galactosidase activity without compromising their acid resistance. There were no major differences in the DNA sequences in the rpoS coding regions among the tested strains. The heterogeneity of rpoS-dependent phenotypes observed for stress-related phenotypes was also evident in the Caco-2 cell adherence assay. Wild-type O157:H7 strains with native rpoS were less adherent than rpoS-complemented counterpart strains, suggesting that rpoS functionality is needed. These results show that some pathogenic E. coli strains can maintain their acid tolerance capability while compromising other RpoS-dependent stress responses. Such adaptation processes may have significant impact on a pathogen's survival in food processing environments, as well in the host's stomach and intestine.

FIG. 1.

Western blot analysis of RpoS expression in pathogenic E. coli strains. Wild-type E. coli strains, rpoS::Ap mutant strain 55, and strain 55-1 carrying pPS4.4 (rpoS) were aerobically grown to the stationary phase in LB-MES medium, and equivalent amounts of protein were loaded into each lane and probed with anti-RpoS antibody to detect RpoS (38 kDa). Individual strains used are indicated above each lane.

FIG. 2.

E. coli strains were grown to the stationary growth phase in LB-MES medium (pH 5.5) and diluted 1:200 in PBS preequilibrated at 58°C. The surviving population was determined at the indicated time points by withdrawing samples and plating appropriate dilutions immediately on LB agar.

FIG. 3.

Glycogen synthesis patterns of pathogenic E. coli strains. The wild-type strains and recombinant strains carrying pPS4.4 were plated on Kornberg agar (containing 50 μg/ml chloramphenicol for recombinant strains) and stained with iodine solution. Dark brown colonies indicate abundant glycogen synthesis, while pale yellow colonies are devoid of glycogen.

FIG. 4.

Phenotypic differences in alkali tolerance among various E. coli strains. E. coli strains were grown to the stationary growth phase in LB (filled bars) or LB-MES (striped bars) medium and diluted 1:200 in 100 mM CAPS buffer (pH 10.2) preequilibrated at 37°C. The surviving population was determined after 4 h by withdrawing samples and plating appropriate dilutions immediately on LB agar.

FIG. 5.

Comparison of β-galactosidase activity with heat and acid survival of various E. coli strains. Heat tolerance data (filled circles) were obtained from Fig. 2, and data on acid resistance by AR1 (open circles) were obtained from Table 1. The regression line (solid) (r² = 0.897) and 99% confidence intervals (broken lines) for heat tolerance and β-galactosidase activity were calculated with SigmaPlot, version 8.0.

FIG. 6.

Adherence phenotypes of pathogenic E. coli strains. Bacteria grown to mid-log phase were used to infect Caco-2 cell monolayers and were allowed to adhere for 1 h at 37°C in a 94% air-6% CO₂ atmosphere. The monolayers were washed three times with Hanks' balanced salt solution and then lysed with 0.1% Triton X-100 in saline. Bacterial adherence (enumerated by spread plate counting) is expressed as the percentage of the inoculum surviving the washing treatment (percent recovery). All assays were conducted in quadruplicate and independently repeated at least twice. Black bars, wild-type E. coli strains; gray bars, strains carrying pPS4.4 (rpoS). The observed differences with 251 versus 251-1, 258 versus 258-1, and 205 versus 205-1 are significant (P < 0.001). Error bars indicate standard deviations.