Global analysis of Escherichia coli gene expression during the acetate-induced acid tolerance response

Abstract

The ability of Escherichia coli to survive at low pH is strongly affected by environmental factors, such as composition of the growth medium and growth phase. Exposure to short-chain fatty acids, such as acetate, proprionate, and butyrate, at neutral or nearly neutral pH has also been shown to increase acid survival of E. coli and Salmonella enterica serovar Typhimurium. To investigate the basis for acetate-induced acid tolerance in E. coli O157:H7, genes whose expression was altered by exposure to acetate were identified using gene arrays. The expression of 60 genes was reduced by at least twofold; of these, 48 encode components of the transcription-translation machinery. Expression of 26 genes increased twofold or greater following treatment with acetate. This included six genes whose products are known to be important for survival at low pH. Five of these genes, as well as six other acetate-induced genes, are members of the E. coli RpoS regulon. RpoS, the stress sigma factor, is known to be required for acid tolerance induced by growth at nonlethal low pH or by entry into stationary phase. Disruption of the rpoS gene by a transposon insertion mutation also prevented acetate-induced acid tolerance. However, induction of RpoS expression did not appear to be sufficient to activate the acid tolerance response. Treatment with either NaCl or sodium acetate (pH 7.0) increased expression of an rpoS::lacZ fusion protein, but only treatment with acetate increased acid survival.

FIG. 1

Effects of NaCl and sodium acetate on growth of E. coli O157:H7. Cultures of E. coli O157:H7 were grown at 37°C in supplemented M63 glucose medium as described in Materials and Methods. Culture growth was monitored by measuring turbidity with a Klett-Summerson colorimeter. When cultures reached ca. 2 × 10⁸ cells ml⁻¹, either NaCl or sodium acetate (pH 7.0) was added to the following final concentrations: 100 mM NaCl (solid diamonds); 25 mM acetate (open circles); 50 mM acetate (solid triangles); or 100 mM acetate (open squares). The arrow indicates the time when NaCl or sodium acetate was added to each culture. Addition of 25 or 50 mM NaCl had the same effect on growth rate as addition of 100 mM NaCl (data not shown).

FIG. 2

Pretreatment with sodium acetate provides protection against oxidative stress. Cultures of E. coli O157:H7 were grown at 37°C in supplemented M63 glucose medium as described in Materials and Methods to ca. 2 × 10⁸ cells ml⁻¹ prior to addition of either deionized water (circles), NaCl to a final concentration of 100 mM (diamonds), or sodium acetate (pH 7.0) to a final concentration of 100 mM (squares). One hour later, a portion of each culture was removed, H₂O₂ was added to a final concentration of 15 mM, and these samples were incubated in a 37°C water bath without aeration. Samples of each culture were diluted, and aliquots were plated onto LB plates to determine the number of viable cells at each time point. One hundred percent survival corresponds to the CFU per milliliter determined immediately before the addition of H₂O₂. The results of a typical experiment are shown.

FIG. 3

Pretreatment with NaCl or sodium acetate provides protection against heat killing at 50°C. Cultures were grown at 37°C in supplemented M63 glucose medium as described in Materials and Methods to ca. 2 × 10⁸ cells ml⁻¹ prior to addition of either deionized water (circles), NaCl to a final concentration of 100 mM (diamonds), or sodium acetate (pH 7.0) to a final concentration of 100 mM (squares). One hour later, a sample of each culture was removed, and cells were diluted into either M63 salts warmed at 50°C or M63 salts at room temperature. Samples of each culture were diluted, and aliquots were plated onto LB plates to determine the number of viable cells at each time point. One hundred percent survival corresponds to the CFU per milliliter in the room temperature control. The results shown are the means (± 1 SD) of three independent experiments.

FIG. 4

Effects of NaCl and sodium acetate treatment on expression of the rpoS742::lacZ fusion protein. β-Galactosidase activity is expressed in Miller units (ΔOD₄₂₀ per OD₆₀₀ per min). The results shown are the means of two independent experiments. Less than 20% variation was seen in the level of β-galactosidase activity between the two experiments.