Acetate and formate stress: opposite responses in the proteome of Escherichia coli

Abstract

Acetate and formate are major fermentation products of Escherichia coli. Below pH 7, the balance shifts to lactate; an oversupply of acetate or formate retards growth. E. coli W3110 was grown with aeration in potassium-modified Luria broth buffered at pH 6.7 in the presence or absence of added acetate or formate, and the protein profiles were compared by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Acetate increased the steady-state expression levels of 37 proteins, including periplasmic transporters for amino acids and peptides (ArtI, FliY, OppA, and ProX), metabolic enzymes (YfiD and GatY), the RpoS growth phase regulon, and the autoinducer synthesis protein LuxS. Acetate repressed 17 proteins, among them phosphotransferase (Pta). An ackA-pta deletion, which nearly eliminates interconversion between acetate and acetyl-coenzyme A (acetyl-CoA), led to elevated basal levels of 16 of the acetate-inducible proteins, including the RpoS regulon. Consistent with RpoS activation, the ackA-pta strain also showed constitutive extreme-acid resistance. Formate, however, repressed 10 of the acetate-inducible proteins, including the RpoS regulon. Ten of the proteins with elevated basal levels in the ackA-pta strain were repressed by growth of the mutant with formate; thus, the formate response took precedence over the loss of the ackA-pta pathway. The similar effects of exogenous acetate and the ackA-pta deletion, and the opposite effect of formate, could have several causes; one possibility is that the excess buildup of acetyl-CoA upregulates stress proteins but excess formate depletes acetyl-CoA and downregulates these proteins.

FIG. 1

Metabolic pathways connecting pyruvate with fermentation acids in E. coli.

FIG. 2

Proteins differentially expressed with acetic acid or in the ackA-pta strain. The layered view superimposes two composite images representing different growth conditions. Each composite image is based on three 2-D gels from replicate cultures. All E. coli cultures were grown at 37°C to an OD₆₀₀ of 0.4 in LBK broth buffered with 50 mM MOPS and 50 mM TES, pH 6.7. (A) Strain W3110 was grown in buffered LBK broth with (pink) or without (green) 50 mM acetic acid. (B) Strain W3110 ackA-pta (pink) or W3110 (green) was grown in buffered LBK broth. In the layered views, pink and green indicate proteins induced and repressed, respectively, in the presence of acetic acid (A) or proteins constitutively induced or repressed in the ackA-pta strain (B), based on pairwise comparison of individual gels (summarized in Table 1). The horizontal axis represents the approximate pH range of the isoelectric focusing first dimension. The vertical axis represents the molecular mass in kilodaltons. Proteins in the 2-D gels were silver stained. For N-terminal sequence identification, the proteins were electroblotted and stained with Coomassie blue.

FIG. 3

Proteins differentially expressed with (pink) or without (green) 20 mM formic acid. (A) Strain W3110; (B) strain W3110 ackA-pta. Other growth and gel conditions were as for Fig. 2.

FIG. 4

Proteins differentially expressed with (pink) or without (green) 50 mM acetic acid. (A) Strain W3110 ackA-pta was grown in LBK broth–50 mM MOPS–50 mM TES, pH 6.7, to an OD₆₀₀ of 0.4. (B) Strain W3110 was grown in glycerol M63 salts–50 mM MOPS–50 mM TES, pH 6.7, to an OD₆₀₀ of 0.2. Other growth and gel conditions were as for Fig. 2.