Transcriptomic analysis of Escherichia coli O157:H7 and K-12 cultures exposed to inorganic and organic acids in stationary phase reveals acidulant- and strain-specific acid tolerance responses

Abstract

The food-borne pathogen Escherichia coli O157:H7 is commonly exposed to organic acid in processed and preserved foods, allowing adaptation and the development of tolerance to pH levels otherwise lethal. Since little is known about the molecular basis of adaptation of E. coli to organic acids, we studied K-12 MG1655 and O157:H7 Sakai during exposure to acetic, lactic, and hydrochloric acid at pH 5.5. This is the first analysis of the pH-dependent transcriptomic response of stationary-phase E. coli. Thirty-four genes and three intergenic regions were upregulated by both strains during exposure to all acids. This universal acid response included genes involved in oxidative, envelope, and cold stress resistance and iron and manganese uptake, as well as 10 genes of unknown function. Acidulant- and strain-specific responses were also revealed. The acidulant-specific response reflects differences in the modes of microbial inactivation, even between weak organic acids. The two strains exhibited similar responses to lactic and hydrochloric acid, while the response to acetic acid was distinct. Acidulant-dependent differences between the strains involved induction of genes involved in the heat shock response, osmoregulation, inorganic ion and nucleotide transport and metabolism, translation, and energy production. E. coli O157:H7-specific acid-inducible genes were identified, suggesting that the enterohemorrhagic E. coli strain possesses additional molecular mechanisms contributing to acid resistance that are absent in K-12. While E. coli K-12 was most resistant to lactic and hydrochloric acid, O157:H7 may have a greater ability to survive in more complex acidic environments, such as those encountered in the host and during food processing.

FIG. 1.

Acid resistance of K-12 (•, ○) and O157:H7 (▪, □) to BHI acidified to pH 3.5 with acetic, lactic, or hydrochloric acid. The percentages of surviving cells of cultures incubated at pH 7 (acid shock) are represented by dashed lines. The percentages of surviving cells of cultures incubated at pH 5.5 (acid adapted; 3 h for K-12 and 2 h for O157:H7) in the absence and presence of chloramphenicol are represented by solid and dotted lines, respectively. The percentages of survivors were determined by plating cells on TYSG agar. Error bars represent standard errors of the means based on counts from three replicate populations; in most cases, their size was smaller than the symbol.

FIG. 2.

Comparison of levels of genome-wide gene expression in K-12 and O157:H7 after induction of the A-ATR, L-ATR, or H-ATR. Bacteria were adapted for 3 h (K-12) and 2 h (O157:H7) in BHI acidified to pH 5.5. The numbers of differentially expressed genes (i.e., genes with a ≥2-fold difference in expression from that of the reference culture) are shown as a Venn diagram. The first two Venn diagrams compare the gene expression responses of K-12 and O157:H7 during adaptation with each of the test acidulants. In the third Venn diagram, labeled “universal acid response,” numbers of genes which are upregulated or downregulated by both strains during adaptation with each acid are compared. Numbers of upregulated genes are shown in bold, and numbers of downregulated genes are shown in italics.

FIG. 3.

Hierarchical cluster analysis of gene expression in K-12 and O157:H7 after induction of the A-ATR, L-ATR, or H-ATR. The hierarchical cluster analysis was performed in GeneSpringGX with the Pearson correlation. Green and red indicate decreased and increased RNA levels, respectively, relative to that of the reference culture.

FIG. 4.

Resistance of K-12 (•, ○) and O157:H7 (▪, □) to heat shock challenge at 50°C. The percentages of survivors of cultures incubated at pH 7 (acid shock) and at pH 5.5 (acid adapted; 3 h for K-12 and 2 h for O157:H7) are represented by dashed lines and solid lines, respectively. The percentages of survivors were determined by plating cells on TYSG agar. Error bars represent standard errors of the means based on counts from three replicate populations.

FIG. 5.

Functional groups of E. coli genes that are differentially expressed during induction of the A-ATR, L-ATR, or H-ATR. Bars indicate percentages of genes in each group that showed significant changes in expression in K-12 and O157:H7 after adaptation for 3 and 2 h, respectively, in BHI, acidified to pH 5.5 with acetic, lactic, or hydrochloric acid (see Table S1 in the supplemental material). The white bars show the percentages of genes upregulated, and the black bars show the percentages of genes downregulated. Genes were divided into functional categories according to the NCBI (http://www.ncbi.nlm.nih.gov/COG/). Functional categories are abbreviated as follows: J, translation, ribosomal structure and biogenesis; A, RNA processing and modification; K, transcription; L, replication, recombination, and repair; D, cell cycle control, cell division, and chromosome partitioning; V, defense mechanisms; T, signal transduction mechanisms; M, cell wall/membrane/envelope biogenesis; N, cell motility; U, intracellular trafficking and secretion; O, posttranslational modification, protein turnover, and chaperoning; C, energy production and conversion; G, carbohydrate transport and metabolism; E, amino acid transport and metabolism; F, nucleotide transport and metabolism genes; H, coenzyme transport and metabolism; I, lipid transport and metabolism; P, inorganic ion transport and metabolism; and Q, secondary-metabolite biosynthesis, transport, and catabolism.