Genome-wide transcriptional profiling of the Escherichia coli responses to superoxide stress and sodium salicylate

Abstract

Escherichia coli responds to oxidative stress by activating sets of coregulated genes that help the cell to maintain homeostasis. Identified previously by genetic and biochemical approaches, the soxRS system mediates the induction of 18 of these redox-inducible genes (including the soxS gene itself). An overlapping set of genes is activated by an assortment of structurally unrelated molecules with antibiotic activities; many genes in this response are controlled by the marRAB system. The activation of either the soxRS or the marRAB system results in enhanced resistance to both superoxide-generating agents and multiple antibiotics. In order to probe the extent of these regulatory networks, we have measured whole-genome transcriptional profiles of the E. coli response to the superoxide-generating agent paraquat (PQ), an inducer of the soxRS system, and to the weak acid salt sodium salicylate (NaSal), an inducer of the marRA system. A total of 112 genes was modulated in response to PQ, while 134 genes were modulated in response to NaSal. We have also obtained transcriptional profiles of the SoxS and MarA regulons in the absence of global stress, in order to establish the regulatory hierarchies within the global responses. Several previously unrelated genes were shown to be under SoxS or MarA control. The genetic responses to both environmental insults revealed several common themes, including the activation of genes coding for functions that replenish reducing potential; regulate iron transport and storage; and participate in sugar and amino acid transport, detoxification, protein modification, osmotic protection, and peptidoglycan synthesis. A large number of PQ- and NaSal-responsive genes have no known function, suggesting that many adaptive metabolic changes that ensue after stress remain uncharacterized.

FIG. 1

Growth of E. coli strain GC4468. OD₆₀₀ as a function of time for strain GC4468 was measured. The cultures were either left untreated (empty circles) or treated with 250 μM PQ (filled circles). PQ was added to log-phase cultures (empty arrow), and cells were harvested after 45 min (filled arrow). For detailed culture conditions, see Materials and Methods.

FIG. 2

Scatter plot of expression levels for the E. coli genome: untreated and PQ-treated cells. The percent total intensity for each gene represented in the arrays is plotted on a log scale. A relatively small number of genes had associated signal intensities that were below average background levels. The subtraction of the background value from these signals resulted in a negative corrected value and therefore a negative percentage of total signal. These genes were given an arbitrary value of 0.000001% of total intensity.

FIG. 3

Comparison of expression ratios from transcriptional profiling and Northern blots. (A) Total RNA was extracted from untreated or PQ-treated cultures (250 μM for 45 min). Aliquots from these preparations were used as a template for cDNA synthesis and hybridization with gene arrays or were run in agarose gels, transferred to Nytran membranes, and probed with labeled gene-specific PCR fragments. The expression ratio of 21 genes is shown for Northern blotting (horizontal axis) or cDNA synthesis and hybridization to gene arrays (vertical axis). The genes tested were cyoD, cysK, dnaE, gshB, inaA, lpxC, nadE, nfo, nupC, pdhR, ptsG, pyrB, rpsS, sodA, speE, uraA, yadJ, ybjC, yhiM, and zwf. See Materials and Methods for detailed protocols. (B) Northern blots of the nfo, yaiA, uraA, sodA, gshB, and ptsG genes. −, absence of PQ; +, presence of PQ. A replicate gel was run and stained with ethidium bromide (EtBr), revealing the 16S and 23S rRNA, which serves as loading control.

FIG. 4

Growth of E. coli GC4468 exposed to NaSal. OD₆₀₀ as a function of time for strain GC4468 was determined. The cultures were either left untreated (empty circles) or treated with 5 mM NaSal (filled circles). NaSal was added to log-phase cultures (empty arrow), and cells were harvested after 45 min (filled arrow). For detailed culture conditions, see Materials and Methods.

FIG. 5

Scatter plot of expression levels for the E. coli genome: untreated and NaSal-treated cells. The percent total intensity for each gene represented in the arrays is plotted on a log scale. A relatively small number of genes had associated signal intensities that were below average background levels. The subtraction of the background value from these signals resulted in a negative corrected value and therefore a negative percentage of total signal. These genes were given an arbitrary value of 0.000001% of total intensity.