Transcript profiling and inference of Escherichia coli K-12 ArcA activity across the range of physiologically relevant oxygen concentrations

Abstract

Oxygen availability is the major determinant of the metabolic modes adopted by Escherichia coli. Although much is known about E. coli gene expression and metabolism under fully aerobic and anaerobic conditions, the intermediate oxygen tensions that are encountered in natural niches are understudied. Here, for the first time, the transcript profiles of E. coli K-12 across the physiologically significant range of oxygen availabilities are described. These suggested a progressive switch to aerobic respiratory metabolism and a remodeling of the cell envelope as oxygen availability increased. The transcriptional responses were consistent with changes in the abundance of cytochrome bd and bo' and the outer membrane protein OmpW. The observed transcript and protein profiles result from changes in the activities of regulators that respond to oxygen itself or to metabolic and environmental signals that are sensitive to oxygen availability (aerobiosis). A probabilistic model (TFInfer) was used to predict the activity of the indirect oxygen-sensing two-component system ArcBA across the aerobiosis range. The model implied that the activity of the regulator ArcA correlated with aerobiosis but not with the redox state of the ubiquinone pool, challenging the idea that ArcA activity is inhibited by oxidized ubiquinone. The amount of phosphorylated ArcA correlated with the predicted ArcA activities and with aerobiosis, suggesting that fermentation product-mediated inhibition of ArcB phosphatase activity is the dominant mechanism for regulating ArcA activity under the conditions used here.

FIGURE 1.

The cytochrome bd and bo′ content of cultures of E. coli MG1655 across the aerobiosis scale. The graphs show the amounts of cytochrome bd (A) and cytochrome bo′ (B) (molecules/cell × 10⁻³ on a log₁₀ scale; open bars) calculated as described under “Experimental Procedures” and the -fold changes in the abundance of the cydA (A) and cyoA (B) transcripts relative to that at 0% aerobiosis (data from Table 1) shown on a log₁₀ scale (closed bars) in cultures grown at the indicated aerobiosis points.

FIGURE 2.

Changes in outer membrane protein profiles across the aerobiosis scale. The Coomassie Blue-stained urea/SDS-polyacrylamide gel shows polypeptides present in outer membrane preparations isolated from equivalent numbers of bacteria sampled from steady-state cultures grown at the indicated points on the aerobiosis scale. The proteins were identified by N-terminal amino acid sequencing.

FIGURE 3.

The redox state of the ubiquinone/ubiquinol pool is not the major determinant of ArcA activity. A, the inferred activity of ArcA from the transcriptomic data assuming 100% activity at 0% aerobiosis (■), the measured percentage of ubiquinol (△), the total amount of oxidized ubiquinone (○), and the calculated ArcA activity assuming that the maximum is reached when ArcA is 50% phosphorylated (17) (♦; bars indicate S.D., n = 4) plotted against the aerobiosis scale. B, a typical Western blot developed using ArcA polyclonal antiserum for steady-state cultures grown at different points on the aerobiosis scale. Lane 1, purified ArcA (15 ng) phosphorylated in vitro using carbamoyl phosphate; lane 2, dephosphorylated ArcA (15 ng); lanes 3–8, extracts from cultures grown at 0, 31, 45, 71, 90, and 109% aerobiosis, respectively. Extracts from an arcA mutant did not produce a signal (not shown). dw, dry weight.