Pseudomonas aeruginosa Ethanol Oxidation by AdhA in Low-Oxygen Environments

Abstract

Pseudomonas aeruginosa has a broad metabolic repertoire that facilitates its coexistence with different microbes. Many microbes secrete products that P. aeruginosa can then catabolize, including ethanol, a common fermentation product. Here, we show that under oxygen-limiting conditions P. aeruginosa utilizes AdhA, an NAD-linked alcohol dehydrogenase, as a previously undescribed means for ethanol catabolism. In a rich medium containing ethanol, AdhA, but not the previously described PQQ-linked alcohol dehydrogenase, ExaA, oxidizes ethanol and leads to the accumulation of acetate in culture supernatants. AdhA-dependent acetate accumulation and the accompanying decrease in pH promote P. aeruginosa survival in LB-grown stationary-phase cultures. The transcription of adhA is elevated by hypoxia and under anoxic conditions, and we show that it is regulated by the Anr transcription factor. We have shown that lasR mutants, which lack an important quorum sensing regulator, have higher levels of Anr-regulated transcripts under low-oxygen conditions than their wild-type counterparts. Here, we show that a lasR mutant, when grown with ethanol, has an even larger decrease in pH than the wild type (WT) that is dependent on both anr and adhA The large increase in AdhA activity is similar to that of a strain expressing a hyperactive Anr-D149A variant. Ethanol catabolism in P. aeruginosa by AdhA supports growth on ethanol as a sole carbon source and electron donor in oxygen-limited settings and in cells growing by denitrification under anoxic conditions. This is the first demonstration of a physiological role for AdhA in ethanol oxidation in P. aeruginosaIMPORTANCE Ethanol is a common product of microbial fermentation, and the Pseudomonas aeruginosa response to and utilization of ethanol are relevant to our understanding of its role in microbial communities. Here, we report that the putative alcohol dehydrogenase AdhA is responsible for ethanol catabolism and acetate accumulation under low-oxygen conditions and that it is regulated by Anr.

Keywords: AdhA; ExaA; Pseudomonas aeruginosa; ethanol; lasR.

FIG 1

Ethanol catabolism in P. aeruginosa. ExaA is a PQQ-linked enzyme that, with a soluble cytochrome c (ExaB), catalyzes the oxidation of ethanol to acetaldehyde. ExaC then oxidizes acetaldehyde to acetate. In this work, we show that AdhA is regulated by Anr in response to oxygen limitation and that AdhA can also catalyze ethanol oxidation as a growth substrate or for conversion to acetate.

FIG 2

Ethanol catabolism by P. aeruginosa leads to lower medium pH through the accumulation of acetate. P. aeruginosa PA14 strains, including wild-type, ΔexaA, exaA::TnM, ΔadhA, ΔadhA adhA, and ΔexaA ΔadhA strains, were grown in LB for 12 h in tubes on a roller drum. (A) pH values of cultures, measured using pH indicator strips, in LB (dotted line) and in LB plus ethanol (bars). (B) Ethanol remaining in culture supernatants relative to that in uninoculated LB plus 1% ethanol (dotted line). (C) Acetate concentrations in supernatants in LB plus 1% ethanol in the same cultures analyzed in panels A and B. (D) Optical density of strains grown in LB (gray) and LB plus 1% ethanol (black). (E) Final culture pH values of P. aeruginosa PAO1 wild-type and ΔadhA strains after 16 h of growth in LB or in LB with 1% ethanol (EtOH). All cultures were grown at 37°C. In panels A to C, samples with different lowercase letters are significantly different, P < 0.05. In panel D, values for ethanol-grown cultures were slightly but significantly higher (P < 0.05) for each strain except WT and the ΔadhA mutant. ****, P < 0.001; ns, not significant.

FIG 3

Ethanol and pH effects on P. aeruginosa stationary-phase survival in LB. The CFU values from 8-h, 24-h, and 48-h cultures were measured for PA14 wild-type (WT), ΔadhA, and ΔexaA ΔadhA strains in LB, LB with 1% ethanol (EtOH), and LB buffered to pH 7 in a single experiment shown in three panels for ease of comparison. *, P < 0.05; **, P < 0.01. Lowercase letters indicate significant differences between 48-h cultures across the three conditions tested; a or b, P < 0.05. Data shown are from a single experiment with three independent replicates at each time point. The experiment was repeated with similar results two additional times.

FIG 4

Culture pH after growth in LB plus 1% ethanol for 16 h. (A) The pH values for cultures of P. aeruginosa PA14 wild-type (WT), ΔadhA, ΔadhA adhA, Δanr, anr-D149A, ΔlasR, ΔlasR Δanr, ΔlasR ΔadhA, and ΔlasR lasR strains after growth in LB (dashed line) or LB with 1% ethanol (bars). Significance for comparisons of the LB plus ethanol cultures was performed using a one-way ANOVA and Tukey’s multiple-comparison test; samples with the same lowercase letter were not significantly different (P > 0.05). (B) The overexpression of adhA in the Δanr mutant was sufficient to restore medium acidification in the presence of ethanol. WT, Δanr, and Δanr strains with an empty vector (EV) or overexpressing adhA were grown without (Control) or with (+EtOH) ethanol for 16 h followed by analysis of the culture pH. The OD values of the strains bearing either vector were, on average, 1 OD₆₀₀ unit lower than the WT or Δanr strains, but the EV and adhA-expressing culture densities were similar.

FIG 5

Growth of P. aeruginosa PA14 strains on ethanol as a sole carbon source. (A) Growth in M63 liquid medium with ethanol as the sole carbon source in 0.2% oxygen. (B) P. aeruginosa strains grown in M63 liquid medium with ethanol as a sole carbon source in anoxia with 50 mM KNO₃. Data are representative of at least three independent experiments, each with three or more biological replicates. Statistics are based on one-way ANOVA and Tukey’s multiple-comparison test; **, P < 0.01; ****, P < 0.001; ns, not significant. Statistics presented indicate differences from the WT unless otherwise indicated.

FIG 6

Anr regulation of adhA expression. P. aeruginosa strains grown in M63 liquid medium with ethanol as the sole carbon source in a 0.2% oxygen atmosphere. The strains included wild-type strain PA14 (WT) and ΔadhA, ΔadhA adhA, Δanr, and Δanr anr mutants as well as a Δanr mutant containing an empty vector (pEV) or a plasmid with adhA under the control of an arabinose-inducible promoter (padhA). Statistics based on one-way ANOVA and Tukey’s multiple-comparison test; unless otherwise indicated, statistical comparisons are to the WT culture. ****, P < 0.0001; **, P < 0.01.