Repression of phenol catabolism by organic acids in Ralstonia eutropha

Abstract

During batch growth of Ralstonia eutropha (previously named Alcaligenes eutrophus) on phenol in the presence of acetate, acetate was found to be the preferred substrate; this organic acid was rapidly metabolized, and the specific rate of phenol consumption was considerably decreased, although phenol consumption was not abolished. This decrease corresponded to a drop in phenol hydroxylase and catechol-2,3-dioxygenase specific activities, and the synthesis of the latter was repressed at the transcriptional level. Studies with a mutant not able to consume acetate indicated that the organic acid itself triggers the repression. Other organic acids were also found to repress phenol degradation. One of these, benzoate, was found to completely block the catabolism of phenol (diauxic growth). A mutant unable to metabolize benzoate was also unable to develop on benzoate-phenol mixtures, indicating that the organic acid rather than a metabolite involved in benzoate degradation was responsible for the repression observed.

FIG. 1

Kinetics of growth and substrate consumption for R. eutropha 335 (wild type) grown in batch cultures on a phenol-acetate mixture. (A) ▪, biomass; ⧫, phenol; •, acetate. (B) ——, q_phenol; –––, q_acetate; ····, μ. The lines for substrate and biomass concentrations were based on interpolation (degree 4) of the raw data. The lines for μ, q_acetate, and q_phenol were directly derived from the lines for substrates and biomass.

FIG. 2

Key enzymes for the catabolism of phenol and acetate during growth of the culture described in the legend to Fig. 1. Symbols: ○, phenol hydroxyase; □, catechol-2,3-dioxygenase; •, acetyl-CoA synthetase; ⧫, malate synthase; ◊, isocitrate lyase.

FIG. 3

Transcription of the catechol-2,3-dioxygenase gene during growth of the culture described in the legend to Fig. 1. RNA isolated from cells grown on phenol alone was used as the control (lane C).

FIG. 4

Kinetics of growth and substrate consumption for R. eutropha T31 (malate synthase mutant) grown in batch cultures on a phenol-acetate mixture. (A) ▪, biomass; ⧫, phenol; •, acetate. (B) ——, q_phenol; ····, μ; ··–··, theoretical washing curve. The lines for substrate and biomass concentrations were based on interpolation (degree 4) of the raw data. The lines for μ and q_phenol were directly derived from the lines for substrates and biomass.

FIG. 5

Kinetics of growth and substrate consumption for R. eutropha 335 (wild type) grown in batch cultures in the presence of phenol-benzoate (▪), phenol (⧫), or benzoate (•).