Acetate Metabolism and the Inhibition of Bacterial Growth by Acetate

Abstract

During aerobic growth on glucose, Escherichia coli excretes acetate, a mechanism called “overflow metabolism.” At high concentrations, the secreted acetate inhibits growth. Several mechanisms have been proposed for explaining this phenomenon, but a thorough analysis is hampered by the diversity of experimental conditions and strains used in these studies. Here, we describe the construction of a set of isogenic strains that remove different parts of the metabolic network involved in acetate metabolism. Analysis of these strains reveals that (i) high concentrations of acetate in the medium inhibit growth without significantly perturbing central metabolism; (ii) growth inhibition persists even when acetate assimilation is completely blocked; and (iii) regulatory interactions mediated by acetyl-phosphate play a small but significant role in growth inhibition by acetate. The major contribution to growth inhibition by acetate may originate in systemic effects like the uncoupling effect of organic acids or the perturbation of the anion composition of the cell, as previously proposed. Our data suggest, however, that under the conditions considered here, the uncoupling effect plays only a limited role. IMPORTANCE High concentrations of organic acids such as acetate inhibit growth of Escherichia coli and other bacteria. This phenomenon is of interest for understanding bacterial physiology but is also of practical relevance. Growth inhibition by organic acids underlies food preservation and causes problems during high-density fermentation in biotechnology. What causes this phenomenon? Classical explanations invoke the uncoupling effect of acetate and the establishment of an anion imbalance. Here, we propose and investigate an alternative hypothesis: the perturbation of acetate metabolism due to the inflow of excess acetate. We find that this perturbation accounts for 20% of the growth-inhibitory effect through a modification of the acetyl phosphate concentration. Moreover, we argue that our observations are not expected based on uncoupling alone.

Enhanced production of heterologous proteins by a synthetic microbial community: Conditions and trade-offs

“…11. The Monod-like function for acetate uptake used in this study was motivated by experimental data describing a monotonic increase of the growth rate with increasing acetate concentration, over the relevant range considered here [16,56]. The non-monotonicity of the function used by Harvey et al, however, is also supported by experimental data (Tomas Gedeon, personal communication).…”

“…In the absence of glucose, E. coli can utilize acetate as an alternative substrate, taken up at a rate r a up [g gDW −1 h −1 ] and converted into biomass with a yield coefficient Y a [gDW g −1 ]. Accumulation of acetate in the medium inhibits bacterial metabolism and growth [15][16][17]. The producer strain differs from a wild-type E. coli strain in that it carries a plasmid for the inducible expression of a heterologous protein.…”

“…The inhibition term with the constant Θ a [g L −1 ] in Eq. 10 represents the inhibitory effect of acetate on bacterial growth [15][16][17], whereas the inhibition term with the constant Θ g [g gDW −1 h −1 ] in Eq. 11 corresponds to carbon catabolite repression (CCR), that is, the repression of enzymes in the metabolism of acetate and many other secondary carbon sources while cells are growing on glucose [34,47].…”

“…Highdensity fermentation in industrial bioreactors may operate at a lower pH though [76], exacerbating the growth-inhibitory effect of acetate. We therefore used a second data set on the effect of acetate on the growth rate of E. coli, carried out at pH 6.4 [16], to adjust the parameter Θ a characterizing acetate inhibition. This decreased the value of Θ a from 3.5 g L −1 to 0.52 g L −1 (S2 Fig.…”

“…Apart from being a model system for studying interactions in naturally occurring microbial communities [14], the results of this evolution experiment also suggest novel applications. The secretion of acetate is not only wasteful but also toxic, as its accumulation in the medium inhibits bacterial growth [15][16][17]. Growth inhibition by acetate and other weak acids is a well-known problem in industrial biotechnology, where it limits the productivity of strains modified to convert a substrate into a recombinant protein or a metabolite of interest [18,19].…”