Toxicity Caused by Hydroxycinnamoyl-Coenzyme A Thioester Accumulation in Mutants of Acinetobacter sp. Strain ADP1

Abstract

Hydroxycinnamates, aromatic compounds that play diverse roles in plants, are dissimilated by enzymes encoded by the hca genes in the nutritionally versatile, naturally transformable bacterium Acinetobacter sp. strain ADP1. A key step in the hca-encoded pathway is activation of the natural substrates caffeate, p-coumarate, and ferulate by an acyl:coenzyme A (acyl:CoA) ligase encoded by hcaC. As described in this paper, Acinetobacter cells with a knockout of the next enzyme in the pathway, hydroxycinnamoyl-CoA hydratase/lyase (HcaA), are extremely sensitive to the presence of the three natural hydroxycinnamate substrates; Escherichia coli cells carrying a subclone with the hcaC gene are hydroxycinnamate sensitive as well. When the hcaA mutation was combined with a mutation in the repressor HcaR, exposure of the doubly mutated Acinetobacter cells to caffeate, p-coumarate, or ferulate at 10 ؊6 M totally inhibited the growth of cells. The toxicity of p-coumarate and ferulate to a ⌬hcaA strain was found to be a bacteriostatic effect. Although not toxic to wild-type cells initially, the diphenolic caffeate was itself converted to a toxin over time in the absence of cells; the converted toxin was bactericidal. In an Acinetobacter strain blocked in hcaA, a secondary mutation in the ligase (HcaC) suppresses the toxic effect. Analysis of suppression due to the mutation of hcaC led to the development of a positive-selection strategy that targets mutations blocking HcaC. An hcaC mutation from one isolate was characterized and was found to result in the substitution of an amino acid that is conserved in a functionally characterized homolog of HcaC.

Chlorophenol Hydroxylases Encoded by Plasmid pJP4 Differentially Contribute to Chlorophenoxyacetic Acid Degradation

“…Intoxication due to unbalanced turnover of chlorophenols produces an immediate effect on chlorophenoxyacetate degradation and should be considered an important factor determining the extent and rate of growth of chlorophenoxyacetate-degrading strains on these xenobiotic compounds. Interestingly, toxicity by an imbalance between the production and degradation of initial intermediates in aromatic metabolism has also been reported for 3-chlorobenzoate (28), hydroxycinnamate (26), and protocatechuate (9).…”

Aromatic degradative pathways in Acinetobacter baylyi underlie carbon catabolite repression

“…To induce expression of the hca genes, p-coumarate was used. It was added in a low concentration (1 mM) because of the known toxic effect caused by the presence of accumulated pcoumaroyl-CoA (Parke & Ornston, 2004). A combination of succinate and acetate in the presence of p-coumarate led to a 14-fold reduced luciferase activity in comparison with lactate (Fig.…”

“…In the hca operon, a transcriptional hcaE-lacZ fusion was used to detect the promoter activity (Parke & Ornston, 2004). The detected b-galactosidase activity at the expense of succinate and p-coumarate decreased by 53 % in comparison with the activity measured on p-coumarate alone.…”

Anaerobic Catabolism of Aromatic Compounds: a Genetic and Genomic View

“…Lignin and suberin are polymers of phenylpropanoid units, and their biodegradation involves the depolymerization and subsequent catabolism of the derived aromatic monomers such as p-coumarate (4-hydroxycinnamate). p-Coumarate can also be generated from the deamination of tyrosine (42,272). Two different routes for aerobic p-coumarate catabolism have been described, i.e., a ␤-oxidative route and a non-␤-oxidative pathway.…”