Toxicity caused by hydroxycinnamoyl-coenzyme A thioester accumulation in mutants of Acinetobacter sp. strain ADP1.
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 DeltahcaA 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.
Parke
Donna
D
Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520-8103, USA. donna.parke@yale.edu
Ornston
L Nicholas
LN
D000150:Acinetobacter / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D002109:Caffeic Acids / Q000378:metabolism; D003066:Coenzyme A Ligases / Q000235:genetics / Q000378:metabolism*; D003373:Coumaric Acids / Q000378:metabolism*; D003470:Culture Media; D004952:Esters / Q000633:toxicity; D062385:Hydroxybenzoates / Q000378:metabolism; D009154:Mutation
D002109:Caffeic Acids; D003373:Coumaric Acids; D003470:Culture Media; D004952:Esters; D062385:Hydroxybenzoates; C009091:protocatechuic acid; C004999:ferulic acid; D003066:Coenzyme A Ligases; C021393:hydroxycinnamate-CoA ligase; C040048:caffeic acid