María Auxiliadora Prieto, JoséL. García
Sep 8, 1997
Citations
0
Influential Citations
34
Citations
Quality indicators
Journal
FEBS Letters
Abstract
The mechanism of uptake of 4‐hydroxyphenylacetate (4‐HPA) by Escherichia coli W. was investigated. The 4‐HPA uptake was induced by 4‐HPA, 3‐hydroxyphenylacetate (3‐HPA) or phenylacetate (PA) and showed saturation kinetics with apparent K t and V max values of 25 μM and 3 nmol/min per 109 cells, respectively. Transport of 4‐HPA was resistant to N,N″‐dimethylcarbodiimide (DCCD), but was completely inhibited by cyanide and 4‐nitrophenol, and, to a lower extent, by arsenate and azide, suggesting that energy is required for the uptake process. Competition studies showed that 4‐HPA uptake was inhibited by 3‐HPA or 3,4‐dihydroxyphenylacetate (3,4‐DHPA) but not by 2‐hydroxyphenylacetate (2‐HPA), l‐tyrosine or other structural analogues, indicating a narrow specificity of the transport system. We have demonstrated, using two experimental approaches, that the hpaX gene of the 4‐HPA catabolic cluster, which encodes a protein of the superfamily of transmembrane facilitators, is responsible for 4‐HPA transport. Aside from the aromatic amino acid transport systems, hpaX is the first transport gene for an aromatic compound of enteric bacteria that has been characterized. A highly sensitive cellular biosensor has been constructed by coupling the 4‐HPA transport system to a regulatory circuit that controls the production of β‐galactosidase. This biosensor has allowed us to demonstrate that the transport system performs efficiently at very low external concentrations of 4‐HPA, similar to levels that would be expected to occur in natural environments.