Identification of the 4-hydroxyphenylacetate transport gene of Escherichia coli W: construction of a highly sensitive cellular biosensor.
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 Kt and Vmax values of 25 microM and 3 nmol/min per 10(9) 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 beta-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.
Prieto
M A
MA
Department of Molecular Microbiology, Centro de Investigaciones Biológicas (CSIC), Madrid, Spain.
García
J L
JL
D001667:Binding, Competitive; D015374:Biosensing Techniques; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000502:physiology*; D005798:Genes, Bacterial; D007700:Kinetics; D005810:Multigene Family; D010648:Phenylacetates / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012680:Sensitivity and Specificity
D002352:Carrier Proteins; D010648:Phenylacetates; D011994:Recombinant Proteins; C026246:4-hydroxyphenylacetate