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 Escherich ia coli W was investigated, The 4-HPA uptake was induced by 4-HPA, 3-h ydroxyphenylacetate (3-HPA) or phenylacetate (PA) and showed saturatio n kinetics with apparent K-t and V-max values of 25 mu M and 3 nmol/mi n per 10(9) cells, respectively, Transport of 4-HPA was resistant to N ,N'-dimethylcarbodiimide (DCCD), but was completely inhibited by cyani de and 4-nitrophenol, and, to a lower extent, by arsenate and azide, s uggesting that energy is required for the uptake process, Competition studies showed that 4-HPA uptake was inhibited by 3-HPA or 3,4-dihydro xyphenylacetate (3,4-DHPA) but not by 2-hydroxyphenylacetate (2-HPA), L-tyrosine or other structural analogues, indicating a narrow specific ity of the transport system, We have demonstrated, using two experimen tal approaches, that the hpaX gene of the 4-HPA catabolic cluster, whi ch encodes a protein of the superfamily of transmembrane facilitators, is responsible for 4-HPA transport, Aside from the aromatic amino aci d transport systems, hpaX is the first transport gene for an aromatic compound of enteric bacteria that has been characterized, A highly sen sitive cellular biosensor has been constructed by coupling the 4-HPA t ransport system to a regulatory circuit that controls the production o f beta-galactosidase, This biosensor has allowed us to demonstrate tha t the transport system performs efficiently at very low external conce ntrations of 4-HPA, similar to levels that would be expected to occur in natural environments. (C) 1997 Federation of European Biochemical S ocieties.