Conserved cytoplasmic loops are important for both the transport and chemotaxis functions of PcaK, a protein from Pseudomonas putida with 12 membrane-spanning regions.

Aromatic compounds are commonly present in the environment as breakdown products of the complex plant polymer lignin and as environmental pollutants. The enzymology and genetic regulation of metabolic pathways required for the aerobic degradation of a variety of aromatic acids and aromatic hydrocarbons by bacteria have been studied extensively (2, 10, 14, 27, 40). By contrast, chemotaxis and transport, two important events that precede degradation, have received little attention. The aromatic acid 4-hydroxybenzoate (4-HBA) is a strong chemoattractant for Pseudomonas putida (15). Metabolism of 4-HBA is not required for chemotaxis because mutants blocked in 4-HBA degradation respond to 4-HBA just as well as the wild-type parent (12). In previous work, we determined that PcaK, a transporter of 4-HBA (13, 25, 26), also acts as a chemoreceptor for this compound. pcaK mutants are nonchemotactic to 4-HBA and structurally related aromatic acids including benzoate (13). The role of PcaK in chemotaxis is not simply to catalyze the intracellular accumulation of 4-HBA because at the concentrations (millimolar) at which chemotaxis is tested, sufficient 4-HBA diffuses into cells that the transport function of PcaK is not needed to allow for wild-type growth rates (13). Although PcaK mediates chemotaxis to 4-HBA, it is not similar to the transmembrane chemoreceptors (methyl-accepting chemotaxis proteins [MCPs]) that have been studied extensively in Escherichia coli and Salmonella typhimurium (37). Rather, PcaK belongs to a large group of transport proteins called the major facilitator superfamily (MFS) (23, 29). The MFS has recently been reevaluated and expanded to include 18 different transporter families that can be loosely grouped according to substrate specificities. PcaK is the founding member of the newly defined family 15 for aromatic acid/H+ symporters within the MFS (29). Permeases from the different MFS families typically share little overall sequence identity. However, all have in common 12 or 14 predicted transmembrane regions and a series of conserved amino acid residues [GXXXD(R/K)XGR(R/K)] in the cytoplasmic hydrophilic loop between the second and third (2-3 loop) membrane-spanning segments. This motif is repeated, although with a lesser degree of conservation, in the cytoplasmic hydrophilic loop between the eighth and ninth (8-9 loop) transmembrane segments of the permease. Certain amino acids within the 2-3 loop have been shown to be required for substrate transport in LacY, the lactose permease of E. coli, and TetA, a tetracycline antiporter of E. coli encoded by transposon Tn10 (19, 32, 41–43). PcaK is predicted to have 12 membrane-spanning regions, and it contains the two conserved stretches of amino acids known to be required for substrate transport by LacY and TetA. To date, PcaK is the only MFS transporter to be implicated in chemoreception. As a start to determining how PcaK functions as both a transporter and a chemoreceptor for 4-HBA, we used site-directed mutagenesis to examine the functional significance of the PcaK 2-3 and 8-9 cytoplasmic loops for these two processes. Our results show that mutations in the 2-3 and 8-9 hydrophilic loops that affect transport also affect chemotaxis.