PcaK, a high-affinity permease for the aromatic compounds 4-hydroxybenzoate and protocatechuate from Pseudomonas putida

Abstract

PcaK is a transporter and chemoreceptor protein from Pseudomonas putida that is encoded as part of the ␤-ketoadipate pathway regulon for aromatic acid degradation. When expressed in Escherichia coli, PcaK was localized to the membrane and catalyzed the accumulation of two aromatic substrates, 4-hydroxybenzoate and protocatechuate, against a concentration gradient. Benzoate inhibited 4-hydroxybenzoate uptake but was not a substrate for PcaK-catalyzed transport. A P. putida pcaK mutant was defective in its ability to accumulate micromolar amounts of 4-hydroxybenzoate and protocatechuate. The mutant was also impaired in growth on millimolar concentrations of these aromatic acids. In contrast, the pcaK mutant grew at wild-type rates on benzoate. The V max for uptake of 4-hydroxybenzoate was at least 25 nmol/min/mg of protein, and the K m was 6 M. PcaK-mediated transport is energized by the proton motive force. These results show that although aromatic acids in the undissociated (uncharged) form can diffuse across bacterial membranes, high-specificity active transport systems probably also contribute to the ability of bacteria to grow on the micromolar concentrations of these compounds that are typically present in soil. A variety of aromatic molecules, including naturally occurring lignin derivatives and xenobiotics, are metabolized by bacteria and may be substrates for transport proteins. The characterization of PcaK provides a foundation for understanding active transport as a critical step in the metabolism of aromatic carbon sources.Aromatic acids, present in soil as degradation products of plant material, are used as carbon and energy sources by many microorganisms. A battery of enzymes is required for aromatic compound degradation, and it seems likely that a corresponding array of transport proteins initiates metabolism. Aromatic acids can diffuse across biological membranes (23), making transport theoretically unnecessary. However, accumulating evidence indicates that active transport of this group of compounds may be widespread among bacteria (1,2,14,15,28,30,38). Recently, the molecular basis for aromatic acid transport has begun to be examined.A permease, designated PcaK, was identified in Pseudomonas putida as a transporter of the aromatic acid 4-hydroxybenzoate (4-HBA) (15). PcaK is a member of the major facilitator superfamily (MFS) (13, 29) of transport proteins. Like other MFS permeases, PcaK has 12 predicted membrane-spanning regions, with several conserved amino acid residues in the hydrophilic loop between the second and third membranespanning segments (15,19,20). Previous work (32) has shown that the pcaK gene is regulated coordinately with genes encoding enzymes of the ␤-ketoadipate pathway, the pathway by which P. putida degrades 4-HBA (Fig. 1) (16).PcaK is unusual among MFS permeases because it is a dual-function protein. In addition to acting as a transporter, PcaK plays a role in chemotaxis to 4-HBA and other aromatic acids (15). The exact function of PcaK in chemotaxis has not yet been deter...

The LysR-type PcaQ protein regulates expression of a protocatechuate-inducible ABC-type transport system in Sinorhizobium meliloti

“…Protocatechuate catabolic genes are organized into two operons (pcaDCHGB and pcaIJF), whose expression is regulated by the transcriptional regulators PcaQ and PcaR, respectively (MacLean et al, 2006. While systems involved in the active transport of aromatic acids such as protocatechuate have been described in many species, including Pseudomonas putida (Harwood et al, 1994;Nichols & Harwood, 1997), a protocatechuate transport system has yet to be identified in S. meliloti or any other member of the a-proteobacteria. Furthermore, S. meliloti appears to lack protein homologues of the PcaK major facilitator superfamily of aromatic acid transport proteins (Collier et al, 1997;Williams & Shaw 1997;D'Argenio et al, 1999;Ledger et al, 2009).…”

3-Chlorobenzoate is taken up by a chromosomally encoded transport system in Cupriavidus necator JMP134

“…Transporter-mediated uptake of aromatic acids has been reported in several bacteria (Allende et al, 1992(Allende et al, , 1993(Allende et al, , 2000(Allende et al, , 2002Chang & Zylstra, 1999;Collier et al, 1997;Harwood et al, 1994;Higgins & Mandelstam, 1972;Nichols & Harwood, 1997;Prieto & García, 1997;Saint & Romas, 1996;Schleissner et al, 1994;Thayer & Wheelis, Fig. 3.…”

“…Establishment and maintenance of concentration gradients requires the intracellular substrate concentration to be kept low relative to that of the external environment, which may be achieved by rapid transformation of the imported compound to metabolic intermediates (Harwood & Gibson, 1986;Merkel et al, 1989;Wong et al, 1994). In this case, uptake is effectively driven by the activity of catabolic enzymes, and this 'metabolic drag' mechanism (Wong et al, 1994) has been proposed for the uptake of benzoate (Harwood & Gibson, 1986) and 4-hydroxybenzoate (4-HB) (Merkel et al, 1989) in Rhodopseudomonas palustris, and for the uptake of 4-HB by Rhizobium leguminosarum (Wong et al, 1994).Transporter-mediated uptake has been reported for some non-chlorinated aromatic acids, such as benzoate (Collier et al, 1997;Thayer & Wheelis, 1982), 4-HB (Allende et al, 1993;Harwood et al, 1994), protocatechuate (Nichols & Harwood, 1997), mandelate (Higgins & Mandelstam, 1972), phenylacetate (Schleissner et al, 1994), 4-hydroxyphenylacetate (Prieto & García, 1997) and phthalate (Chang & Zylstra, 1999). Only a few of these permease-type transport proteins have been biochemically characterized, and the corresponding genes described.…”

4-Chlorobenzoate Uptake in Comamonas sp. Strain DJ-12 Is Mediated by a Tripartite ATP-Independent Periplasmic Transporter

“…For instance, Chang and Zylstra (5) reported that a phthalate permease knockout mutant strain could take up phthalate at the same rate as the wild type, suggesting the existence of a second phthalate transport system. PcaK in Pseudomonas putida is characterized as a multifunctional transporter for 4-hydroxybenzoate as well as protocatechuate (22). Metabolic enzymes can accelerate the simple diffusion of the undissociated form of benzoate and 4-hydroxybenzoate across biological membranes (6,34).…”

“…Several transporters are known to facilitate the movement of aromatic compounds across the membrane: BenK for benzoate (6), OphD for phthalate (5), PcaK for 4-hydroxybenzoate and protocatechuate (22), TfdK for 2,4-dichlorophenoxyacetate (18), StyE for styrene (21), and XylN for m-xylene (15). However, little is known about the transporter for 4-chlorobenzoate (4CBA), which is a metabolite in the microbial breakdown of certain chloroaromatic pollutants.…”