Transport of 4-hydroxyphenylacetic acid in Klebsiella pneumoniae

Allende, J.L.; Gibello, Alicia; Martín, Margarita; Garrido-Pertierra, Amando

doi:10.1016/0003-9861(92)90034-t

Cited by 21 publications

(14 citation statements)

References 13 publications

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“…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.…”

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confidence: 99%

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

1997

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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...

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confidence: 99%

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

1997

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“…However, very little is known about the characteristics of the presumed receptor proteins that are responsible for initial attractant recognition. Similarly, although a number of studies have inferred the existence of specific transport systems for aromatic acids and related compounds, only a few detailed studies of aromatic compound permeases have been reported (1,2,14,30), and no molecular analyses of bacterial genes that encode such proteins have been presented. 20 pug/ml; kanamycin, 100 jxg/ml; and tetracycline, 100 iug/ml.…”

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Identification of the pcaRKF gene cluster from Pseudomonas putida: involvement in chemotaxis, biodegradation, and transport of 4-hydroxybenzoate

et al. 1994

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Aromatic compounds are abundant in the biosphere as components of the complex polymer lignin and as environmental pollutants. The bacterial biodegradation of structurally simple, readily degradable aromatic compounds has been studied with the expectation that this will facilitate work on more recalcitrant members of the group. As a result, much information has been obtained about the enzymology and molecular regulation of aerobic pathways of aromatic compound degradation (6,16,35,53). An aspect of aromatic biology that has received almost no attention, however, is the question of how bacteria sense and respond to the presence of aromatic compounds in their environment. Chemotaxis and transport are two physiological functions that operate in this capacity, and a number of studies have shown that aromatic acids such as benzoate and 4-hydroxybenzoate are strong chemoattractants for Pseudomonas putida, as well as a number of other species of gram-negative bacteria including Agrobacterium spp. and rhizobia (19,20,31,39,40). However, very little is known about the characteristics of the presumed receptor proteins that are responsible for initial attractant recognition. Similarly, although a number of studies have inferred the existence of specific transport systems for aromatic acids and related compounds, only a few detailed studies of aromatic compound permeases have been reported (1, 2, 14, 30), and no molecular analyses of bacterial genes that encode such proteins have been presented.

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“…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.…”

Section: Resultsmentioning

confidence: 94%

“…In most cases, however, the description of transport is limited to assessment of uptake of the radiolabelled substrate and the proposition of the type of transporter involved. For two of the compounds and organisms described above, uptake was proposed to be mediated by an ABC-type primary transporter (energized by ATP hydrolysis): 4-hydroxyphenylacetate in K. pneumoniae strain M5a1 (Allende et al, 1992) and 4-HB in Acinetobacter sp. strain BEM2 (Allende et al, 2000).…”

Section: Resultsmentioning

confidence: 99%

“…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.…”

Section: Introductionmentioning

confidence: 99%

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

2009

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Cupriavidus necator JMP134(pJP4) is able to grow on 3-chlorobenzoate (3-CB), a model chloroaromatic pollutant. Catabolism of 3-CB is achieved via the expression of the chromosomally encoded benABCD genes and the tfd genes from plasmid pJP4. Since passive diffusion of benzoic acid derivatives at physiological pH is negligible, the uptake of this compound should be facilitated by a transport system. However, no transporter has so far been described to perform this function, and identification of chloroaromatic compound transporters has been limited. In this work, uptake experiments using 3-[ring-UL-14C]CB showed an inducible transport system in strain JMP134, whose expression is activated by 3-CB and benzoate. A similar level of 3-CB uptake was found for a mutant strain of JMP134, defective in chlorobenzoate degradation, indicating that metabolic drag is not an important component of the measured uptake rate. Competitive inhibitor assays showed that uptake of 3-CB was inhibited by benzoate and, to a lesser degree, by 3-CB and 3,5-dichlorobenzoate, but not by any of 12 other substituted benzoates tested. The expression of several gene candidates for this transport function was analysed by RT-PCR, including both permease-type and ABC-type ATP-dependent transporters. Induction of a chromosomally encoded putative permease transporter (benP gene) was found specifically in the presence of 3-CB or benzoate. A benP knockout mutant of strain JMP134 displayed an almost complete loss of 3-CB transport activity. This is to our knowledge the first report of a 3-CB transporter.

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Transport of 4-hydroxyphenylacetic acid in Klebsiella pneumoniae

Cited by 21 publications

References 13 publications

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

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

Identification of the pcaRKF gene cluster from Pseudomonas putida: involvement in chemotaxis, biodegradation, and transport of 4-hydroxybenzoate

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

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