4-Hydroxybenzoate Uptake in Klebsiella pneumoniae Is Driven by Electrical Potential

Allende, J.L.; Suárez, Mónica; Gallego, Manuel Requena; Garrido-Pertierra, Amando

doi:10.1006/abbi.1993.1020

Cited by 11 publications

(13 citation statements)

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“…We have previously shown that E. coli does not modify or metabolize 4-HBA following its transport in the PcaK expression strain (15). Physiological evidence for active transport of aromatic compounds has been found in other bacteria (1,2,14,28,30), and candidate genes have been identified as encoding probable transporters of other aromatic compounds. A pcaK homolog has been found in A. calcoaceticus (25), and mopB from Burkholderia cepacia was recently shown to encode a 4-methylphthalate transporter (38).…”

Section: Discussionmentioning

confidence: 99%

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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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Section: Discussionmentioning

confidence: 99%

“…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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“…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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confidence: 99%

“…Cells that were heated that specific carriers for this class of compounds need not for 5 min failed to accumulate label, indicating that necessarily be evoked (18). However, the existence of trans-;ybenzoate was not simply sticking to the surfaces of porters for several aromatic acids, including 4-chlorobenzoate, addition, labeled 4-hydroxybenzoate, extracted from 4-hydroxybenzoate, and mandelate, has been inferred from lls and analyzed by thin-layer chromatography, was physiological studies (2,14,23). In addition, at least three ) be unmodified, confirming that E. coli did not groups have described aromatic acid permease mutants.…”

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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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“…Recently, it has been recognized that the microbial processes of chemotaxis and solute transport operate as early steps in benzoate and 4-HBA utilization (4,7,18,19,45) and, as such, are important adjuncts to pathway degradation enzymes. In P. putida, chemotactic responses to benzoate and 4-HBA are induced by ␤-ketoadipate (19), and transport of 4-HBA into intact cells is also a ␤-ketoadipate pathway-associated trait (18).…”

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Repression of 4-hydroxybenzoate transport and degradation by benzoate: a new layer of regulatory control in the Pseudomonas putida beta-ketoadipate pathway

Nichols

Harwood

1995

J Bacteriol

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Pseudomonas putida PRS2000 degrades the aromatic acids benzoate and 4-hydroxybenzoate via two parallel sequences of reactions that converge at ␤-ketoadipate, a derivative of which is cleaved to form tricarboxylic acid cycle intermediates. Structural genes (pca genes) required for the complete degradation of 4-hydroxybenzoate via the protocatechuate branch of the ␤-ketoadipate pathway have been characterized, and a specific transport system for 4-hydroxybenzoate has recently been described. To better understand how P. putida coordinates the processes of 4-hydroxybenzoate transport and metabolism to achieve complete degradation, the regulation of pcaK, the 4-hydroxybenzoate transport gene, and that of pcaF, a gene required for both benzoate and 4-hydroxybenzoate degradation, were compared. Primer extension analysis and lacZ fusions showed that pcaK and pcaF, which are adjacent on the chromosome, are transcribed independently. PcaR, a transcriptional activator of several genes of the ␤-ketoadipate pathway, is required for expression of both pcaF and pcaK, and the pathway intermediate ␤-ketoadipate induces both genes. In addition to these expected regulatory elements, expression of pcaK, but not pcaF, is repressed by benzoate. This previously unrecognized layer of regulatory control in the ␤-ketoadipate pathway appears to extend to the first two steps of 4-hydroxybenzoate degradation, since levels of 4-hydroxybenzoate hydroxylase and protocatechuate 3,4-dioxygenase activities were also depressed when cells were grown on a mixture of 4-hydroxybenzoate and benzoate. The apparent consequence of benzoate repression is that cells degrade benzoate in preference to 4-hydroxybenzoate. These findings indicate that 4-hydroxybenzoate transport is an integral feature of the ␤-ketoadipate pathway in P. putida and that transport plays a role in establishing the preferential degradation of benzoate over 4-hydroxybenzoate. These results also demonstrate that there is communication between the two branches of the ␤-ketoadipate pathway.

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4-Hydroxybenzoate Uptake in Klebsiella pneumoniae Is Driven by Electrical Potential

Cited by 11 publications

References 0 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

Repression of 4-hydroxybenzoate transport and degradation by benzoate: a new layer of regulatory control in the Pseudomonas putida beta-ketoadipate pathway

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