This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by D'Argenio, D. A. Articles by Ornston, L. N. Search for Related Content PubMed PubMed Citation Articles by D'Argenio, D. A. Articles by Ornston, L. N.

Journal of Bacteriology, June 1999, p. 3505-3515, Vol. 181, No. 11
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

The Physiological Contribution of Acinetobacter PcaK, a Transport System That Acts upon Protocatechuate, Can Be Masked by the Overlapping Specificity of VanK

David A. D'Argenio, Ana Segura, Wayne M. Coco,^§ Patricia V. Bünz, and L. Nicholas Ornston^*

Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, Connecticut 06520-8103

Received 21 December 1998/Accepted 30 March 1999

VanK is the fourth member of the ubiquitous major facilitator superfamily of transport proteins to be identified that, together with PcaK, BenK, and MucK, contributes to aromatic catabolism in Acinetobacter sp. strain ADP1. VanK and PcaK have overlapping specificity for p-hydroxybenzoate and, most clearly, for protocatechuate: inactivation of both proteins severely impairs growth with protocatechuate, and the activity of either protein alone can mask the phenotype associated with inactivation of its homolog. Furthermore, vanK pcaK double-knockout mutants appear completely unable to grow in liquid culture with the hydroaromatic compound quinate, although such cells on plates convert quinate to protocatechuate, which then accumulates extracellularly and is readily visible as purple staining. This provides genetic evidence that quinate is converted to protocatechuate in the periplasm and is in line with the early argument that quinate catabolism should be physically separated from aromatic amino acid biosynthesis in the cytoplasm so as to avoid potential competition for intermediates common to both pathways. Previous studies of aromatic catabolism in Acinetobacter have taken advantage of the ability to select directly strains that contain a spontaneous mutation blocking the -ketoadipate pathway and preventing the toxic accumulation of carboxymuconate. By using this procedure, strains with a mutation in structural or regulatory genes blocking degradation of vanillate, p-hydroxybenzoate, or protocatechuate were selected. In this study, the overlapping specificity of the VanK and PcaK permeases was exploited to directly select strains with a mutation in either vanK or pcaK. Spontaneous mutations identified in vanK include a hot spot for frameshift mutation due to contraction of a G₆ mononucleotide repeat as well as point mutations producing amino acid substitutions useful for analysis of VanK structure and function. Preliminary second-site suppression analysis using transformation-facilitated PCR mutagenesis in one VanK mutant gave results similar to those using LacY, the prototypic member of the major facilitator superfamily, consistent with the two proteins having a similar mechanism of action. The selection for transport mutants described here for Acinetobacter may also be applicable to Pseudomonas putida, where the PcaK permease has an additional role in chemotaxis.

---

^* Corresponding author. Mailing address: Department of Molecular, Cellular and Developmental Biology, Yale University, P.O. Box 208103, New Haven, CT 06520-8103. Phone: (203) 432-3498. Fax: (203) 432-3497. E-mail: nicholas.ornston{at}yale.edu.

Publication 20 from the Biological Transformation Center in the Yale Biospherics Institute.

Present address: Department of Biochemistry, Consejo Superior de Investigaciones Científicas, Estación Experimental de Zaidín, 18012 Granada, Spain.

^§ Present address: Energy Biosystems, The Woodlands, Tex.

Present address: Institut für Allgemeine Botanik, Abteilung Mikrobiologie, Hamburg, Germany.

---

Journal of Bacteriology, June 1999, p. 3505-3515, Vol. 181, No. 11
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

This article has been cited by other articles:

• Chaudhry, M. T., Huang, Y., Shen, X.-H., Poetsch, A., Jiang, C.-Y., Liu, S.-J. (2007). Genome-wide investigation of aromatic acid transporters in Corynebacterium glutamicum. Microbiology 153: 857-865 [Abstract] [Full Text]  
• Siehler, S. Y., Dal, S., Fischer, R., Patz, P., Gerischer, U. (2007). Multiple-Level Regulation of Genes for Protocatechuate Degradation in Acinetobacter baylyi Includes Cross-Regulation. Appl. Environ. Microbiol. 73: 232-242 [Abstract] [Full Text]  
• Providenti, M. A., O'Brien, J. M., Ruff, J., Cook, A. M., Lambert, I. B. (2006). Metabolism of Isovanillate, Vanillate, and Veratrate by Comamonas testosteroni Strain BR6020. J. Bacteriol. 188: 3862-3869 [Abstract] [Full Text]  
• Buchan, A., Ornston, L. N. (2005). When Coupled to Natural Transformation in Acinetobacter sp. Strain ADP1, PCR Mutagenesis Is Made Less Random by Mismatch Repair. Appl. Environ. Microbiol. 71: 7610-7612 [Abstract] [Full Text]  
• Peng, X., Masai, E., Kasai, D., Miyauchi, K., Katayama, Y., Fukuda, M. (2005). A Second 5-Carboxyvanillate Decarboxylase Gene, ligW2, Is Important for Lignin-Related Biphenyl Catabolism in Sphingomonas paucimobilis SYK-6. Appl. Environ. Microbiol. 71: 5014-5021 [Abstract] [Full Text]  
• Dal, S., Trautwein, G., Gerischer, U. (2005). Transcriptional Organization of Genes for Protocatechuate and Quinate Degradation from Acinetobacter sp. Strain ADP1. Appl. Environ. Microbiol. 71: 1025-1034 [Abstract] [Full Text]  
• Barbe, V., Vallenet, D., Fonknechten, N., Kreimeyer, A., Oztas, S., Labarre, L., Cruveiller, S., Robert, C., Duprat, S., Wincker, P., Ornston, L. N., Weissenbach, J., Marliere, P., Cohen, G. N., Medigue, C. (2004). Unique features revealed by the genome sequence of Acinetobacter sp. ADP1, a versatile and naturally transformation competent bacterium. Nucleic Acids Res 32: 5766-5779 [Abstract] [Full Text]  
• Parke, D., Ornston, L. N. (2003). Hydroxycinnamate (hca) Catabolic Genes from Acinetobacter sp. Strain ADP1 Are Repressed by HcaR and Are Induced by Hydroxycinnamoyl-Coenzyme A Thioesters. Appl. Environ. Microbiol. 69: 5398-5409 [Abstract] [Full Text]  
• Brzostowicz, P. C., Reams, A. B., Clark, T. J., Neidle, E. L. (2003). Transcriptional Cross-Regulation of the Catechol and Protocatechuate Branches of the {beta}-Ketoadipate Pathway Contributes to Carbon Source-Dependent Expression of the Acinetobacter sp. Strain ADP1 pobA Gene. Appl. Environ. Microbiol. 69: 1598-1606 [Abstract] [Full Text]  
• Smith, M. A., Weaver, V. B., Young, D. M., Ornston, L. N. (2003). Genes for Chlorogenate and Hydroxycinnamate Catabolism (hca) Are Linked to Functionally Related Genes in the dca-pca-qui-pob-hca Chromosomal Cluster of Acinetobacter sp. Strain ADP1. Appl. Environ. Microbiol. 69: 524-532 [Abstract] [Full Text]  
• Ditty, J. L., Harwood, C. S. (2002). Charged Amino Acids Conserved in the Aromatic Acid/H+ Symporter Family of Permeases Are Required for 4-Hydroxybenzoate Transport by PcaK from Pseudomonas putida. J. Bacteriol. 184: 1444-1448 [Abstract] [Full Text]  
• Parke, D., Garcia, M. A., Ornston, L. N. (2001). Cloning and Genetic Characterization of dca Genes Required for {beta}-Oxidation of Straight-Chain Dicarboxylic Acids in Acinetobacter sp. Strain ADP1. Appl. Environ. Microbiol. 67: 4817-4827 [Abstract] [Full Text]  
• Trautwein, G., Gerischer, U. (2001). Effects Exerted by Transcriptional Regulator PcaU from Acinetobacter sp. Strain ADP1. J. Bacteriol. 183: 873-881 [Abstract] [Full Text]  
• Morawski, B., Segura, A., Ornston, L. N. (2000). Substrate Range and Genetic Analysis of Acinetobacter Vanillate Demethylase. J. Bacteriol. 182: 1383-1389 [Abstract] [Full Text]  
• Parke, D., D'Argenio, D. A., Ornston, L. N. (2000). Bacteria Are Not What They Eat: That Is Why They Are So Diverse. J. Bacteriol. 182: 257-263 [Full Text]  
• D'Argenio, D. A., Vetting, M. W., Ohlendorf, D. H., Ornston, L. N. (1999). Substitution, Insertion, Deletion, Suppression, and Altered Substrate Specificity in Functional Protocatechuate 3,4-Dioxygenases. J. Bacteriol. 181: 6478-6487 [Abstract] [Full Text]