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

 Previous Article  |  Next Article 

Journal of Bacteriology, October 1998, p. 5058-5069, Vol. 180, No. 19
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Mutation Analysis of PobR and PcaU, Closely Related Transcriptional Activators in Acinetobacterdagger

Ruben G. Kok, David A. D'Argenio, and L. Nicholas Ornston*

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

Received 23 April 1998/Accepted 23 July 1998

Acinetobacter PobR and PcaU are transcriptional activators that closely resemble each other in primary structure, DNA-binding sites, metabolic modulators, and physiological function. PobR responds to the inducer-metabolite p-hydroxybenzoate and activates transcription of pobA, the structural gene for the enzyme that converts p-hydroxybenzoate to protocatechuate. This compound, differing from p-hydroxybenzoate only in that it contains an additional oxygen atom, binds to PcaU and thereby specifically activates transcription of the full set of genes for protocatechuate catabolism. Particular experimental attention has been paid to PobR and PcaU from Acinetobacter strain ADP1, which exhibits exceptional competence for natural transformation. This trait allowed selection of mutant strains in which pobR function had been impaired by nucleotide substitutions introduced by PCR replication errors. Contrary to expectation, the spectrum of amino acids whose substitution led to loss of function in PobR shows no marked similarity to the spectrum of amino acids conserved by the demand for continued function during evolutionary divergence of PobR, PcaU, and related proteins. Surface plasmon resonance was used to determine the ability of mutant PobR proteins to bind to DNA in the pobA-pobR intergenic region. Deleterious mutations that strongly affect DNA binding all cluster in and around the PobR region that contains a helix-turn-helix motif, whereas mutations causing defects in the central portion of the PobR primary sequence do not seem to have a significant effect on operator binding. PCR-generated mutations allowing PobR to mimic PcaU function invariably caused a T57A amino acid substitution, making the helix-turn-helix sequence of PobR more like that of PcaU. The mutant PobR depended on p-hydroxybenzoate for its activity, but this dependence could be relieved by any of six amino acid substitutions in the center of the PobR primary sequence. Independent mutations allowing PcaU to mimic PobR activity were shown to be G222V amino acid substitutions in the C terminus of the 274-residue protein. Together, the analyses suggest that PobR and PcaU possess a linear domain structure similar to that of LysR transcriptional activators which largely differ in primary structure.

* Corresponding author. Mailing address: Department of Molecular, Cellular and Developmental Biology, P.O. Box 208103, Yale University, New Haven, CT 06520-8103. Phone: (203) 432-3498. Fax: (203) 432-6161. E-mail: nicholas.ornston{at}.yale.edu.

dagger Publication 18 from the Biological Transformation Center in the Yale Biospherics Institute.

Journal of Bacteriology, October 1998, p. 5058-5069, Vol. 180, No. 19
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


This article has been cited by other articles:

  • Fillet, S., Velez, M., Lu, D., Zhang, X., Gallegos, M.-T., Ramos, J. L. (2009). TtgV Represses Two Different Promoters by Recognizing Different Sequences. J. Bacteriol. 191: 1901-1909 [Abstract] [Full Text]  
  • Guazzaroni, M.-E., Gallegos, M.-T., Ramos, J. L., Krell, T. (2007). Different Modes of Binding of Mono- and Biaromatic Effectors to the Transcriptional Regulator TTGV: ROLE IN DIFFERENTIAL DEREPRESSION FROM ITS COGNATE OPERATOR. J. Biol. Chem. 282: 16308-16316 [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]  
  • Shen, X.-H., Jiang, C.-Y., Huang, Y., Liu, Z.-P., Liu, S.-J. (2005). Functional Identification of Novel Genes Involved in the Glutathione-Independent Gentisate Pathway in Corynebacterium glutamicum. Appl. Environ. Microbiol. 71: 3442-3452 [Abstract] [Full Text]  
  • Guazzaroni, M.-E., Krell, T., Felipe, A., Ruiz, R., Meng, C., Zhang, X., Gallegos, M.-T., Ramos, J. L. (2005). The Multidrug Efflux Regulator TtgV Recognizes a Wide Range of Structurally Different Effectors in Solution and Complexed with Target DNA: EVIDENCE FROM ISOTHERMAL TITRATION CALORIMETRY. J. Biol. Chem. 280: 20887-20893 [Abstract] [Full Text]  
  • Tropel, D., van der Meer, J. R. (2004). Bacterial Transcriptional Regulators for Degradation Pathways of Aromatic Compounds. Microbiol. Mol. Biol. Rev. 68: 474-500 [Abstract] [Full Text]  
  • Parke, D., Ornston, L. N. (2004). Toxicity Caused by Hydroxycinnamoyl-Coenzyme A Thioester Accumulation in Mutants of Acinetobacter sp. Strain ADP1. Appl. Environ. Microbiol. 70: 2974-2983 [Abstract] [Full Text]  
  • Jones, R. M., Williams, P. A. (2003). Mutational Analysis of the Critical Bases Involved in Activation of the AreR-Regulated {sigma}54-Dependent Promoter in Acinetobacter sp. Strain ADP1. Appl. Environ. Microbiol. 69: 5627-5635 [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]  
  • Zhang, R.-g., Kim, Y., Skarina, T., Beasley, S., Laskowski, R., Arrowsmith, C., Edwards, A., Joachimiak, A., Savchenko, A. (2002). Crystal Structure of Thermotoga maritima 0065, a Member of the IclR Transcriptional Factor Family. J. Biol. Chem. 277: 19183-19190 [Abstract] [Full Text]  
  • Popp, R., Kohl, T., Patz, P., Trautwein, G., Gerischer, U. (2002). Differential DNA Binding of Transcriptional Regulator PcaU from Acinetobacter sp. Strain ADP1. J. Bacteriol. 184: 1988-1997 [Abstract] [Full Text]  
  • Diaz, E., Ferrandez, A., Prieto, M. A., Garcia, J. L. (2001). Biodegradation of Aromatic Compounds by Escherichia coli. Microbiol. Mol. Biol. Rev. 65: 523-569 [Abstract] [Full Text]  
  • Young, D. M., Ornston, L. N. (2001). Functions of the Mismatch Repair Gene mutS from Acinetobacter sp. Strain ADP1. J. Bacteriol. 183: 6822-6831 [Abstract] [Full Text]  
  • Bertani, I., Kojic, M., Venturi, V. (2001). Regulation of the p-hydroxybenzoic acid hydroxylase gene (pobA) in plant-growth-promoting Pseudomonas putida WCS358. Microbiology 147: 1611-1620 [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]  
  • 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]  
  • D'Argenio, D. A., Segura, A., Coco, W. M., Bünz, P. V., Ornston, L. N. (1999). The Physiological Contribution of Acinetobacter PcaK, a Transport System That Acts upon Protocatechuate, Can Be Masked by the Overlapping Specificity of VanK. J. Bacteriol. 181: 3505-3515 [Abstract] [Full Text]  
  • Kok, R. G., Young, D. M., Ornston, L. N. (1999). Phenotypic Expression of PCR-Generated Random Mutations in a Pseudomonas putida Gene after Its Introduction into an Acinetobacter Chromosome by Natural Transformation. Appl. Environ. Microbiol. 65: 1675-1680 [Abstract] [Full Text]  


Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»