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 Johnson, D. C.
Right arrow Articles by Dean, D. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Johnson, D. C.
Right arrow Articles by Dean, D. R.

 Previous Article  |  Next Article 

Journal of Bacteriology, November 2006, p. 7551-7561, Vol. 188, No. 21
0021-9193/06/$08.00+0     doi:10.1128/JB.00596-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Controlled Expression and Functional Analysis of Iron-Sulfur Cluster Biosynthetic Components within Azotobacter vinelandii{triangledown}

Deborah C. Johnson, Mihaela-Carmen Unciuleac, and Dennis R. Dean*

Department of Biochemistry, Virginia Tech, Blacksburg, Virginia 24061

Received 27 April 2006/ Accepted 13 August 2006

A system for the controlled expression of genes in Azotobacter vinelandii by using genomic fusions to the sucrose catabolic regulon was developed. This system was used for the functional analysis of the A. vinelandii isc genes, whose products are involved in the maturation of [Fe-S] proteins. For this analysis, the scrX gene, contained within the sucrose catabolic regulon, was replaced by the contiguous A. vinelandii iscS, iscU, iscA, hscB, hscA, fdx, and iscX genes, resulting in duplicate genomic copies of these genes: one whose expression is directed by the normal isc regulatory elements (Pisc) and the other whose expression is directed by the scrX promoter (PscrX). Functional analysis of [Fe-S] protein maturation components was achieved by placing a mutation within a particular Pisc-controlled gene with subsequent repression of the corresponding PscrX-controlled component by growth on glucose as the carbon source. This experimental strategy was used to show that IscS, IscU, HscBA, and Fdx are essential in A. vinelandii and that their depletion results in a deficiency in the maturation of aconitase, an enzyme that requires a [4Fe-4S] cluster for its catalytic activity. Depletion of IscA results in a null growth phenotype only when cells are cultured under conditions of elevated oxygen, marking the first null phenotype associated with the loss of a bacterial IscA-type protein. Furthermore, the null growth phenotype of cells depleted of HscBA could be partially reversed by culturing cells under conditions of low oxygen. Conserved amino acid residues within IscS, IscU, and IscA that are essential for their respective functions and/or whose replacement results in a partial or complete dominant-negative growth phenotype were also identified using this system.

* Corresponding author. Mailing address: Department of Biochemistry, Virginia Tech, Blacksburg, VA 24061. Phone: (540) 231-5895. Fax: (540) 231-7126. E-mail: deandr{at}vt.edu.

{triangledown} Published ahead of print on 25 August 2006.

Journal of Bacteriology, November 2006, p. 7551-7561, Vol. 188, No. 21
0021-9193/06/$08.00+0     doi:10.1128/JB.00596-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Setubal, J. C., dos Santos, P., Goldman, B. S., Ertesvag, H., Espin, G., Rubio, L. M., Valla, S., Almeida, N. F., Balasubramanian, D., Cromes, L., Curatti, L., Du, Z., Godsy, E., Goodner, B., Hellner-Burris, K., Hernandez, J. A., Houmiel, K., Imperial, J., Kennedy, C., Larson, T. J., Latreille, P., Ligon, L. S., Lu, J., Maerk, M., Miller, N. M., Norton, S., O'Carroll, I. P., Paulsen, I., Raulfs, E. C., Roemer, R., Rosser, J., Segura, D., Slater, S., Stricklin, S. L., Studholme, D. J., Sun, J., Viana, C. J., Wallin, E., Wang, B., Wheeler, C., Zhu, H., Dean, D. R., Dixon, R., Wood, D. (2009). Genome Sequence of Azotobacter vinelandii, an Obligate Aerobe Specialized To Support Diverse Anaerobic Metabolic Processes. J. Bacteriol. 191: 4534-4545 [Abstract] [Full Text]  
  • Boyd, J. M., Sondelski, J. L., Downs, D. M. (2009). Bacterial ApbC Protein Has Two Biochemical Activities That Are Required for in Vivo Function. J. Biol. Chem. 284: 110-118 [Abstract] [Full Text]  
  • Boyd, J. M., Lewis, J. A., Escalante-Semerena, J. C., Downs, D. M. (2008). Salmonella enterica Requires ApbC Function for Growth on Tricarballylate: Evidence of Functional Redundancy between ApbC and IscU. J. Bacteriol. 190: 4596-4602 [Abstract] [Full Text]  
  • Raulfs, E. C., O'Carroll, I. P., Dos Santos, P. C., Unciuleac, M.-C., Dean, D. R. (2008). In vivo iron-sulfur cluster formation. Proc. Natl. Acad. Sci. USA 105: 8591-8596 [Abstract] [Full Text]  
  • Bandyopadhyay, S., Naik, S. G., O'Carroll, I. P., Huynh, B.-H., Dean, D. R., Johnson, M. K., Dos Santos, P. C. (2008). A Proposed Role for the Azotobacter vinelandii NfuA Protein as an Intermediate Iron-Sulfur Cluster Carrier. J. Biol. Chem. 283: 14092-14099 [Abstract] [Full Text]  
  • Ayala-Castro, C., Saini, A., Outten, F. W. (2008). Fe-S Cluster Assembly Pathways in Bacteria. Microbiol. Mol. Biol. Rev. 72: 110-125 [Abstract] [Full Text]  
  • Shimomura, Y., Kamikubo, H., Nishi, Y., Masako, T., Kataoka, M., Kobayashi, Y., Fukuyama, K., Takahashi, Y. (2007). Characterization and Crystallization of an IscU-type Scaffold Protein with Bound [2Fe 2S] Cluster from the Hyperthermophile, Aquifex aeolicus. J Biochem 142: 577-586 [Abstract] [Full Text]  
  • Loiseau, L., Gerez, C., Bekker, M., Ollagnier-de Choudens, S., Py, B., Sanakis, Y., Teixeira de Mattos, J., Fontecave, M., Barras, F. (2007). ErpA, an iron sulfur (Fe S) protein of the A-type essential for respiratory metabolism in Escherichia coli. Proc. Natl. Acad. Sci. USA 104: 13626-13631 [Abstract] [Full Text]  
  • Justino, M. C., Almeida, C. C., Teixeira, M., Saraiva, L. M. (2007). Escherichia coli Di-iron YtfE Protein Is Necessary for the Repair of Stress-damaged Iron-Sulfur Clusters. J. Biol. Chem. 282: 10352-10359 [Abstract] [Full Text]  
  • Dos Santos, P. C., Johnson, D. C., Ragle, B. E., Unciuleac, M.-C., Dean, D. R. (2007). Controlled Expression of nif and isc Iron-Sulfur Protein Maturation Components Reveals Target Specificity and Limited Functional Replacement between the Two Systems. J. Bacteriol. 189: 2854-2862 [Abstract] [Full Text]  
  • Ding, H., Yang, J., Coleman, L. C., Yeung, S. (2007). Distinct Iron Binding Property of Two Putative Iron Donors for the Iron-Sulfur Cluster Assembly: IscA AND THE BACTERIAL FRATAXIN ORTHOLOG CyaY UNDER PHYSIOLOGICAL AND OXIDATIVE STRESS CONDITIONS. J. Biol. Chem. 282: 7997-8004 [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»