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 Augustus, A. M.
Right arrow Articles by Misra, R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Augustus, A. M.
Right arrow Articles by Misra, R.

 Previous Article  |  Next Article 

Journal of Bacteriology, March 2004, p. 1851-1860, Vol. 186, No. 6
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.6.1851-1860.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Antibiotic-Sensitive TolC Mutants and Their Suppressors

Anne Marie Augustus,1,{dagger} Teresa Celaya,2,3 Fasahath Husain,3,4 Matthew Humbard,3 and Rajeev Misra2,3,4*

Department of Chemistry and Biochemistry,1 Molecular and Cellular Biology Graduate Program,2 Microbiology Graduate Program,4 School of Life Sciences, Arizona State University, Tempe, Arizona 852873

Received 1 December 2003/ Accepted 4 December 2003

The TolC protein of Escherichia coli, through its interaction with AcrA and AcrB, is thought to form a continuous protein channel that expels inhibitors from the cell. Consequently, tolC null mutations display a hypersensitive phenotype. Here we report the isolation and characterization of tolC missense mutations that direct the synthesis of mutant TolC proteins partially disabled in their efflux role. All alterations, consisting of single amino acid substitutions, were localized within the periplasmic {alpha}-helical domain. In two mutants carrying an I106N or S350F substitution, the hypersensitivity phenotype may be in part due to aberrant TolC assembly. However, two other alterations, R367H and R390C, disrupted efflux function by affecting interactions among the helices surrounding TolC's periplasmic tunnel. Curiously, these two TolC mutants were sensitive to a large antibiotic, vancomycin, and exhibited a Dex+ phenotype. These novel phenotypes of TolCR367H and TolCR390C were likely the result of a general influx of molecules through a constitutively open tunnel aperture, which normally widens only when TolC interacts with other proteins during substrate translocation. An intragenic suppressor alteration (T140A) was isolated from antibiotic-resistant revertants of the hypersensitive TolCR367H mutant. T140A also reversed, either fully (R390C) or partially (I106N and S350F), the hypersensitivity phenotype of other TolC mutants. Our data suggest that this global suppressor phenotype of T140A is the result of impeded antibiotic influx caused by tapering of the tunnel passage rather than by correcting individual mutational defects. Two extragenic suppressors of TolCR367H, mapping in the regulatory region of acrAB, uncoupled the AcrR-mediated repression of the acrAB genes. The resulting overexpression of AcrAB reduced the hypersensitivity phenotype of all the TolC mutants. Similar results were obtained when the chromosomal acrR gene was deleted or the acrAB genes were expressed from a plasmid. Unlike the case for the intragenic suppressor T140A, the overexpression of AcrAB diminished hypersensitivity towards only erythromycin and novobiocin, which are substrates of the TolC-AcrAB efflux pump, but not towards vancomycin, which is not a substrate of this pump. This showed that the two types of suppressors produced their effects by fundamentally different means, as the intragenic suppressor decreased the general influx while extragenic suppressors increased the efflux of TolC-AcrAB pump-specific antibiotics.

* Corresponding author. Mailing address: School of Life Sciences, Arizona State University, LSE407, Tempe, AZ 85287-4501. Phone: (480) 965-3320. Fax: (480) 965-6899. E-mail: rajeev.misra{at}asu.edu.

{dagger} Present address: Department of Biochemistry, Duke University, Durham, NC 27710.

Journal of Bacteriology, March 2004, p. 1851-1860, Vol. 186, No. 6
0021-9193/04/$08.00+0     DOI: 10.1128/JB.186.6.1851-1860.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Masi, M., Duret, G., Delcour, A. H., Misra, R. (2009). Folding and trimerization of signal sequence-less mature TolC in the cytoplasm of Escherichia coli. Microbiology 155: 1847-1857 [Abstract] [Full Text]  
  • Fenosa, A., Fuste, E., Ruiz, L., Veiga-Crespo, P., Vinuesa, T., Guallar, V., Villa, T. G., Vinas, M. (2009). Role of TolC in Klebsiella oxytoca resistance to antibiotics. J Antimicrob Chemother 63: 668-674 [Abstract] [Full Text]  
  • Vuong, P., Bennion, D., Mantei, J., Frost, D., Misra, R. (2008). Analysis of YfgL and YaeT Interactions through Bioinformatics, Mutagenesis, and Biochemistry. J. Bacteriol. 190: 1507-1517 [Abstract] [Full Text]  
  • Bagai, I., Liu, W., Rensing, C., Blackburn, N. J., McEvoy, M. M. (2007). Substrate-linked Conformational Change in the Periplasmic Component of a Cu(I)/Ag(I) Efflux System. J. Biol. Chem. 282: 35695-35702 [Abstract] [Full Text]  
  • Eguchi, Y., Itou, J., Yamane, M., Demizu, R., Yamato, F., Okada, A., Mori, H., Kato, A., Utsumi, R. (2007). B1500, a small membrane protein, connects the two-component systems EvgS/EvgA and PhoQ/PhoP in Escherichia coli. Proc. Natl. Acad. Sci. USA 104: 18712-18717 [Abstract] [Full Text]  
  • Masi, M., Vuong, P., Humbard, M., Malone, K., Misra, R. (2007). Initial Steps of Colicin E1 Import across the Outer Membrane of Escherichia coli. J. Bacteriol. 189: 2667-2676 [Abstract] [Full Text]  
  • Lobedanz, S., Bokma, E., Symmons, M. F., Koronakis, E., Hughes, C., Koronakis, V. (2007). A periplasmic coiled-coil interface underlying TolC recruitment and the assembly of bacterial drug efflux pumps. Proc. Natl. Acad. Sci. USA 104: 4612-4617 [Abstract] [Full Text]  
  • Cescau, S., Debarbieux, L., Wandersman, C. (2007). Probing the In Vivo Dynamics of Type I Protein Secretion Complex Association through Sensitivity to Detergents. J. Bacteriol. 189: 1496-1504 [Abstract] [Full Text]  
  • van Amsterdam, K., Bart, A., van der Ende, A. (2005). A Helicobacter pylori TolC Efflux Pump Confers Resistance to Metronidazole. Antimicrob. Agents Chemother. 49: 1477-1482 [Abstract] [Full Text]  
  • Husain, F., Humbard, M., Misra, R. (2004). Interaction between the TolC and AcrA Proteins of a Multidrug Efflux System of Escherichia coli. J. Bacteriol. 186: 8533-8536 [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»