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The Haemophilus influenzae hFbpABC Fe3+ transporter: analysis of the membrane permease and development of a gallium-based screen for mutants.

Publication ,  Journal Article
Anderson, DS; Adhikari, P; Weaver, KD; Crumbliss, AL; Mietzner, TA
Published in: Journal of bacteriology
July 2007

The obligate human pathogen Haemophilus influenzae utilizes a siderophore-independent (free) Fe(3+) transport system to obtain this essential element from the host iron-binding protein transferrin. The hFbpABC transporter is a binding protein-dependent ABC transporter that functions to shuttle (free) Fe(3+) through the periplasm and across the inner membrane of H. influenzae. This investigation focuses on the structure and function of the hFbpB membrane permease component of the transporter, a protein that has eluded prior characterization. Based on multiple-sequence alignments between permease orthologs, a series of site-directed mutations targeted at residues within the two conserved permease motifs were generated. The hFbpABC transporter was expressed in a siderophore-deficient Escherichia coli background, and effects of mutations were analyzed using growth rescue and radiolabeled (55)Fe(3+) transport assays. Results demonstrate that mutation of the invariant glycine (G418A) within motif 2 led to attenuated transport activity, while mutation of the invariant glycine (G155A/V/E) within motif 1 had no discernible effect on activity. Individual mutations of well-conserved leucines (L154D and L417D) led to attenuated and null transport activities, respectively. As a complement to site-directed methods, a mutant screen based on resistance to the toxic iron analog gallium, an hFbpABC inhibitor, was devised. The screen led to the identification of several significant hFbpB mutations; V497I, I174F, and S475I led to null transport activities, while S146Y resulted in attenuated activity. Significant residues were mapped to a topological model of the hFbpB permease, and the implications of mutations are discussed in light of structural and functional data from related ABC transporters.

Duke Scholars

Published In

Journal of bacteriology

DOI

EISSN

1098-5530

ISSN

0021-9193

Publication Date

July 2007

Volume

189

Issue

14

Start / End Page

5130 / 5141

Related Subject Headings

  • Sequence Homology, Amino Acid
  • Mutation
  • Mutagenesis, Site-Directed
  • Molecular Sequence Data
  • Models, Biological
  • Microbiology
  • Membrane Transport Proteins
  • Leucine
  • Iron
  • Haemophilus influenzae
 

Citation

APA
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ICMJE
MLA
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Anderson, D. S., Adhikari, P., Weaver, K. D., Crumbliss, A. L., & Mietzner, T. A. (2007). The Haemophilus influenzae hFbpABC Fe3+ transporter: analysis of the membrane permease and development of a gallium-based screen for mutants. Journal of Bacteriology, 189(14), 5130–5141. https://doi.org/10.1128/jb.00145-07
Anderson, Damon S., Pratima Adhikari, Katherine D. Weaver, Alvin L. Crumbliss, and Timothy A. Mietzner. “The Haemophilus influenzae hFbpABC Fe3+ transporter: analysis of the membrane permease and development of a gallium-based screen for mutants.Journal of Bacteriology 189, no. 14 (July 2007): 5130–41. https://doi.org/10.1128/jb.00145-07.
Anderson DS, Adhikari P, Weaver KD, Crumbliss AL, Mietzner TA. The Haemophilus influenzae hFbpABC Fe3+ transporter: analysis of the membrane permease and development of a gallium-based screen for mutants. Journal of bacteriology. 2007 Jul;189(14):5130–41.
Anderson, Damon S., et al. “The Haemophilus influenzae hFbpABC Fe3+ transporter: analysis of the membrane permease and development of a gallium-based screen for mutants.Journal of Bacteriology, vol. 189, no. 14, July 2007, pp. 5130–41. Epmc, doi:10.1128/jb.00145-07.
Anderson DS, Adhikari P, Weaver KD, Crumbliss AL, Mietzner TA. The Haemophilus influenzae hFbpABC Fe3+ transporter: analysis of the membrane permease and development of a gallium-based screen for mutants. Journal of bacteriology. 2007 Jul;189(14):5130–5141.

Published In

Journal of bacteriology

DOI

EISSN

1098-5530

ISSN

0021-9193

Publication Date

July 2007

Volume

189

Issue

14

Start / End Page

5130 / 5141

Related Subject Headings

  • Sequence Homology, Amino Acid
  • Mutation
  • Mutagenesis, Site-Directed
  • Molecular Sequence Data
  • Models, Biological
  • Microbiology
  • Membrane Transport Proteins
  • Leucine
  • Iron
  • Haemophilus influenzae