Item Details

abstract

The opportunistic pathogen Pseudomonas aeruginosa secretes the exopolysaccharide alginate during chronic lung infection of cystic fibrosis (CF) patients. The genes encoding the alginate biosynthetic enzymes are clustered in a single operon, and the first gene of this operon, algD, is under tight transcriptional control. One essential activator of algD is AlgZ, a proposed ribbon-helix-helix (RHH) DNA-binding protein that shares 30% amino acid identity with the Arc and Mnt repressors of Salmonella typhimurium bacteriophage P22. In order to elucidate the mechanism of AlgZ-mediated activation of algD transcription, we sought to determine what region of AlgZ was responsible for its DNA-binding activity, whether AlgZ regulated other genes using this DNA-binding domain, and how this DNA-binding domain contributed to AlgZ-mediated gene regulation. From these studies we found that the DNA-binding domain of AlgZ is located between residues 1-26, and amino acid residues 18, 20 and 22 are essential for algD transcription and alginate production. AlgZ utilized amino acid residues 18 and 22 to repress algZ transcription by binding to two sites upstream of the algZ promoter, suggesting that AlgZ-mediated gene regulation is modulated by a single DNA-binding domain. Comparison of the known AlgZ binding sites revealed a partially palindromic consensus sequence, 5’-gGCCAttAcCagcc-3’. To further determine the role of the AlgZ DNA-binding domain in structure-function relationships, glutaraldehyde cross-linking studies of wild type or truncated AlgZ were performed. Cross-linking of wild type AlgZ showed the formation of several oligomeric species in solution, with dimeric and tetrameric forms of AlgZ predominating. Loss of the DNA-binding domain of AlgZ led to the formation of monomers and dimers only. Together these studies suggest that AlgZ utilizes a single DNA-binding domain to mediate either activation or repression of its target genes. To our knowledge, this is the first report of a RHH DNA-binding protein that exhibits both activation and repression of gene transcription in vivo through a common DNA-binding mechanism.

subject

algD

alginate

AlgZ

Pseudomonas aeruginosa

transcription

contributor

Ramsey, Deborah Michele (author)

Leslie B. Poole (committee chair)

Sean Reid (committee member)

Rajendar Deora (committee member)

W. Edward Swords (committee member)

Daniel J. Wozniak (committee member)

creator

Ramsey, Deborah Michele

date

2008-09-28T10:55:22Z (accessioned)

2010-06-18T18:58:10Z (accessioned)

2006-06-04 (available)

2008-09-28T10:55:22Z (available)

2010-06-18T18:58:10Z (available)

2005 (issued)

degree

null (defenseDate)

Microbiology & Immunology (discipline)

Wake Forest University (grantor)

PHD (level)

identifier

Thesis_Ramsey.pdf

http://hdl.handle.net/10339/14738 (uri)

migration

etd-05132005-163550 (oldETDId)

rights

Release the entire work for access only to the Wake Forest University system for one year from the date of approval. After one year, release the entire work for access worldwide, unless I send notification to delay release. (accessRights)

I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to Wake Forest University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. (license)

title

DNA Binding Activity, Ligand Recognition, and Oligomerization of the Pseudomonas Aeruginosa Transcriptional Regulator AlgZ