Identification of a specific target sequence of a potential regulatory protein in Pseudomonas aeruginosa

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Title: Identification of a specific target sequence of a potential regulatory protein in Pseudomonas aeruginosa
Author: Luna, Ann Marie
Abstract: Exotoxin A is one of the most toxic virulence factor produced by Pseudomonas aeruginosa . Exotoxin A production by P . aeruginosa is regulated by several genes in response to different environmental conditions . We have previously characterized the P . aeruginosa genes , ptxR and ptxS that are divergently transcribed . However , the mechanism that regulates the expression of either gene in not known . Preliminary analysis suggested that presence of several potential regulatory proteins that specifically bind to the ptxRIptxS intergenic region . The present study was conducted to characterize one of these proteins and to identify the specific nucleotide sequence to which it binds . Initial DNA gel shift experiments revealed the presence of a specific gel shift band when the lysate of P . aeruginosa was incubated with a 201 -bp fragment within iht ptxRIptxS intergenic region . Additional experiments , using several overlapping probes within the 201 -bp fragment , localized the binding to a 150 -bpfragment . The potential binding protein was identified using the Heparin -Sepharose columns . One eluted column fraction , which produced the gel shift band , contained only two proteins , which were 9 -kDa and 16 -kDa in molecular weight . The amino acid sequence of the first 15 amino acids of each protein was determined . Computer analysis of the sequences confirmed that one protein is the previously characterized P . aeruginosa HU protein , while the other is an uncharacterized protein that carries an 82 % homology to the Pseudomonas mevalonii transcriptional activator , MvaT . DNase I protection experiments using the Heparin -Sepharose fraction that contains both proteins , produces a 25 -bp protected region . Analysis of this region revealed the presence of an 8 -bp palindromic sequence . Beside the MvaT homologue . P . aeruginosa carries a hypothetical protein that has 647r homology to the MvaT . However , P . aeruginosa does not contain a homologue of the mvaAB operon , which is the target of the MvaT protein . Instead , a hypothetical protein that has limited homology to the mvaB was detected . This suggests that the HU protein , the P . aeruginosa MvaT homologue , or both regulate ptxS expression by specifically binding to the ptxS upstream region .
URI: http : / /hdl .handle .net /2346 /20124
Date: 2001-12

Citation

Identification of a specific target sequence of a potential regulatory protein in Pseudomonas aeruginosa. Master's thesis, Texas Tech University. Available electronically from http : / /hdl .handle .net /2346 /20124 .

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