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



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Texas Tech University


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.



Deoxyribonucleic acid (DNA)-Binding Proteins, Pseudomonas aeruginosa