Nucleotide sequence and the gene product of Pseudomonas pseudomallei in Hemolysin-expressing Escherichia coli

Abstract

Pseudomonas pseudomallei is a gram negative-bacterium which is the causative agent of melioidosis. Among several potential virulence factors, whichinclude exotoxin, protease, and endotoxin, hemolysin is also implicated as a virulence determinant in contributing to pathogenesis. In order to study the role of hemolysin in molecular pathogenesis of melioidosis, the E. coli JM109 earring P. pseudomallei DNA in pUC18 that expresses hemolytic activity of 2 hemolytic units/ml was studied. The nucleotide sequence of insert DNA was determined by dideoxy chain termination method. It revealed that this insert DNA was of 1,188 bp and encoded a truncated polypeptide of 43.5 kDa which was a fusion protein with c-terminus of ᵦ-galactocidase (αfragment). The expression of the cloned gene was under the control of lac promoter. The predicted isoelectric point (pI) of protein product is 5.65 and the N-ter- minal signal peptides of 31 amino acid was found. The G+C content of this insert DNA is 66.6%. For in vitro gene expression, a protein of 45 kDa was detected. The nucleotide and amino acid sequence were demonstrated to have no homology to any known hemolysin. This is the first gene of P. pseudomallei to be sequenced. The available data will assist in the further study of complete characterization and the role of hemolysin at the molecular level in pathogenesis.