Molecular Cloning, Nucleotide Sequence, and Expression in Escherichia coli of a Hemolytic Toxin (Aerolysin) Gene from Aeromonas trota

Abstract

ABSTRACT Aeromonas trota AK2, which was derived from ATCC 49659 and produces the extracellular pore-forming hemolytic toxin aerolysin, was mutagenized with the transposon mini-Tn 5Km1 to generate a hemolysin-deficient mutant, designated strain AK253. Southern blotting data indicated that an 8.7-kb Not I fragment of the genomic DNA of strain AK253 contained the kanamycin resistance gene of mini-Tn 5Km1 . The 8.7-kb Not I DNA fragment was cloned into the vector pGEM5Zf(−) by selecting for kanamycin resistance, and the resultant clone, pAK71, showed aerolysin activity in Escherichia coli JM109. The nucleotide sequence of the aerA gene, located on the 1.8-kb Apa I- Eco RI fragment, was determined to consist of 1,479 bp and to have an ATG initiation codon and a TAA termination codon. An in vitro coupled transcription-translation analysis of the 1.8-kb region suggested that the aerA gene codes for a 54-kDa protein, in agreement with nucleotide sequence data. The deduced amino acid sequence of the aerA gene product of A. trota exhibited 99% homology with the amino acid sequence of the aerA product of Aeromonas sobria AB3 and 57% homology with the amino acid sequences of the products of the aerA genes of Aeromonas salmonicida 17-2 and A. sobria 33.