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Appl Environ Microbiol, July 1998, p. 2473-2478, Vol. 64, No. 7
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

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

Ashraf A. Khan,* Eungbin Kim, and Carl E. Cerniglia

Division of Microbiology, Food and Drug Administration, National Center for Toxicological Research, Jefferson, Arkansas 72079

Received 22 January 1998/Accepted 16 April 1998

Aeromonas trota AK2, which was derived from ATCC 49659 and produces the extracellular pore-forming hemolytic toxin aerolysin, was mutagenized with the transposon mini-Tn5Km1 to generate a hemolysin-deficient mutant, designated strain AK253. Southern blotting data indicated that an 8.7-kb NotI fragment of the genomic DNA of strain AK253 contained the kanamycin resistance gene of mini-Tn5Km1. The 8.7-kb NotI 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 ApaI-EcoRI 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.

* Corresponding author. Mailing address: Division of Microbiology, U.S. Food and Drug Administration, National Center for Toxicological Research, Jefferson, AR 72079. Phone: (870) 543-7601. Fax: (870) 543-7307. E-mail: AKHAN{at}NCTR.FDA.GOV.

Appl Environ Microbiol, July 1998, p. 2473-2478, Vol. 64, No. 7
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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