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Infection and Immunity, August 1999, p. 4008-4013, Vol. 67, No. 8
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Cloning, Sequencing, and Role in Virulence of Two Phospholipases (A1 and C) from Mesophilic Aeromonas sp. Serogroup O:34

Susana Merino,1 Alicia Aguilar,1 Maria Mercedes Nogueras,1 Miguel Regue,2 Simon Swift,3 and Juan M. Tomás1,*

Departamento de Microbiología, Facultad de Biología, Universidad de Barcelona, 08071 Barcelona,1 and Departamento de Microbiología y Parasitología Sanitarias, Facultad de Farmacia, Universidad de Barcelona, 08028 Barcelona,2 Spain, and School of Pharmaceutical Sciences, University of Nottingham, Nottingham NG7 2RD, United Kingdom3

Received 29 March 1999/Returned for modification 20 April 1999/Accepted 13 May 1999

Two different representative recombinant clones encoding Aeromonas hydrophila lipases were found upon screening on tributyrin (phospholipase A1) and egg yolk agar (lecithinase-phospholipase C) plates of a cosmid-based genomic library of Aeromonas hydrophila AH-3 (serogroup O34) introduced into Escherichia coli DH5alpha . Subcloning, nucleotide sequencing, and in vitro-coupled transcription-translation experiments showed that the phospholipase A1 (pla) and C (plc) genes code for an 83-kDa putative lipoprotein and a 65-kDa protein, respectively. Defined insertion mutants of A. hydrophila AH-3 defective in either pla or plc genes were defective in phospholipase A1 and C activities, respectively. Lecithinase (phospholipase C) was shown to be cytotoxic but nonhemolytic or poorly hemolytic. A. hydrophila AH-3 plc mutants showed a more than 10-fold increase in their 50% lethal dose on fish and mice, and complementation of the plc single gene on these mutants abolished this effect, suggesting that Plc protein is a virulence factor in the mesophilic Aeromonas sp. serogroup O:34 infection process.

* Corresponding author. Mailing address: Departamento de Microbiología, Facultad de Biología, Universidad de Barcelona, Diagonal 645, 08071 Barcelona, Spain. Phone: 34-93-4021486. Fax: 34-93-4110592. E-mail: juant{at}bio.ub.es.

Infection and Immunity, August 1999, p. 4008-4013, Vol. 67, No. 8
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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