CLONING AND CHARACTERIZATION OF 2 IMMUNOPHILIN-LIKE GENES, ILPA AND FKPA, ON A SINGLE 3.9-KILOBASE FRAGMENT OF AEROMONAS-HYDROPHILA GENOMICDNA

Antiserum to Aeromonas hydrophila A6 cell envelopes was shown in a pre vious study (C. Y. F. Wong, G. Mayrhofer, M. W. Heuzenroeder, H. M. At kinson, D. M. Quinn, and R. L. P. Flower, FEMS Immunol. Med. Microbiol , 15:233-241, 1996) to protect mice against lethal infection by this o rganism. In this study, colony blot analysis of an A. hydrophila genom ic library using antiserum to A; hydrophila A6 cell envelopes revealed a cosmid clone expressing a 30-kDa protein which has not been describ ed previously in aeromonads. The nucleotide sequence of a 3.9-kb fragm ent derived from this cosmid which expressed the 30-kDa protein reveal ed two potential open reading frames (ORFs) with homology to known imm unophilin proteins. ORF1 encoded a 212-amino-acid protein (molecular m ass, 22.4 kDa) with 56% identity to the immunophilin SlyD protein of E scherichia coli. ORF1 was subsequently designated ilpA (immunophilin-l ike protein). ORF3 encoded a potential gene product of 268 amino acids with a typical signal sequence and a predicted molecular size of 28.7 kDa. The inferred amino acid sequence showed 46% identity with the se quence of the FkpA protein of E. coli and 40% identity with the sequen ce of the macrophage infectivity potentiator (Mip) protein of Legionel la pneumophila. ORF3 was designated fkpA (FK506 binding protein) by an alogy with the E. coli FkpA protein. Expression of the FkpA protein wa s confirmed by Western blot (immunoblot) analysis, which detected a 30 -kDa protein, with antiserum to the Mip protein of Legionella longbeac hae and a specific antiserum to an A. hydrophila 30-kDa membrane prote in. PCR and Southern analysis showed that a DNA sequence encoding FkpA was found in all 178 aeromonads of diverse origins tested. A nonpolar insertion mutation in the fkpA gene did not attenuate virulence in a suckling mouse model nor did it affect the expression of hemolysins or DNase. This suggests that either the fkpA gene is not essential in th e virulence of A. hydrophila under these conditions or there are other genes in A. hydrophila coding for proteins with similar functions.