Article Figures & Data
Figures
- Fig. 1.
Restriction maps of the ahpB locus and construction of the ahpA::Kanrcassette, the base of allele exchange. Black boxes represent A. hydrophila AG2 cloned DNA; the thicker black box represents theA. hydrophila ahpB gene, which is oriented from 5′ (left) to 3′ (right). The shaded box represents the Kanr cassette. Horizontal lines represent different plasmid vectors or A. hydrophila ahpA mutant chromosomal DNA.
- Fig. 2.
Nucleotide sequence of the ahpB gene and amino acid sequence deduced from its open reading frame. DNA bases (top line) and amino acids (one-letter code) are numbered at the right. The ATG initiation codon (boldface) is preceded by a potential Shine-Dalgarno (boldface and underlined). Initiation of prepro-AhpB, pro-AhpB, and mature AhpB proteins is underlined by an arrow. The symbol—indicates the TGA termination codon; underlined boldfaced amino acid positions 184 to 196 correspond to the amino-terminal sequence determined for both the purified AhpB mature protease and the purified 43.4-kDa intermediate; double-underlined and boldfaced amino acid positions 318 to 327 correspond to a zinc-binding region signature.
- Fig. 3.
Amino acids sequence alignment of the AhpB protease of A. hydrophila AG2 (AHPB), the hemagglutinin/proteinase precursor of V. cholerae (HAPT), the elastase, a zinc-metalloprotease of P. aeruginosa(LASB), and the hemagglutinin/proteinase fragment of H. pylori (HAP). Amino acids highlighted in black boxes are identical in three out of four proteins. Shaded boxes correspond to residues specifically conserved with AhpB protease of A. hydrophilaAG2.
- Fig. 4.
SDS-PAGE of purified AhpB protease from culture supernatant of A. hydrophila AG2 (lane 1), and molecular weight markers (lane 2); from top to bottom: phosphorylaseb, bovine serum albumin, ovalbumin, carbonic anhydrase, soybean trypsin inhibitor, and lysozyme). Numbers at left and right are molecular sizes in kilodaltons.
- Fig. 5.
Secretion and processing of AhpB protease in A. hydrophila AG2. SDS-PAGE and immunoblotting with antibodies to AhpB protease were performed as detailed in the text. (A) Whole-cell extracts; (B) cell culture supernatants. Samples were removed at 10- or 20-min intervals. Numbers at the left are molecular sizes in kilodaltons.
- Fig. 6.
SDS-PAGE and immunoblotting with antibodies to AhpB protease from cell culture supernatants of A. hydrophila AG2 (A), A. hydrophila ahpB mutant (B), and A. salmonicida masoucida containing plasmid pAHE6 (C) in a long-lived experiment. Lanes are culture supernatant samples taken at different hours. Lanes a and b are filtered culture supernatants after incubation 48 and 72 h, respectively, at 37°C. Numbers at the left are molecular masses in kilodaltons.
- Fig. 7.
Proteolytic activity detected on solid media. Macrocolonies of A. hydrophila strains and A. salmonicida masoucida were grown on LB medium supplemented with elastin (A, C4, and C5) or casein (B, C6, and C7) and incubated for 48 h at 28°C. 1, A. hydrophila ahpA mutant; 2,A. hydrophila ahpB mutant; 3, A. hydrophila AG2 wild type; 4 and 6, A. salmonicida masoucida containing plasmid pJRD215; 5 and 7, A. salmonicida masoucidacontaining plasmid pAHE6. The medium for the later was also supplemented with kanamycin.
Tables
- Table 1.
Characteristics of bacterial strains and plasmids used in this study
Strain or plasmid Relevant properties Reference or source Strains E. coli C600 Transformation recipient for plasmids 38 S17-1 Mobilizing donor for conjugation 45 A. hydrophilaAG2 Virulent strain rainbow trout isolate 17 A. salmonicida subsp.masoucida Nonproteolytic strain CECT 896 Plasmids pUC18 Apr; cloning vector 50 pJRD215 Kanr, Smr; broad-host-range mobilizable vector 42 pSUP202 Tcr, Apr, Cmr, ColE1 ori, Mob+; broad-host-range mobilizable suicide vector 45 pSUP202-1 Tcr, Cmr, ColE1 ori, Mob+; broad-host-range mobilizable suicide vector This study pAHE5 Apr; pUC18 containing ahpBgene This study pAHE6 pJRD215 with 2.5-kbSalI-XhoI fragment from pAHE5 This study pAHE7 pAHE5 with a Kanr cassette ligated intoBglII site This study pAHE8 pSUP202-1 with 3.8-kb SalI-XhoI fragment from pAHE7 ligated intoSalII site This study - Table 2.
Proteolytic activities of Aeromonas strains
Strains % Caseinolytic activity % Elastolytic activity A. hydrophila AG2 100 100 AG2 ahpAmutant 15 80 A. salmonicida subsp.masoucida 80 10 AG2 ahpBmutant 0 0 A. salmonicida subsp. masoucida ahpA 50 17 A. salmonicida subsp.masoucida ahpB 16 90 - Table 3.
Calculatons of LD50 strain AG2 and theaphB mutant
Bacteria/0.1 m.l No. of fish that died AG2 aphB mutant Exp 1 Exp 2 Exp 2 Exp 1 109 10 10 9 10 108 9 10 6 5 107 8 8 3 3 106 6 6 2 2 105 3 3 1 1 104 0 0 0 0 103 0 0 0 0 LD50 7 × 105 6 × 105 2.9 × 107 3.3 × 107
