Secretion of haemolysins and proteases by Aeromonas hydrophila EO63: separation and characterization of the serine protease (caseinase) and the metalloprotease (elastase).

AIMS: To determine the haemolysins and proteases excreted by the virulent strain EO63 of Aeromonas hydrophila grown in complex media and to then fractionate and characterize them, in particular those with elastolytic activity. METHODS AND RESULTS: The amount of haemolytic and proteolytic activity in EO63 culture supernatants was dependent on the culture media used. In all media, haemolysins appeared during the phase of active growth and haemolytic activity decreased quickly thereafter, as previously described for aerolysin. In contrast, proteases were mainly released during the stationary phase. Serine protease activity in EO63 culture supernatants was four times greater than that caused by metalloproteases. Two main proteases were partially purified from EO63 culture supernatants by isoelectrophoresis: a serine protease (68 kDa) active against casein; a mixture of different protein bands (60, 44 and 31 kDa) representing a thermostable metalloprotease active against elastin and casein. This metallo-elastase was also inhibited by dithiothreitol and showed a pH optimum of 8.0. Both exoenzymes were toxic for eels at LD50 doses of 1.1 and 3.5 microg (g fish)(-1), respectively. CONCLUSIONS: A serine caseinase and a metallo-elastase may play a role in the pathogenicity of EO63 for eels. These toxins are excreted in vitro by EO63 in the ratio of 4:1 during the stationary phase of growth. Strain EO63 also produced beta-haemolysins in vitro which could correspond to aerolysin. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on the purification of a metallo-elastase excreted by a wild-type A. hydrophila strain.

Dual diode-laser fiber-optic diagnostic for water-vapor measurements.

We present a novel diode-laser diagnostic for water-vapor measurements based on absorption spectroscopy. Two InGaAsP diode lasers were used to record H(2)O absorption line shapes near 1395 nm at a 3-kHz repetition rate along a single path through a water-vapor flow field generated in a shock tube. The use of two lasers permits simultaneous line-shape measurements in different spectral regions and facilitates the selection of appropriate line pairs for thermometry. The wavelength tuning rate was determined by use of a fiber-optic device known as a ring interferometer. Flow-field conditions (1320 K, 0.3 atm) inferred from measured line shapes compared favorably with calculated values.