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Applied and Environmental Microbiology, August 1999, p. 3555-3560, Vol. 65, No. 8
Department of Biochemistry, Microbiology, and
Molecular Genetics, University of Rhode Island, Kingston, Rhode
Island 02881
Received 16 February 1999/Accepted 26 May 1999
The effect of gastrointestinal mucus on protease activity in
Vibrio anguillarum was investigated. Protease activity was
measured by using an azocasein hydrolysis assay. Cells grown to
stationary phase in mucus (200 µg of mucus protein/ml) exhibited
ninefold-greater protease activity than cells grown in Luria-Bertani
broth plus 2% NaCl (LB20). Protease induction was examined with cells
grown in LB20 and resuspended in mucus, LB20, nine-salts solution (NSS [a carbon-, nitrogen-, and phosphorus-free salt solution]), or marine
minimal medium (3M) (~109 CFU/ml). Induction of protease
activity occurred 60 to 90 min after addition of mucus and was
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Induction of Protease Activity in Vibrio
anguillarum by Gastrointestinal Mucus
70-fold greater than protease activity measured in cells incubated in
either LB20 or 3M. Mucus was fractionated into aqueous and
chloroform-methanol-soluble fractions. The aqueous fraction supported
growth of V. anguillarum cells, but did not induce protease
activity. The chloroform-methanol-soluble fraction did not support
growth, nor did it induce protease activity. When the two fractions
were mixed, protease activity was induced. The
chloroform-methanol-soluble fraction did not induce protease activity
in cells growing in LB20. EDTA (50 mM) inhibited the protease induced
by mucus. Upon addition of divalent cations, Mg2+ (100 mM)
was more effective than equimolar amounts of either Ca2+ or
Zn2+ in restoring activity, suggesting that the
mucus-inducible protease was a magnesium-dependent metalloprotease. An
empA mutant strain of V. anguillarum did not
exhibit protease activity after exposure to mucus, but did grow in
mucus. Southern analysis and PCR amplification confirmed that V. anguillarum M93 contained empA. These data
demonstrate that the empA metalloprotease of V. anguillarum is specifically induced by gastrointestinal mucus.
*
Corresponding author. Mailing address: Department of
Biochemistry, Microbiology, and Molecular Genetics, 117 Morrill Hall, University of Rhode Island, Kingston, RI 02881. Phone: (401) 874-5902. Fax: (401) 874-2202. E-mail: dnelson{at}uri.edu.
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