Affiliation:
1. Department of Microbiology and Immunology, The University of Texas Medical Branch, Galveston, Texas 77555-1070
Abstract
ABSTRACT
The cytotoxic enterotoxin Act from a diarrheal isolate, SSU, of
Aeromonas hydrophila
is aerolysin related and crucial to the pathogenesis of
Aeromonas
infections. To elucidate the role of environmental signals which influence the expression of the cytotoxic enterotoxin gene (
act
), a portion of the
act
gene, including the putative promoter region, was fused in frame to a truncated alkaline phosphatase gene (
phoA
) of
Escherichia coli
. The
act
::
phoA
reporter gene was then introduced into the chromosome of
A. hydrophila
by using the suicide vector pJQ200SK, allowing the fusion protein to be secreted out into the culture medium. Western blot analysis demonstrated the presence of a correctly size 110-kDa fusion protein in the culture supernatant, which reacted with both anti-Act and anti-alkaline phosphatase antibodies. Based on alkaline phosphatase (PhoA) activity in the culture supernatant, we demonstrated that calcium significantly increased the activity of the
act
promoter but that glucose and iron repressed its activity in a dose-dependent fashion. The
act
promoter exhibited optimal activity at pH 7.0 and at 37°C, and maximal PhoA activity was noted when the culture was aerated. Using a
Vibrio cholerae
iron uptake regulator gene (
fur
) as a probe, a 2.6-kb
Sal
I/
Hin
dIII DNA fragment from an
A. hydrophila
chromosome was cloned and sequenced. The DNA sequence revealed a 429-bp open reading frame that exhibited 69% homology at the DNA level with the
fur
gene and 79% homology at the amino acid level with the iron uptake regulator (Fur) protein of
V. cholerae
. Complementation experiments demonstrated that the
A. hydrophila fur
gene could restore iron regulation in an
E. coli fur
-minus mutant. Using the suicide vector pDMS197, we generated a
fur
isogenic mutant of wild-type
A. hydrophila
SSU. Northern blot analysis data indicated that the repression in the transcription of the
act
gene by iron was relieved in the
fur
isogenic mutant. Further, iron regulation in the
fur
isogenic mutant of
A. hydrophila
could be restored by complementation. These results are important in understanding the regulation of the
act
gene under in vivo conditions.
Publisher
American Society for Microbiology
Subject
Infectious Diseases,Immunology,Microbiology,Parasitology
Cited by
33 articles.
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