Affiliation:
1. Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland 212011;
2. Unité de Pathogénie Bactérienne des Muqueuses, Institut Pasteur, Paris 75724, France2; and
3. School of Biochemistry and Molecular Genetics, University of New South Wales, Sydney, New South Wales 2052, Australia3
Abstract
ABSTRACT
Arginase of the
Helicobacter pylori
urea cycle hydrolyzes
l
-arginine to
l
-ornithine and urea.
H. pylori
urease hydrolyzes urea to carbon dioxide and ammonium, which neutralizes acid. Both enzymes are involved in
H. pylori
nitrogen metabolism. The roles of arginase in the physiology of
H. pylori
were investigated in vitro and in vivo, since arginase in
H. pylori
is metabolically upstream of urease and urease is known to be required for colonization of animal models by the bacterium. The
H. pylori
gene
hp1399
, which is orthologous to the
Bacillus subtilis rocF
gene encoding arginase, was cloned, and isogenic allelic exchange mutants of three
H. pylori
strains were made by using two different constructs: 236-2 and
rocF::aphA3
. In contrast to wild-type (WT) strains, all
rocF
mutants were devoid of arginase activity and had diminished serine dehydratase activity, an enzyme activity which generates ammonium. Compared with WT strain 26695 of
H. pylori
, the
rocF::aphA3
mutant was ∼1,000-fold more sensitive to acid exposure. The acid sensitivity of the
rocF::aphA3
mutant was not reversed by the addition of
l
-arginine, in contrast to the WT, and yielded a ∼10,000-fold difference in viability. Urease activity was similar in both strains and both survived acid exposure equally well when exogenous urea was added, indicating that
rocF
is not required for urease activity in vitro. Finally,
H. pylori
mouse-adapted strain SS1 and the 236-2
rocF
isogenic mutant colonized mice equally well: 8 of 9 versus 9 of 11 mice, respectively. However, the
rocF::aphA3
mutant of strain SS1 had moderately reduced colonization (4 of 10 mice). The geometric mean levels of
H. pylori
recovered from these mice (in log
10
CFU) were 6.1, 5.5, and 4.1, respectively. Thus,
H. pylori rocF
is required for arginase activity and is crucial for acid protection in vitro but is not essential for in vivo colonization of mice or for urease activity.
Publisher
American Society for Microbiology
Subject
Molecular Biology,Microbiology