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Journal of Bacteriology, December 1999, p. 7314-7322, Vol. 181, No. 23
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Helicobacter pylori rocF Is Required for Arginase Activity and Acid Protection In Vitro but Is Not Essential for Colonization of Mice or for Urease Activity

David J. McGee,1,* Fiona J. Radcliff,2,dagger George L. Mendz,3 Richard L. Ferrero,2 and Harry L. T. Mobley1

Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland 212011; Unité de Pathogénie Bactérienne des Muqueuses, Institut Pasteur, Paris 75724, France2; and School of Biochemistry and Molecular Genetics, University of New South Wales, Sydney, New South Wales 2052, Australia3

Received 1 June 1999/Accepted 21 September 1999

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 log10 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.

* Corresponding author. Mailing address: University of Maryland School of Medicine, Department of Microbiology and Immunology, Baltimore, MD 21201. Phone: (410) 706-0466. Fax: (410) 706-6751. E-mail: dmcge001{at}umaryland.edu.

dagger Present address: Royal Children's Hospital, Tumour Immunology Laboratory, Department of Hematology and Oncology, Parkville, Victoria 3052, Australia.

Journal of Bacteriology, December 1999, p. 7314-7322, Vol. 181, No. 23
0021-9193/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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