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Infection and Immunity, May 1999, p. 2433-2440, Vol. 67, No. 5
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Identification of Virulence Genes of Helicobacter pylori by Random Insertion Mutagenesis

J. J. E. Bijlsma,1 C. M. J. E. Vandenbroucke-Grauls,1 S. H. Phadnis,2,3 and J. G. Kusters1,*

Department of Medical Microbiology, Faculty of Medicine, Vrije Universiteit Amsterdam, The Netherlands,1 and Department of Pathology, Medical College of Wisconsin,2 and Pathology and Laboratory Medicine Service, Department of Veterans Affairs, Clement J. Zablocki Medical Center,3 Milwaukee, Wisconsin

Received 2 November 1998/Returned for modification 18 December 1998/Accepted 18 February 1999

The complete genome of the gram-negative bacterial pathogen Helicobacter pylori, an important etiological agent of gastroduodenal disease in humans, has recently been published. This sequence revealed that the putative products of roughly one-third of the open reading frames (ORFs) have no significant homology to any known proteins. To be able to analyze the functions of all ORFs, we constructed an integration plasmid for H. pylori and used it to generate a random mutant library in this organism. This integration plasmid, designated pBCalpha 3, integrated randomly into the chromosome of H. pylori. To test the capacity of this library to identify virulence genes, subsets of this library were screened for urease-negative mutants and for nonmotile mutants. Three urease-negative mutants in a subset of 1,251 mutants (0.25%) and 5 nonmotile mutants in a subset of 180 mutants (2.7%) were identified. Analysis of the disrupted ORFs in the urease-negative mutants revealed that two had disruptions of genes of the urease locus, ureB and ureI, and the third had a disruption of a unrelated gene; a homologue of deaD, which encodes an RNA helicase. Analysis of the disrupted ORFs in the nonmotile mutants revealed one ORF encoding a homologue of the paralyzed flagellar protein, previously shown to be involved in motility in Campylobacter jejuni. The other four ORFs have not been implicated in motility before. Based on these data, we concluded that we have generated a random insertion library in H. pylori that allows for the functional identification of genes in H. pylori.

* Corresponding author. Mailing address: Department of Medical Microbiology, Faculty of Medicine, Vrije Universiteit Amsterdam, Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands. Phone: 31-20-4448319. Fax: 31-20-4448318. E-mail: JG.Kusters.mm{at}med.vu.nl.

Infection and Immunity, May 1999, p. 2433-2440, Vol. 67, No. 5
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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