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J. Biol. Chem., Vol. 279, Issue 12, 11156-11162, March 19, 2004

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Isolation and Characterization of a HpyC1I Restriction-Modification System in Helicobacter pylori^*

Tzu-Lung Lin, Chia-Tun Shun, Kai-Chih Chang, and Jin-Town Wang¶||

From the Graduate Institute of Microbiology, National Taiwan University, College of Medicine, Taipei 10016, Taiwan, the Department of Forensic Medicine, National Taiwan University Hospital, Taipei 10016, Taiwan, and the ^¶Department of Internal Medicine, National Taiwan University Hospital, Taipei 10016, Taiwan

Using transposon shuttle mutagenesis, we identified six Helicobacter pylori mutants from the NTUH-C1 strain that exhibited decreased adherence and cell elongation. Inverse polymerase chain reaction and DNA sequencing revealed that the same locus was interrupted in these six mutants. Nucleotide and amino acid sequences showed no homologies with H. pylori 26695 and J99 strains. This novel open reading frame contained 1617 base pairs. The amino acid sequence shared 24% identity with a putative nicking enzyme in Bacillus halodurans and 23 and 20% identity with type IIS restriction endonucleases PleI and MlyI, respectively. The purified protein, HpyC1I, showed endonuclease activity with the recognition and cleavage site 5'-CCATC(4/5)-3'. Two open reading frames were located upstream of the gene encoding HpyC1I. Together, HpyC1I and these two putative methyltransferases (M1.HpyC1I and M2.HpyC1I) function as a restriction-modification (R-M) system. The HpyC1I R-M genes were found in 9 of the 15 H. pylori strains tested. When compared with the full genome, significantly lower G + C content of HpyC1I R-M genes implied that these genes might have been acquired by horizontal gene transfer. Plasmid DNA transformation efficiencies and chromosomal DNA digestion assays demonstrated protection from HpyC1I digestion by the R-M system. In conclusion, we have identified a novel R-M system present in 60% of H. pylori strains. Disruption of this R-M system results in cell elongation and susceptibility to HpyC1I digestion.

Received for publication, October 23, 2003 , and in revised form, January 6, 2004.

^* This work was supported by the National Science Council, Taiwan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ/GenBank^TM/EBI Data Bank with accession number(s) AB118944.

^|| To whom correspondence should be addressed: Graduate Institute of Microbiology, College of Medicine, National Taiwan University, 1, Sec 1, Jen-Ai Rd., Taipei 10016, Taiwan. Tel.: 886-2-23123456 (ext. 8292); Fax: 886-2-23948718 or 886-2-23778111; E-mail: wangjt{at}ccms.ntu.edu.tw.

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