| Summary: | 博士 === 國立臺灣大學 === 微生物學研究所 === 91 === Abstract
To study whether the capability of horizontal DNA transfer is associated
with metronidazole resistance in Helicobacter pylori, a total of 81 clinical
isolates were tested for MICs of metronidazole (MTZ) and the capability of
natural transformation. The MIC assays were performed by using E-testing and
reconfirmed by the agar dilution method. Natural competence assays were
performed by transferring a chloramphenicol acetyltransferase (CAT) cassette
and a 23S rRNA gene from a clarithromycin-resistant strain (with A-to-G
mutation at nucleotide 2143) using natural transformation. Of the 81 isolates,
65 (80.3 %) were naturally competent while 16 were not. Among the 65
naturally competent strains, 39 (60.0%) were highly resistant to MTZ (MICs >32
µg/mL) while only 2 of 16 (12.5%) noncompetent strains were highly
MTZ-resistant (p <0.1). Therefore, there is an association between natural
competence and MTZ resistance. Next, we used bioinformatics database
search to find genes involved in natural competence, and two
transformation-related open reading frames were found: a comE3 homologue
(HP1361) of Bacillus subtilis and a comL homologue (HP1378) of Neisseria
gonorrhoea. However, we were unable to obtain HP1378 knock-out mutant.
HP1361 knock-out mutant was obtained by transposon shuttle mutagenesis.
For DNA transformation experiments, 3 donor DNAs were used: theCAT-cassette-inserted HP0691, the clarithromycin resistant 23S rRNA gene
and an Escherichia coli-H. pylori shuttle vector pHel2. DNA transformation
ability was severely impaired (frequency <1.0x10-9) in HP1361 knock-out
mutant, both by natural transformation and electroporation. Complementation
with a plasmid or chromosomal integration restored partial capabilities of
natural competence (to frequencies 2.8x10-7-8.2x10-6) and electroporation (to
frequencies 3.0x10-7-1.0x10-5). Isotope-labeled DNA assays revealed that DNA
binding and uptake capabilities were impaired in HP1361 knock-out mutant and
restored after complementation. The results suggest that HP1361, a comE3
homologue, is required for DNA binding and uptake during DNA transformation
of H. pylori.
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