HP0333, a member of the dprA family, is involved in natural transformation in Helicobacter pylori

Abstract

Helicobacter pylori is naturally competent for DNA transformation, but the mechanism by which transformation occurs is not known. For Haemophilus influenzae, dprA is required for transformation by chromosomal but not plasmid DNA, and the complete genomic sequence of H. pylori 26695 revealed a dprA homolog (HP0333). Examination of genetic databases indicates that DprA homologs are present in a wide variety of bacterial species. To examine whether HP0333 has a function similar to dprA of H. influenzae, HP0333, present in each of 11 strains studied, was disrupted in two H. pylori isolates. For both mutants, the frequency of transformation by H. pylori chromosomal DNA was markedly reduced, but not eliminated, compared to their wild-type parental strains. Mutation of HP0333 also resulted in a marked decrease in transformation frequency by a shuttle plasmid (pHP1), which differs from the phenotype described in H. influenzae. Complementation of the mutant with HP0333 inserted in trans in the chromosomal ureAB locus completely restored the frequency of transformation to that of the wild-type strain. Thus, while dprA is required for high-frequency transformation, transformation also may occur independently of DprA. The presence of DprA homologs in bacteria known not to be naturally competent suggests a broad function in DNA processing.

FIG. 1

Phylogram showing relatedness of putative proteins with homology to HP0333. Organisms in which related putative proteins were found are indicated. GenBank accession numbers for complete DNA sequence submissions are as follows: H. influenzae, U18657; Synchocystis, D90905; B. subtilis, Z99112; M. tuberculosis, Z74024; S. coelicolor, AL023797; B. burgdorferi, AE001137; E. coli, X65946; T. pallidum, AE001217; S. aureus AB015195; and A. aeolicus AE000728. Sequences from unfinished microbial genomes are from the following databases: C. jejuni, The Sanger Centre (42a); C. tepidum, The Institute for Genomic Research (TIGR) (23a); Y. pestis, The Sanger Centre; N. gonorrhoeae, University of Oklahoma’s Advanced Center for Genome Technology (OU-AGCT) (1); P. gingivalis, TIGR; D. radiodurans, TIGR; A. actinomycetemcomitans, OU-AGCT; S. pneumoniae, TIGR; P. aeruginosa, Pseudomonas Genome Project (40a); S. pyogenes, OU-AGCT; S. typhi, The Sanger Centre; E. faecalis, TIGR; T. maritima, TIGR. The accession number for the R. fascians homolog is AF001836.

FIG. 2

Multiple sequence alignment of predicted H. pylori HP0333 product and 24 homologs. Alignment includes region of conservation between HP0333 product and proteins identified by BLAST search. Consensus amino acids, i.e., those which are conserved among greater than 60% of the sequences, are indicated by black boxes (and the corresponding amino acids on the consensus line), whereas gray boxes (and a + on the consensus line) indicate similar amino acids. Alignment was performed with the Genetics Computer Group program Pileup and shaded with MacBoxshade.

FIG. 3

Map of the region of HP0332 to HP0334 showing PCR primers in relation to HP0333 and the inserted aphA cassette.

FIG. 4

Conservation of chromosomal organization flanking HP0333 in three H. pylori strains. (A) Map of 4.7-kb region from nadE (HP0329) through HP0335 in strain 26695. Arrowheads refer to PCR primers (Table 2); arrows reflect the direction of transcription. (B) Products of PCR using these primer pairs for strains 84-183 (lanes 1), HPK5 (lanes 2), and 26695 (lanes 3). Lanes 4 are no-DNA controls.

FIG. 5

Construction of H. pylori mutants involving HP0333. For each strain, the loci surrounding HP0333 and ureAB are shown. A Kan^r (aphA) cassette was introduced into HP0333 of HPK5 to create HPK5/0333::aphA. The insert of pANDO2 was introduced into the ureAB locus of HPK5 downstream of the ureAB promoter (P_ure) to create C5. An aphA cassette was introduced into strain C5 to create either C5G9 or C5G10.