Recombination frequencies in NER-deficient H. pylori mutants after natural transformation We next examined the role of the H. pylori NER system in recombination. Each mutant strain was individually transformed with genomic DNA extracted from H. pylori strain J99-R3. This strain contains a point mutation (A1618T) that confers Rif click here resistance

which can be used as a selection marker to recover recombinant clones (Additional file 2: Figure S2). Recombinant clones were distinguished from spontaneous mutants by partial rpoB sequence analysis. The uvrA mutant exhibited a highly significant decrease of the recombination frequency in comparison to the wild type (Figure 2B). A decreased mean recombination frequency was also determined for the uvrB deficient mutant, MEK162 ic50 however, the difference between the uvrB mutant and wild type did not reach statistical significance (BF =14, “strong evidence”). There was no significant

difference between the recombination frequency of the uvrC mutant and the wild type (Figure 2B). The introduction of an intact copy of the uvrA gene into the uvrA mutant restored the recombination frequency to wild type levels. In contrast, the uvrD deletion mutant (ΔuvrD) showed a hyper-recombinational phenotype (Figure 2B) that is in agreement with previous studies in E. coli[26] and in H. pylori[23]. Characterization of the donor DNA imports after recombination in NER-deficient mutants One of the characteristics of H. pylori is the import of relatively short fragments of donor DNA into the recipient chromosome after natural transformation. see more In order to understand whether components of the NER system play a role in the control of the length of DNA fragments replaced after natural transformation, and in the

formation of interspersed ID-8 sequences of the recipient (ISR), single recombination events were further characterized. For this, Rif resistant clones obtained using the in vitro transformation assay were randomly selected and a 1663 bp fragment in the rpoB locus was sequenced. Recombinant nucleotide sequences were aligned with both donor and recipient sequences to identify the different import parameters used for graphic comparisons of the polymorphisms (Figure 3). Maximum likelihood estimations (MLE) of the import size were calculated and the total number of ISR found among the isolates was counted. Statistical significance of the results was evaluated using a Bayesian approach (see Methods). Since the uvrA mutant showed a strongly reduced recombination frequency, an allele-specific PCR was used in a pre-screening step to distinguish between spontaneous mutants and recombinant clones. Figure 3 Import patterns after transformation of recipient strain 26695 wild type (wt, left panel) and  uvrC  mutant (right panel) with DNA of Rif resistant strain J99-R3. Each row represents a 1663 bp partial rpoB sequence.