Functional Characterization of Excision Repair and RecA-Dependent Recombinational DNA Repair in Campylobacter jejuni

Abstract

The presence and functionality of DNA repair mechanisms in Campylobacter jejuni are largely unknown. In silico analysis of the complete translated genome of C. jejuni NCTC 11168 suggests the presence of genes involved in methyl-directed mismatch repair (MMR), nucleotide excision repair, base excision repair (BER), and recombinational repair. To assess the functionality of these putative repair mechanisms in C. jejuni, mutS, uvrB, ung, and recA knockout mutants were constructed and analyzed for their ability to repair spontaneous point mutations, UV irradiation-induced DNA damage, and nicked DNA. Inactivation of the different putative DNA repair genes did not alter the spontaneous mutation frequency. Disruption of the UvrB and RecA orthologues, but not the putative MutS or Ung proteins, resulted in a significant reduction in viability after exposure to UV irradiation. Assays performed with uracil-containing plasmid DNA showed that the putative uracil-DNA glycosylase (Ung) protein, important for initiation of the BER pathway, is also functional in C. jejuni. Inactivation of recA also resulted in a loss of natural transformation. Overall, the data indicate that C. jejuni has multiple functional DNA repair systems that may protect against DNA damage and limit the generation of genetic diversity. On the other hand, the apparent absence of a functional MMR pathway may enhance the frequency of on-and-off switching of phase variable genes typical for C. jejuni and may contribute to the genetic heterogeneity of the C. jejuni population.

Functional Analysis of the RdxA and RdxB Nitroreductases of Campylobacter jejuni Reveals that Mutations in rdxA Confer Metronidazole Resistance

“…While these experiments are not perfect in their execution, they provide evidence that C. jejuni does undergo spontaneous mutation of rdxA resulting in Mtz r . Some of the spontaneous mutations that resulted in disruption of rdxA to give the Mtz r phenotype are likely caused by the absence in C. jejuni of a complete methyl-directed mismatch repair (MMR) system consisting of MutL, MutH, and MutS, usually present in other bacteria (9,10,39). MMR normally corrects base pair mismatches and insertions or deletions of small pieces of DNA, usually under four nucleotides in length (29).…”

Oxidative and nitrosative stress defences ofHelicobacterandCampylobacterspecies that counteract mammalian immunity

“…MutL along with MutS, which recognises and binds to the misincorporated nucleotide, activates MutH that cleaves specific unmethylated daughter strand DNA and allows access for single-strand exonucleases that remove the defective DNA region (Harfe and Jinks-Robertson 2000). Helicobacter pylori and C. jejuni lack the classical SOS system, and mutL and mutH genes, but do encode a mutS homologue (Wang et al 2005a;Gaasbeek, van der Wal et al 2009). However, the MutS protein is not thought to be functionally involved in methyldirected mismatch repair.…”

Change Is Good: Variations in Common Biological Mechanisms in the Epsilonproteobacterial GeneraCampylobacterandHelicobacter

“…Overall, the H. pylori RecA sequence is very similar to those of other bacterial RecA proteins and shares 75% identity with C. jejuni RecA (524,598). Like that of E. coli, RecA proteins in both H. pylori and C. jejuni are required for homologous recombination and DNA repair, and RecA activity may play a role in pH resistance in H. pylori (193,234,524,598). H. pylori recA mutants are deficient in colonization in a murine model, which highlights the importance of H. pylori RecA in vivo (15).…”