doi: 10.1371/journal.ppat.1001026.
DNA damage triggers genetic exchange in Helicobacter pylori
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- PMID:20686662
- PMCID: PMC2912397
- DOI: 10.1371/journal.ppat.1001026
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DNA damage triggers genetic exchange in Helicobacter pylori
Marion S Dorer et al. PLoS Pathog..
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Abstract
Many organisms respond to DNA damage by inducing expression of DNA repair genes. We find that the human stomach pathogen Helicobacter pylori instead induces transcription and translation of natural competence genes, thus increasing transformation frequency. Transcription of a lysozyme-like protein that promotes DNA donation from intact cells is also induced. Exogenous DNA modulates the DNA damage response, as both recA and the ability to take up DNA are required for full induction of the response. This feedback loop is active during stomach colonization, indicating a role in the pathogenesis of the bacterium. As patients can be infected with multiple genetically distinct clones of H. pylori, DNA damage induced genetic exchange may facilitate spread of antibiotic resistance and selection of fitter variants through re-assortment of preexisting alleles in this important human pathogen.
Conflict of interest statement
The authors have declared that no competing interests exist.
Figures
A. Ciprofloxacin exposure andaddA mutation lead to similar transcriptional changes. A comparison of log2 ratios of gene expression changes measured by microarray for ciprofloxacin treatment (2.5 hours, 10× MIC) vs untreated and untreated ΔaddA mutant vs wild-type cells during logarithmic growth. Each condition is represented as mean values measured from multiple microarray experiments from three independent cultures. Regression correlation = 0.9.B. RecA is required for induction of DNA damage responsive genes. Heat map showing 471 genes with 1.6 fold change upon treatment with ciprofloxacin (column 1) or in the ΔaddA mutant (column 2). The enlarged panel shows genes with statistically significant induction in both ciprofloxacin-treated cells and the ΔaddA mutant, as determined by SAM (DNA damage responsive genes), with annotation from strain G27 . Untreated wild-type cells compared to either wild-type cells treated with ciprofloxacin (WT+C), untreatedΔaddA mutant (aA−), untreated ΔrecA mutant (rA−) or the ΔrecA mutant treated with ciprofloxacin (+C). Bottom, scale bar for heat maps in fold change. OMP = outer membrane protein, genes with no annotation are left blank.
A. Wild-type cells were treated at or above the MIC of ciprofloxacin (cipro), ampicillin (amp) (0.016 ug/ml [50]) or gentamicin (1 ug/ml, data not shown) for 2.5 hours and transformed with 20ng genomic DNA harboring an antibiotic resistance cassette and the fraction of cells transformed per CFU was determined.B. Wild-type cells were treated with the minimum inhibitory concentration (MIC) of ciprofloxacin for 2.5 hours and the fraction of cells transformed by genomic DNA per CFU was determined. Wild-type transformation frequency ranges between 10−7 and 10−4, depending on DNA concentration and experiment and for each panel, error bars are the standard deviation of the mean with at least three replicates for each point and a representative from two experiments is shown.C. Wild-type cells were treated with 10× MIC gentamicin for the indicated time and the fraction of cells transformed by genomic DNA per CFU was determined either by plating directly to selective medium or by plating to non-selective medium to allow translation of the selective marker prior to selection.D. Increased expression ofcomB4 (comB4IE) increases natural transformation. The fraction of wild-type cells andcomB4IE cells transformed by genomic DNA was determined.
A. ThecomB4 merodiploid (comB4IE) induces expression of DNA damage responsive genes. Untreated wild-type cells are compared to either wild-type cells treated with ciprofloxacin (WT+C) or untreatedcomB4IE cells and the mean of fold change in RNA expression measured by microarray from three independent cultures is shown. Bottom, scale bar indicates fold change for heat maps in A,B,C.B. Increased expression of DNA damage responsive genes incomB4IE cells requiresrecA and thecom T4SS. Untreated wild-type cells are compared to eithercomB4IE ΔcomB10 (comB4IE B10) cells, orcomB4IE ΔrecA cells and data is represented as in Figure 1A.C. Full induction of DNA damage responsive genes by DNA damage requires thecom T4SS. Untreated wild-type cells are compared to ciprofloxacin treated ΔcomB10 mutant cells (B10- +C) and untreated ΔaddA ΔcomB10 double mutant cells (B10- addA-). Data is represented as in Figure 1A.
A. Log-phase recipient cells were mixed with stationary phase donor cells and the frequency of transformation was determined.B. The fraction of cells of the indicated genotype transformed by genomic DNA per CFU was determined using 10 ng genomic DNA. Error bars are the standard deviation of the mean with at least three replicates for each point and a representative of two independent experiments is shown.
RecA binds DSBs and transcription of DNA damage responsive genes is induced that includes a lysozyme-like protein, which may be used to acquire DNA and thecom T4SS, which transports DNA in the cell. Once in the cytosol, transforming DNA is bound by RecA and further induces transcription of DNA damage responsive genes.
Each data point shows the competitive index of mutant cells vs wild-type cells or the indicated double mutant compared to either single mutant for a single mouse after one-week stomach colonization (A, B) or for a single well during co-culture in broth (C) and bars indicate the geometric mean.A. Competition between the ΔcomB10 mutant and wild-type cells shows no defect during stomach colonization; however stomach colonization of the ΔaddA mutant is improved by disruption of natural competence.B. Competition between the ΔrecR mutant and wild-type cells shows a strong defect during stomach colonization, but is unaffected by disruption of natural competence.C. Competence does not affect growth of the ΔaddA mutant in broth culture. The ΔaddA ΔcomB10 double mutant and the ΔaddA mutant were maintained in logarithmic growth for three days in broth culture by dilution.
References
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- Chey WD, Wong BC. American College of Gastroenterology guideline on the management of Helicobacter pylori infection. Am J Gastroenterol. 2007;102:1808–1825. - PubMed
- Alm RA, Ling LS, Moir DT, King BL, Brown ED, et al. Genomic-sequence comparison of two unrelated isolates of the human gastric pathogen Helicobacter pylori. Nature. 1999;397:176–180. - PubMed
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