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. 2016 May 11;12(5):e1005626.
doi: 10.1371/journal.ppat.1005626. eCollection 2016 May.

Genetic Diversity as Consequence of a Microaerobic and Neutrophilic Lifestyle

Nora-Johanna Krüger  1 , Marie-Theres Knüver  1 , Anna Zawilak-Pawlik  2 , Bernd Appel  1 , Kerstin Stingl  1
Affiliations

Genetic Diversity as Consequence of a Microaerobic and Neutrophilic Lifestyle

Nora-Johanna Krüger et al. PLoS Pathog. .

Abstract

As a neutrophilic bacterium, Helicobacter pylori is growth deficient under extreme acidic conditions. The gastric pathogen is equipped with an acid survival kit, regulating urease activity by a pH-gated urea channel, opening below pH 6.5. After overcoming acid stress, the bacterium's multiplication site is situated at the gastric mucosa with near neutral pH. The pathogen exhibits exceptional genetic variability, mainly due to its capability of natural transformation, termed competence. Using single cell analysis, we show here that competence is highly regulated in H. pylori. DNA uptake complex activity was reversibly shut down below pH 6.5. pH values above 6.5 opened a competence window, in which competence development was triggered by the combination of pH increase and oxidative stress. In contrast, addition of sublethal concentrations of the DNA-damaging agents ciprofloxacin or mitomycin C did not trigger competence development under our conditions. An oxygen-sensitive mutant lacking superoxide dismutase (sodB) displayed a higher competent fraction of cells than the wild type under comparable conditions. In addition, the sodB mutant was dependent on adenine for growth in broth and turned into non-cultivable coccoid forms in its absence, indicating that adenine had radical quenching capacity. Quantification of periplasmically located DNA in competent wild type cells revealed outstanding median imported DNA amounts of around 350 kb per cell within 10 min of import, with maximally a chromosomal equivalent (1.6 Mb) in individual cells, far exceeding previous amounts detected in other Gram-negative bacteria. We conclude that the pathogen's high genetic diversity is a consequence of its enormous DNA uptake capacity, triggered by intrinsic and extrinsic oxidative stress once a neutral pH at the site of chronic host colonization allows competence development.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Transformation rate in H. pylori correlated with the fraction of cells with active outer membrane DNA transport.
Cell suspensions were derived from the experiments illustrated in Fig 4. Bacteria with different competent fractions of cells were taken during or after competence development; non-competent cells were taken before competence development. H. pylori N6 were incubated in parallel with either Cy3-labelled λ DNA or a PCR fragment of rpsL(A128G), conferring streptomycin resistance. The data show the principle suitability of measuring the fraction of cells with active outer membrane DNA transport for the evaluation of competence development. Cumulative data from 13 independent experiments (n = 42).
Fig 2
Fig 2. Quantification of imported DNA into H. pylori cells.
A, Cy3 image of single Cy3-labelled λ DNA molecules, stained in yellow; B, DIC/Cy3 overlay image of H. pylori J99 after 10 min of DNA uptake under microaerobic conditions; C, results of the quantitative analysis of DNA uptake. For H. pylori, fluorescence intensity in single DNA foci (red) and in whole cells (blue) was determined in two different wild type strains (N6 and J99) after microaerobic growth in TSB-FBS into competent phase. D, histogram of the number of foci per cell. Grey bars, strain N6; black bars, strain J99. Fluorescence intensity was compared to that of single λ DNA molecules and expressed as imported kb. The images were acquired using a Zeiss Axio Observer Z1 and ZEN software, blue edition, version 2.0.0.0. ncells = 928 or 1483 and nfoci = 1903 or 1740 were analyzed for J99 or N6, respectively.
Fig 3
Fig 3. DNA uptake complexes are reversibly shut down at acidic pH values.
Competent bacteria were incubated in BB-FBS that had been titrated with HCl or NaOH to the indicated pH values. Uptake of Cy3-labelled λ DNA occurred for 10 min under aerobic conditions. Cells were washed once in the respective pH medium before DNase treatment for 5 min in TSB-FBS at pH 7.5. The fraction of competent cells relative to the control condition incubated in TSB-FBS at pH 7.5 are depicted. Values stem from at least three experiments; error bars, standard deviation.
Fig 4
Fig 4. Competence development during microaerobic growth.
Competence development was monitored by the fraction of cells with active outer membrane DNA uptake at distinct growth phases under microaerobic atmosphere at 37°C (n = 16; large graph, circles, with left y-axis, sigmoidal curve fit using Sigma Plot 11.0). pH was monitored during growth (large graph, triangles). H. pylori N6 was grown overnight in BB-FBS to an OD600 of 0.19 ± 0.05 (t0) at which only a minor fraction of cells displayed competence (2.2 ± 2.86%). The onset of competence development was defined at t1 at which cells exhibited a mean OD600 of 0.39 ± 0.05 and 4 ± 2.8% of competent cells (~ t0 + 3–4 hours). At t2 (~ t0 + 6–8 hours) upon switch into competent state (50.6 ± 15.6% of cells with active outer membrane DNA uptake) cells exhibited a mean OD600 of 0.7 ± 0.11. At t0, either effectors (0.5 mM adenine or 0.5 mM glutamine) were added or the medium was exchanged by fresh BB-FBS medium with our without supplementation of 0.125 μg/ml ciprofloxacin or 0.025 μg/ml mitomycin C or temperature was decreased for 5°C or the cell suspension was exposed to aerobic conditions. Differences in fraction of competent cells at t1 or t2 due to change in incubation conditions are shown in the inserted diagram (at least three experiments for each condition; error bars, standard deviation). For aerobic stress conditions data are depicted from time point 1h after t0.
Fig 5
Fig 5. Kinetics of competence development under aerobic conditions.
H. pylori N6 was grown microaerobically in BB-FBS overnight at t0 before competence development (OD600~0.2). Cells were exposed to aerobic conditions at 37°C for 120 min. At the indicated time points the competence fraction of the cells was monitored. Control cells were kept under microaerobic atmosphere. Upper panel, DIC/Cy3 images of bacteria at indicated timepoints, with DNA stained in yellow; lower panel (left), fraction of competent cells; lower panel (right), number of distinct DNA foci per competent cell. Data stem from at least three experiments; error bars, standard deviation.
Fig 6
Fig 6. Neutral pH opens the opportunity for competence development triggered by oxidative stress.
H. pylori N6 was grown microaerobically in BB-FBS overnight until growth phase before competence development (OD600~0.2). Cells were exposed to different pH and atmospheric conditions for 1 hour. Data stem from at least three experiments; error bars, standard deviation.
Fig 7
Fig 7. Phenotype of an oxygen-sensitive mutant sodB lacking superoxide dismutase.
H. pylori were grown in BB-FBS at reduced oxygen atmosphere (1% O2, 10% CO2, 89% N2) in the absence and presence of 0.5 mM adenine. A and B, overlay images of DIC/Cy3 of sodB after 10 min of DNA uptake with DNA stained in yellow; arrowheads indicate coccoid formation of the sodB mutant in the absence of adenine (A), while cells kept their rod-shaped morphology in the presence of adenine (B). C, log CFU/ml after 18–24 h of growth of sodB, the wild type and the sodBcompl. D, competence development occurred with lower OD600 values in the sodB mutant compared to the wild type; the sodBcompl showed an intermediate phenotype. Addition of adenine reduced competence development. Data in C and D stem from at least three independent experiments; datapoints of the respective strain/condition were highlighted in D for better visualization.
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Grants and funding

NJK was financed by the Deutsche Forschungsgemeinschaft (DFG) project STI 201/1-3. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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