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Imprint
Molecular mechanism of horizontal gene transfer in pathogenic epsilon-proteobacteria (Gene transfer)
Project
Project code: BfR-BIOS-08-1350-009, 175974972, FKZ STI 201/3-1
Contract period: 01.10.2011
- 31.03.2015
Purpose of research: Project preparation activity
The uptake of DNA from the environment is the first step of the acquisition of novel characteristics, such as antibiotic resistances or virulence factors, which enable adaptation towards altered environmental conditions. In this project, financed by the Deutsche Forschungsgemeinschaft (DFG) and performed in the department Biological Safety of the BfR (research grant), the molecular mechanisms of DNA uptake in ε-Proteobacteria are investigated in vivo and in vitro. As model organims the two gram-negative human pathogenic bacteria, Helicobacter pylori and Campylobacter jejuni are considered, which are close relatives but use different DNA uptake systems. Using newly developed fluorescence-based detection methods, transport of DNA over the outer and inner membrane of the bacteria can be visualized for the analysis of the role of different gene products in natural transformation of theses bacteria. The goal is a better understanding of the different steps of natural transformation as well as the development of inhibitors, which e. g. can be used for the reduction of the spread of resistance genes.
As a neutrophilic bacterium, 'Helicobacter pylori' is growth deficient under extremely acidic conditions. After overcoming the acid stress in the gastric lumen, the bacterium reaches its natural habitat with near neutral pH where it causes chronic gastritis. The bacterium shows an exceptionally high genetic variability that is mainly due to its natural competence, characterized by the capability to take up free DNA. During micro-aerobic growth significant competence development was observed. Comparative cultural and microscopic analysis showed that 'H. pylori' was capable of regulating its transformation rate by several log levels. The dynamics were controlled by variation of the amount of cells exhibiting active DNA uptake over the outer membrane. The single cell analysis is based on the monitoring of uptake of covalently labelled fluorescent DNA in living cells. Single cell analysis showed that natural transformation of 'H. pylori' was strongly influenced by pH. In this regard, pH values above 6.5 opened a competence window, in which competence development was triggered by the level of oxidative stress. These conclusions were drawn from the fact that the kinetics of competence development was influenced by different oxygen levels as well as by the presence of adenine. Furthermore, it was observed that previously active DNA uptake complexes were reversibly shut down below pH 6.5. In contrast, the addition of sub-lethal concentrations of the DNA-damaging agent ciprofloxacin or mitomycin C did not trigger competence development under the studied conditions. An oxygen-sensitive mutant lacking the gene coding for superoxide dismutase (sodB) displayed a higher competent fraction of cells than the wildtype under comparable conditions. In addition, the 'sodB' mutant showed substantial growth problems and conversion into non-cultivable coccoid forms even under reduced oxygen concentration in liquid media, which was compensated by addition of adenine. This indicated that adenine acted as a radical quencher. Quantification of periplasmically located DNA in competent J99 wildtype cells revealed an outstanding medial imported DNA amount of around 350 kb per cell within 10 min, with maximal amounts of a chromosomal equivalent (1.6 Mb) in individual cells. The values for strain N6 were somewhat lower, but still impressive compared to other Gram-negative bacteria. It could be concluded that the pathogen's high genetic diversity is a consequence of its extremely high DNA uptake capacity, triggered by oxidative stress at neutral pH values. This might reflect the successful adaption of 'H. pylori' to its natural environment, the gastric mucosa with near neutral pH values and probably increased oxygen stress exerted by the immune response of the host.
Section overview
Subjects
- Biotechnology
