Prokaryotic organisms represent by far the most abundant living organisms populating our planet. They play important function for the ecology of our ecosystems and environments and without prokaryotes living on earth would not be possible. However, certain bacterial species are associated with severer kinds of human diseases such as tuberculosis or cholera. In addition, invasive infections such as soft tissue infections or bloodstream infection represent a constant threat for human health. Bacterial infections are commonly treated by antibiotics. However, antibiotic therapy becomes more and more challenging as resistant bacterial strains arise. The adaption of bacteria towards antibiotic pressure is a good example of the remarkable ability of prokaryotes to adapt to changing environmental conditions and to secure the thriving of the population. However, environmental pressure is manifold and ranges from the availability of different nutrient sources in the environment to the destructive ability of the human immune system when a pathogen enters the human soft tissue. In all these cases a bacteria community needs to adapt to the new environment which can happen by multiple routs. A prominent way is the so-called horizontal gene transfer were additional genetic material is acquired, transferring novel capabilities. However, it relays on the availability of exogenous genetic material (DNA) in the environment.
A mechanism that is independent of exogenous DNA is “gene duplication and amplification” (GDA). GDA uses a genetic mechanism dependent on the SOS recombinase RecA. RecA can promote chromosomal recombination during cell division leading to the duplication of genes within a single chromosome (two copies of an identical gene in a row). Following this duplication, the process can be repeated to extend or to contract the gene array in an accordion-like manner. Long gene arrays can thereby be created and removed rapidly. The amplification of a gene results in strong overexpression of the target protein. GDAs are described to be able transfer antibiotic resistance (overexpression of drug efflux pumps) or to allow better usage of unusual carbon sources. However, it is unclear how frequent GDAs are in natural populations of pathogens and whether GDAs might be important for the adaption of pathogens to certain environmental niches or during the transition from colonization to infectious disease. This lack of knowledge has to be attributed to the difficulty of GDA detection. Even in times of Next Generation Sequencing (NGS) when entire bacterial genomes are sequenced within days, GDAs are only rarely reported. We hypothesized that this is due to the difficulties of their detection. The individual gene copies of GDAs are frequently identical on the DNA level and the short reads (100-200bp) created by NGS technology do therefore fail to indicate that several copies of the same gene are present in a genome. However indications about GDAs can be gained from NGS datasets if analysed accordingly. If more copies of the same gene are present, more individual NGS reads covering this gene will be created (the scaffolding will increase). However, this needs special attention during analysis. In this project we sought to investigate whether NGS technology can be used to conveniently detect GDAs to show how our community can gain additional knowledge from NGS. Furthermore, we wanted to identify the frequency of GDAs in clinical populations and the effects of GDAs on phenotypic characteristics to demonstrate effects of theses mechanism on pathogen evolution.
