B4

State of the art

Epilithic photoautotrophic biofilms represent very complex communities exhibiting a high phenotypic plurality regarding substrate and nutrient utilization, production of extracellular polymeric substances (EPS), and cell/cell communication (Hoagland et al. 1993). In the littoral zone of Lake Constance, such biofilms contribute considerably to primary production. Recent data show that biofilms may consist of defined communities of photoautotrophic algae and bacteria (Bruckner et al. 2011). We have shown previously that specific bacteria can influence carbohydrate secretion, growth, and colony formation of benthic diatoms, and thus strongly affect biofilm formation, while the bacteria feed on the carbohydrates (Leinweber and Kroth 2015; Buhmann et al. 2016). This tight interdependence of the organisms in the biofilms raises questions about the benefits of such communities with regard to resilience against environmental changes, e.g. increasing temperatures, light avalability or changing nutrient levels. Further questions include the reversibility of biofilm composition after conditions return to the previous state, and how this reversal effects interspecific communication. Therefore, in this project we plan to study the impact of changing external conditions on biofilm communities as well as on individual key partners

Preparatory work

We established defined laboratory communities of isolated diatom and bacteria strains from biofilms. Effects of changed conditions on biofilm formation will be monitored automatically in specially designed culture chambers (Buhmann et al. 2011). Previous work demonstrated that bacteria such as Dyadobacter strain 32 may specifically induce diatom behaviour including capsule formation, movement etc. (Windler et al. 2015). We established a transcriptomic library of the model diatom Achnanthidium minutissimum, which is commonly found in littoral biofilms of Lake Constance, and we currently try to establish a genetic transformation system for this diatom. We furthermore have created transposon mutated bacterial strains to identify bacterial genes involved in diatom/bacteria communication. These approaches will allow us to understand and verify interspecies communication as a response to changing external cues.

Proposed project and role within the RTG

The goal of this project is to understand as to whether biofilms respond to external cues with a slow recovery or with a regime shift (Dai et al., 2012). Therefore, responses of mixed bacterial and algal communities to changes in light intensity, temperature, nutrient availability (eutrophication), will be analysed using the laboratory model system A. minutissimum and Dyadobacter strain 32 in order to identify physiological and chemical adaptations. Identified responses will later be analysed in natural bacterial/algal communities in Lake Constance and matched to the data collected in project A2 by Schleheck/Kroth. Our model system will be cultured in flasks or agar plates in climate chambers, in biofilm chambers, or in mesocosms under controlled conditions in order to identify the impact of a selected influencing factor. Metabolic changes will be identified by differential metabolite profiling of supernatants and extracts comparing different growth conditions (planktonic cells, biofilms) by liquid chromatography connected to a high resolution tandem mass spectrometer (LC-HR-MS/MS) and gas chromatography connected to a mass spectrometer (GC-MS). Xenic and axenic cultures, as well as cultures with bacterial transposon tagged mutants, will be compared in order to identify the impact of interspecies interactions on the response to changing environmental conditions. Metabolomic tools will be used in order to identify these differences between different sample treatments. In addition, bioassays will be performed in order to identify the ecological function of potentially induced secondary metabolites. Agar diffusion assays will be performed in order to identify chemical signals, algicides, antibiotic or antifungal compounds. Bioactive compounds that are induced under certain growth conditions will be isolated by bioassay-guided fractionation using extraction, column chromatography and high performance liquid chromatography (HPLC). The purified secondary metabolites will be identified by high resolution mass spectrometry (MS) and nuclear magnetic resonance (NMR). Such identified compounds, involved in shaping the interactions in dynamic growth conditions will be thoroughly studied for their biological function in the ecosystem. In detailed bioassays, we will try to monitor the compounds directly in natural samples using sensitive and selective tandem mass spectrometry techniques.

The project will cooperate with project A2 regarding the composition of algal/bacterial communities. Furthermore, studies of inter-species metabolic exchange as well as quantitative community analysis will be performed in collaboration with project C1.