Dans cette thèse, les applications des plasmas à basse température à la pression atmosphérique sont discutées. En particulier, la bio-décontamination des bactéries planctoniques et biofilms bactériens sur des surfaces planes et complexe sont réalisées par des décharges corona de l'air et de plasma d'argon. Dans ce travail, nous caractérisons trois sources de plasma qui sont utilisées pour la décontamination d'Escherichia coli. CC corona décharges dans l'air - corona streamer positive et négative des impulsions Trichel ont été utilisés pour la décontamination des bactéries planctoniques et biofilms bactériens. Dans certaines expériences de l'eau a été électro-pulvérisée sur des échantillons de haute tension électrode. Bio-décontamination des biofilms bactériens a été réalisée sur des lames en verre, pendant 15 min le traitement de plasma a été rendu majorités des bactéries incultivables. Selon la microscopie confocale à balayage laser de biofilms colorées par kit de la viabilité, une partie de ces bactéries incultivables restait viables, seulement les couches les plus supérieures du biofilm ont été tuées. La deuxième source de plasma est corona décharge pulsée propagé à l'intérieur du tube de quartz longue et étroite où l'argon sec ou l'argon avec de la vapeur d'eau coulait à la pression atmosphérique. Ce type de décharge et a une application potentielle dans la décontamination des surfaces intérieures des cathéters ou d'autres dispositifs longs et tubulaires ou pourrait fournir un plasma à basse température sur des distances longues à l'intérieur du corps humain. Tout d'abord, cette source de plasma à basse température a été caractérisée par ses paramètres électriques, et ensuite, une spectroscopie d'émission optique identifiés l'émission de plasma UV-B de radical hydroxyle excité en particulier avec l'argon humide. L'effet de cette UV-B a été testé sur des bactéries planctoniques et a été découvert pour causer jusqu'à un dommage substantiel encore plus loin en aval du tube. La source de plasma d'argon dernière doit aller qui utilise l'argon saturé sec, humide ou de l'eau en tant que gaz de travail. Cette décharge a été principalement utilisée pour la décontamination biofilm, et nous avons reçu des résultats similaires comme décharges corona CC. / In this PhD thesis, applications of lowtemperature plasmas at atmospheric pressure are discussed. In particular, bio-decontamination of planktonic bacteria and bacterial biofilms on flat and complex surfaces by air corona discharges and argon plasma. In this work, we characterize three plasma sources which are used for decontamination of Escherichia coli. DC corona discharges in air - positive streamer corona and negative Trichel pulses were used for decontamination of planktonic bacteria and bacterial biofilms. In some experiments water was electrosprayed onto samples from high voltage electrode. Bio-decontamination of bacterial biofilms was carried out on glass cover slides, within 15 min plasma treatment most of the bacteria were rendered uncultivable. Part from these uncultivable bacteria remained viable only top layers of the biofilm were killed, according to confocal laser scanning microscopy of biofilms stained by live/dead viability kit. The secondplasma source was pulsed corona discharge propagated inside the long narrow quartz tube in which dry argon or argon with water vapor was flowing at atmospheric pressure. This type of discharge has a potential application in decontamination of inner surfaces of catheters or other long tubular devices or could able to deliver low-temperature plasma on longer distances inside the human body. Firstly, this low-temperature plasma source was characterized by its electrical parameters, then, an optical emission spectroscopy of plasma identified UV B emission form excited hydroxyl radical especially with humid argon working gas. The effect of this UV B was tested on planktonic bacteria and was found out to cause up to a substantial damage even further downstream the tube. The last plasma source has argon jet which used dry, humid or water saturated argon as a working gas. This discharge was predominantly used for biofilm decontamination, where we received similar results as with DC corona discharges.

Les pertes alimentaires mondiales se chiffrent à environ un tiers des aliments destinés à la consommation humaine, soit environ 1,3 milliards de tonnes par an (FAO). Une large fraction de ces pertes est due aux altérations microbiologiques des denrées alimentaires. L’utilisation de produits phytosanitaires reste aujourd’hui la solution la plus largement utilisée en agriculture pour limiter ces pertes. Cependant, avec le plan EcoPhyto 2, le gouvernement français a pour objectif de réduire de 50% l’usage des pesticides chimiques d’ici 2025, en particulier en promouvant l’émergence du biocontrôle. Pour développer cette approche, il est cependant nécessaire de comprendre, pour mieux les maitriser, les mécanismes sous-jacents. Les différents modes d’action de biocontrôle par les microorganismes décrits sont la stimulation des défenses naturelles des plantes, la production de substances antimicrobienne et la compétition nutritionnelle. L'originalité de ce projet est d'intégrer le mode de vie en biofilm dans les mécanismes de bioprotection (compétition spatiale et nutritionnelle, libération de principes antimicrobiens). Dans la filière Française des champignons de couche (Agaricus bisporus), l’agent de biocontrôle utilisé depuis 2008 par plus de 80 % de la filière, est Bacillus subtilis QST713. Ce biofongicide montre une nette efficacité contre Trichoderma aggressivum, la principale moisissure à l’origine de pertes économiques lors de la culture d’A. bisporus. Afin d’accompagner la filière dans cette voie biologique, nous avons entrepris de séquencer et étudier le génome de cette souche, afin de déterminer son potentiel de biocontrôle et sa capacité à former des biofilms. Nous avons également évalué l’impact de ce biofongicide sur la dynamique des communautés microbiennes du compost de culture d’A. bisporus exposé ou non à T. aggressivum. Enfin, l'étude de la reprogrammation cellulaire de cet agent de biocontrôle lors de sa culture en micromodèles axéniques, nous a permis une meilleure compréhension des phénomènes de colonisation des substrats et d'inhibition des flores indésirables. Ce projet a permis d’enrichir les connaissances vis-à-vis des mécanismes de biocontrôle dans la filière des champignons et pourra permettre une possible application à d’autres filières agricoles. / Worldwide, food losses amount for about one-third of food for human consumption, 1.3 billion tons per year (FAO). A large fraction of these losses are due to microbiological alterations. The use of phytosanitary products remains today the most widely used solution in agriculture to limit these losses. However, with the EcoPhyto 2 plan, the French government aims to reduce the use of chemical pesticides by 50% by 2025, in particular by promoting the emergence of biocontrol. To develop this approach, it is necessary to understand the underlying mechanisms. The different modes of action of biocontrol by the microorganisms described are the stimulation of the natural defenses of the plants, the production of antimicrobial substances and the nutritional competition. The originality of this project is to integrate the biofilm mode of life into bioprotection mechanisms (spatial and nutritional competition, release of antimicrobial principles). In the French sector of the button mushrooms (Agaricus bisporus) culture, the biocontrol agent used since 2008 by more than 80% of the sector, is Bacillus subtilis QST713. This biofungicide shows a clear efficacy against Trichoderma aggressivum, the main mold causing economic losses during the cultivation of A. bisporus. To accompany the sector in this biological pathway, we have sequenced and studied the genome of this strain, in order to determine its biocontrol potential and its ability to form biofilms. We also evaluated the impact of this biofungicide on the dynamics of microbial communities in A. bisporus culture compost exposed or not to T. aggressivum. Finally, the study of the cellular reprogramming of this biocontrol agent during the culture in axenic micromodels allowed us a better understanding of the substrates colonization phenomenon and the inhibition of undesirable flora. This project will enrich the knowledge of the biocontrol mechanisms used in the mushroom industry and may allow a possible application to other agricultural sectors.

With the rise in global energy prices, as well as energy consumption being the largest source of greenhouse gas emissions, biofilm-based systems utilized for wastewater treatment, such as moving bed biofilm reactors (MBBRs), have grown in popularity for their lower energy consumption compared to conventional activated sludge processes. However, this technology requires large amounts of energy to constantly distribute and suspend its biofilm carrier within the reactor by either aerators or mechanical mixers. Many studies have been done on optimizing the aeration systems, but limited research has been focused on the mechanical mixing systems. This master’s thesis project aims to narrow the research and data gaps in MBBR mechanical mixing by conducting scaled-down experiments to study the influence of different mixer configurations on carrier’s flow fields in a reactor tank. The main objective is to determine the conditions for good carrier mixing and their energy use efficiency. Other objectives include determining the effects of scaling on carrier flow fields and whether the experimental results can be used to help develop and validate MBBR computational fluid dynamics (CFD) models. The results showed that good carrier mixing occurred in conditions where (1) mixer height was 3 cm from the bottom of the tank, had no inclinations and positioned along a long wall; (2) mixer flows could maintain their momentum; (3) sufficient length was given for mixer jet streams to develop and widen; (4) there was a dual presence of vertical flow loops and horizontal bulk flow loops; (5) 2 mixers did not result in counter- rotating flows; and (6) 2 mixers were not positioned in one corner of the tank. The effects of scaling did not impact the carrier flow fields and was determine by comparing the experimental results from this study with the results from a previous experiment that utilized a smaller tank. The results from this study could qualitatively match with the results of the CFD model. Limitations that occurred during the study when trying to define “good mixing” were also discussed. Lastly, the thesis ends with stating future work and recommendations. / Med stigande globala energipriser och en energiförbrukning som är den största källan till utsläpp av växthusgaser, har biofilmbaserade system för avloppsvattenrening, t.ex. biofilmsreaktorer med rörlig bädd (MBBR), ökat i popularitet tack vare sin lägre energiförbrukning jämfört med konventionella processer med aktivt slam. Denna teknik kräver dock stora mängder energi för att ständigt distribuera och suspendera biofilmbäraren i reaktorn med hjälp av antingen luftare eller mekaniska blandare. Många studier har gjorts för att optimera luftningssystemen, men begränsad forskning har fokuserats på de mekaniska blandningssystemen. Detta examensarbete syftar till att minska forsknings- och dataluckorna inom mekanisk blandning i MBBR genom att genomföra nedskalade experiment för att studera hur olika blandarkonfigurationer påverkar bärarens flödesfält i en reaktortank.Huvudsyftet är att fastställa villkoren för god blandning av bärare och deras energianvändningseffektivitet. Andra mål är att fastställa effekterna av skalning på bärarnas flödesfält och om de experimentella resultaten kan användas för att utveckla och validera CFD-modeller (Computational Fluid Dynamics) för MBBR. Resultaten visade att god bärarblandning inträffade under förhållanden där (1) blandarhöjden var 3 cm från tankens botten, utan lutningar och placerad längs en lång vägg; (2) blandarflöden kunde behålla sitt momentum; (3) tillräcklig längd gavs för blandarjetströmmar att utvecklas och breddas; (4) det fanns en dubbel närvaro av vertikala flödesslingor och horisontella bulkflödesslingor; (5) 2 blandare inte resulterade i motroterande flöden; och (6) 2 blandare inte placerades i ett hörn av tanken. Effekterna av skalning påverkade inte bärarens flödesfält och fastställdes genom att jämföra de experimentella resultaten från denna studie med resultaten från ett tidigare experiment som använde en mindre tank. Resultaten från denna studie kunde kvalitativt matchas med resultaten från CFD-modellen. Begränsningar som uppstod under studien när man försökte definiera "bra blandning" diskuterades också. Slutligen avslutas avhandlingen med att ange framtida arbete och rekommendationer.

No description available.

Exposure to herbicides poses a threat to aquatic biofilms by affecting their community structure, physiology and function. These changes render biofilms to become more tolerant, but on the downside community tolerance has ecologic costs. A concept that addresses induced community tolerance to a pollutant (PICT) was introduced by Blanck and Wängberg (1988). The basic principle of the concept is that microbial communities undergo pollution-induced succession when exposed to a pollutant over a long period of time, which changes communities structurally and functionally and enhancing tolerance to the pollutant exposure. However, the mechanisms of tolerance and the ecologic consequences were hardly studied up to date. This thesis addresses the structural and functional changes in biofilm communities and applies modern molecular methods to unravel molecular tolerance mechanisms. Two different freshwater biofilm communities were cultivated for a period of five weeks, with one of the communities being contaminated with 4 μg L-1 diuron. Subsequently, the communities were characterized for structural and functional differences, especially focusing on their crucial role of photosynthesis. The community structure of the autotrophs was assessed using HPLC-based pigment analysis and their functional alterations were investigated using Imaging-PAM fluorometry to study photosynthesis and community oxygen profiling to determine net primary production. Then, the molecular fingerprints of the communities were measured with meta-transcriptomics (RNA-Seq) and GC-based community metabolomics approaches and analyzed with respect to changes in their molecular functions. The communities were acute exposed to diuron for one hour in a dose-response design, to reveal a potential PICT and uncover related adaptation to diuron exposure. The combination of apical and molecular methods in a dose-response design enabled the linkage of functional effects of diuron exposure and underlying molecular mechanisms based on a sensitivity analysis. Chronic exposure to diuron impaired freshwater biofilms in their biomass accrual. The contaminated communities particularly lost autotrophic biomass, reflected by the decrease in specific chlorophyll a content. This loss was associated with a change in the molecular fingerprint of the communities, which substantiates structural and physiological changes. The decline in autotrophic biomass could be due to a primary loss of sensitive autotrophic organisms caused by the selection of better adapted species in the course of chronic exposure. Related to this hypothesis, an increase in diuron tolerance has been detected in the contaminated communities and molecular mechanisms facilitating tolerance have been found. It was shown that genes of the photosystem, reductive-pentose phosphate cycle and arginine metabolism were differentially expressed among the communities and that an increased amount of potential antioxidant degradation products was found in the contaminated communities. This led to the hypothesis that contaminated communities may have adapted to oxidative stress, making them less sensitive to diuron exposure. Moreover, the photosynthetic light harvesting complex was altered and the photoprotective xanthophyll cycle was increased in the contaminated communities. Despite these adaptation strategies, the loss of autotrophic biomass has been shown to impair primary production. This impairment persisted even under repeated short-term exposure, so that the tolerance mechanisms cannot safeguard primary production as a key function in aquatic systems.:1. The effect of chemicals on organisms and their functions .............................. 1 1.1 Welcome to the anthropocene .......................................................................... 1 1.2 From cellular stress responses to ecosystem resilience ................................... 3 1.2.1 The individual pursuit for homeostasis ....................................................... 3 1.2.2 Stability from diversity ................................................................................. 5 1.3 Community ecotoxicology - a step forward in monitoring the effects of chemical pollution? ................................................................................................................. 6 1.4 Functional ecotoxicological assessment of microbial communities ................... 9 1.5 Molecular tools – the key to a mechanistic understanding of stressor effects from a functional perspective in microbial communities? ...................................... 12 2. Aims and Hypothesis ......................................................................................... 14 2.1 Research question .......................................................................................... 14 2.2 Hypothesis and outline .................................................................................... 15 2.3 Experimental approach & concept .................................................................. 16 2.3.1 Aquatic freshwater biofilms as model community ..................................... 16 2.3.2 Diuron as model herbicide ........................................................................ 17 2.3.3 Experimental design ................................................................................. 18 3. Structural and physiological changes in microbial communities after chronic exposure - PICT and altered functional capacity ................................................. 21 3.1 Introduction ..................................................................................................... 21 3.2 Methods .......................................................................................................... 23 3.2.1 Biofilm cultivation ...................................................................................... 23 3.2.2 Dry weight and autotrophic index ............................................................. 23 3.2.4 Pigment analysis of periphyton ................................................................. 23 3.2.4.1 In-vivo pigment analysis for community characterization ....................... 24 3.2.4.2 In-vivo pigment analysis based on Imaging-PAM fluorometry ............... 24 3.2.4.3 In-vivo pigment fluorescence for tolerance detection ............................. 26 3.2.4.4 Ex-vivo pigment analysis by high-pressure liquid-chromatography ....... 27 3.2.5 Community oxygen metabolism measurements ....................................... 28 3.3 Results and discussion ................................................................................... 29 3.3.1 Comparison of the structural community parameters ............................... 29 3.3.2 Photosynthetic activity and primary production of the communities after selection phase ................................................................................................. 33 3.3.3 Acquisition of photosynthetic tolerance .................................................... 34 3.3.4 Primary production at exposure conditions ............................................... 36 3.3.5 Tolerance detection in primary production ................................................ 37 3.4 Summary and Conclusion ........................................................................... 40 4. Community gene expression analysis by meta-transcriptomics ................... 41 4.1 Introduction to meta-transcriptomics ............................................................... 41 4.2. Methods ......................................................................................................... 43 4.2.1 Sampling and RNA extraction................................................................... 43 4.2.2 RNA sequencing analysis ......................................................................... 44 4.2.3 Data assembly and processing................................................................. 45 4.2.4 Prioritization of contigs and annotation ..................................................... 47 4.2.5 Sensitivity analysis of biological processes .............................................. 48 4.3 Results and discussion ................................................................................... 48 4.3.1 Characterization of the meta-transcriptomic fingerprints .......................... 49 4.3.2 Insights into community stress response mechanisms using trend analysis (DRomic’s) ......................................................................................................... 51 4.3.3 Response pattern in the isoform PS genes .............................................. 63 4.5 Summary and conclusion ................................................................................ 65 5. Community metabolome analysis ..................................................................... 66 5.1 Introduction to community metabolomics ........................................................ 66 5.2 Methods .......................................................................................................... 68 5.2.1 Sampling, metabolite extraction and derivatisation................................... 68 5.2.2 GC-TOF-MS analysis ............................................................................... 69 5.2.3 Data processing and statistical analysis ................................................... 69 5.3 Results and discussion ................................................................................... 70 5.3.1 Characterization of the metabolic fingerprints .......................................... 70 5.3.2 Difference in the metabolic fingerprints .................................................... 71 5.3.3 Differential metabolic responses of the communities to short-term exposure of diuron ............................................................................................................ 73 5.4 Summary and conclusion ................................................................................ 78 6. Synthesis ............................................................................................................. 79 6.1 Approaches and challenges for linking molecular data to functional measurements ...................................................................................................... 79 6.2 Methods .......................................................................................................... 83 6.2.1 Summary on the data ............................................................................... 83 6.2.2 Aggregation of molecular data to index values (TELI and MELI) .............. 83 6.2.3 Functional annotation of contigs and metabolites using KEGG ................ 83 6.3 Results and discussion ................................................................................... 85 6.3.1 Results of aggregation techniques ........................................................... 85 6.3.2 Sensitivity analysis of the different molecular approaches and endpoints 86 6.3.3 Mechanistic view of the molecular stress responses based on KEGG functions ............................................................................................................ 89 6.4 Consolidation of the results – holistic interpretation and discussion ............... 93 6.4.1 Adaptation to chronic diuron exposure - from molecular changes to community effects.............................................................................................. 93 6.4.2 Assessment of the ecological costs of Pollution-induced community tolerance based on primary production ............................................................. 94 6.5 Outlook ............................................................................................................ 97

Dricksvatten är en essentiell del av ett hållbart samhälle. Därför är det viktigt att säkerställa säkert dricksvatten genom fungerande vattenreningsverk. En viktig förorening att behandla är NOM, som i sig är ofarligt men som kan producera farliga föroreningar. En teknik som används för behandling av NOM är kolfiltrering. Hur ökad flödeshastighet och ökad kontakttid påverkade kolfiltrens effektivitet undersöktes vid dricksvattenreningsverket Norrvatten. De undersökta parametrarna var partiklar, ultraviolett absorbans vid 254 nm, turbiditet, konduktivitet, fluorescent löst organiskt material, totalt organiskt kol, kemisk syreförbrukning, odlingsbara mikroorganismer och lukt. Tre kolfilter studerades vid olika flödeshastigheter; 190, 220, 250 och 280 L/s under ett dygn var. Samtidigt hade två filter ökad kontakttid på 60 och 76 % under sex veckor, medan ett filter fortsatte med den vanliga flödeshastigheten på 190 L/s. Utgående vatten från filtren analyserades för att se om dessa ändringar hade någon effekt på vattenreningen. Denna preliminära studie fann ingen signifikant effekt på kolfiltreringens rening på grund av ökad flödeshastighet eller uppehållstid. Detta kan vara en indikation på att kolfiltreringen kan hantera en framtida flödesökning och därmed vara en väsentlig del av en framtida expansionen av vattenreningsverket. De tecken som visade på att kolfiltren påverkades av ökningen av flödeshastigheter kunde förklaras av fluktuationer i inkommande vatten och skillnader mellan de olika filtren. I framtiden bör effekten av inkommande vatten studeras i detalj. En mer ingående analys av både inkommande och utgående vatten till kolfiltren bör utföras, där provtagning sker oftare för att bättre förstå fluktuationerna i inkommande föroreningskoncentrationer. Dessutom bör testerna upprepas för att se hur reningen skiljer sig från dag till dag. Hur kolfiltren hanterar ökade flödeshastigheter över längre tidsperioder bör också undersökas vidare. / Drinking water is an essential part of a sustainable society. In the future, the demand for drinking water will increase and contaminants in the water sources are also predicted to increase. Therefore, it is essential to ensure safe drinking water through functioning drinking water treatment plants (DWTPs). One important contaminant to treat is natural organic matter (NOM), which is harmless in itself but can produce harmful products. One technique to use for treating NOM is carbon filters (CFs). The effect of increased flow rate and increased empty bed contact time (EBCT) on the CF efficiency was investigated at a DWTP. The investigated parameters were particles, ultraviolet absorbance at 254 nm, turbidity, conductivity, cultivable microorganisms, fluorescent dissolved organic matter, total organic carbon, chemical oxygen demand, and odour. Three CFs were studied at different flow rates; 190, 220, 250, and 280 L/s for 24 hours each. Additionally, two filters had increased EBCT of 60 and 76 %, while one filter continued with the regular flow rate of 190 L/s for six weeks. Outgoing water from the filters was analysed to see if the change had any effect on the DWTP. This preliminary study did not find any significant effect on the CF treatment caused by increased flow rate or EBCT. This could be an indication that the CFs can handle a future increase in flow rate and thus be an essential part of a future expansion of the DWTP. The indications of CFs being affected by the increase in flow rates for some of the parameters could be explained by fluctuations in incoming water or differences between the separate filters. In the future, a more thorough analysis of both incoming and outgoing water to the CFs should be done, where sampling occurs more frequently to better understand the fluctuations in incoming contaminant concentrations. The measurements should also be repeated to see how the treatment differs from day to day. How the CFs handle increased flow rates over longer time periods should also be investigated further.

Background: Bioadhesion and surface interactions on enamel are of essential relevance for initiation, progression and prevention of caries and erosions. Salivary proteins on and within initial carious and erosive lesions can facilitate or aggravate de- and remineralization. This applies for the pellicle layer, the subsurface pellicle and for proteins within initial carious lesions. Little is known about these proteinaceous structures related to initial caries and erosion. Accordingly, there is a considerable demand for an understanding of the underlying processes occurring at the interface between the tooth surface and the oral cavity in order to develop novel agents that limit and modulate caries and erosion. Objectives and findings: The present paper depicts the current knowledge of the processes occurring at the interface of the tooth surface and the oral fluids. Proteinaceous layers on dental hard tissues can prevent or aggravate demineralization processes, whereas proteins within initial erosive or carious lesions might hinder remineralization considerably and restrict the entry of ions into lesions. Conclusions: Despite the fact that organic–inorganic surface interactions are of essential relevance for de- and remineralization processes at the tooth surface, there is limited knowledge on these clinically relevant phenomena. Accordingly, intensive research is necessary to develop new approaches in preventive dentistry.

The rising number of antibiotic resistant bacteria (ARB) may introduce to the post antibiotic era because they cause a loss of the therapeutic potential of antibiotics. For many years the important role of the natural environment as reservoir and dissemination pathway for ARB and responsible genes has been largely overlooked. However, especially aquatic ecosystems provide optimal conditions for the antibiotic resistance (AR) evolution: first, aquatic ecosystems are frequently affected by anthropogenic activities that cause multiple pollutions for example with heavy metals, that potentially cause co-selection of antibiotic- and heavy metal resistance. Second, aquatic ecosystems feature a dissemination pathway between human populations and natural environments via the urban water cycle. Water cycles between human associated environments (e.g. house holds and clinics) via waste water through waste water treatment plants into natural ecosystems (e.g. water bodies) and back as drinking water after purification. Third, ecosystem internal biotic interactions such as competition between bacteria and predation by the natural consumers seem to impact AR evolution sustainably. The present doctoral thesis focuses on the impact of abiotic and biotic factors on the proliferation of AR and responsible genes in natural aquatic environments, with special emphasis on (i) heavy metal driven co-selection of antibiotic and heavy metal resistance and (ii) on the impact of competition and predation on the evolution of AR. In order to quantify the risk of heavy metal driven co-selection for AR spread, I provide a first risk assessment based on literature values of environmental heavy metal loadings and related AR. Additionally, I developed a limit value named minimum co-selective concentration (MCC), which is the lowest concentration of a heavy metal that can potentially cause coselection in nature. It turned out that Cu, Zn, Ni, Hg, and Cd are suspected to be the main co-selecting heavy metals in the aquatic environment. I further investigated heavy metal driven co-selection of AR in a river ecosystem, the Western Bug River (Ukraine). I found indications for co-selection of resistance to five antibiotics (ciprofloxacin, gentamicin, amikacin, tobramycin, and cefepime) and two metals (Ni and Cd) caused by Ni- and Cd-levels. Both metals exceed their MCC for water samples and Cd additionally in sediments. As a second focal point the present work emphasis on ecological interactions effecting AR evolution. Currently three possible effects of ecological interactions on AR spread are discussed. First, environmental antibiotic levels are rather low, however they might favour ARB due to a competitive advantage. The reason is that even sublethal antibiotic levels reduce the growth of sensitive bacteria while resistant cells remain unaffected by the antibiotic action. Second, predation by protozoa is believed to impact conjugation between prey bacteria (and thus the transfer of DNA and potential resistance genes) by keeping bacteria in a growing stage that favours conjugation. Third, in order to escape predation by protozoa, bacteria evolved grazing defence mechanisms such as the formation of inedible biofilms, which can feedback on the evolution of AR. With an ordinary differential equation model, I tested the effect of low antibiotic levels and losses (e.g. due to predation) on the proliferation of ARB in a modelled planktonic system. In case that the model contains the mechanism that conjugation frequencies are highest during exponential growth, I found that (i) (grazing) losses enhance conjugation frequencies between bacteria and that (ii) medium levels of antibiotics and (grazing) losses favour resistant cells in the competition to sensitive bacteria. Biofilms are thought to be \'hot spots\' for conjugation but some plasmids have lower conjugation frequencies in biofilms compared to planktonic systems. As a first step, in order to discover predation effects on plasmid spread in plankton - biofilm systems I investigated grazing resistance of bacteria in grazing experiments. Both plankton and biofilm phenotypes were consumed, when exposed to their specialized grazer (either plankton-feeder or biofilmfeeder), whereas the other phenotype remained grazing-resistant and thus became the dominant prey type. Both predators together effectively control planktonic and biofilm prey. With regards to the spread of AR-genes via conjugation, I speculate that the feeding preference of the present predator can affect the invasion success of resistance plasmids in planktonic - biofilm systems. For dynamic systems, I assume that dynamics of predator and prey traits (plankton vs. biofilm-feeder and biofilm vs. planktonic prey) will lead to dynamics of conjugation frequencies in planktonic or biofilm bacteria. I assume that conjugation events are more frequent in the dominant prey type (plankton or biofilm). However, other factors such as pili-type of the plasmid (short and rigid pili, prefers conjugation in biofilms or long and flexible pili, prefers conjugation in plankton) might additionally influence plasmid invasion success in plankton - biofilm morphotypes.

Für die E. coli K12 Biofilmbildung ist die Expression des Masterregulators CsgD essentiell. Dies erfordert das Signalmolekül c-di-GMP, dessen Auf- und Abbau durch 12 Diguanylatzyklasen (DGCs mit GGDEF-Domänen) und 13 Phosphodiesterasen (PDEs mit EAL-Domänen) erfolgt. DgcE ist mit einer MASE1-umfassenden Transmembranregion (TM), drei PAS-, einer GGDEF- und einer degenerierten EAL-Domäne die strukturell komplexeste DGC und notwendig für die Biofilmbildung. Diese Arbeit zeigt, dass die Aktivität von DgcE einer hoch komplexen Regulation unterliegt. Einzelnen DgcE-Domänen konnten aktivierende bzw. inhibierende Rollen hinsichtlich der Biofilmmatrixsynthese zugeordnet werden. Die Biofilmbildung hängt von DgcE-produziertem c-di-GMP ab, wobei die DgcE-Dimerisierung v.a. durch die PAS-Region vermittelt wird. Die EAL-Domäne wirkt einer aktiven DgcE-Form entgegen. Für die DgcE-vermittelte Matrixproduktion sind die GTPase YjdA und sein Partnerprotein YjcZ nötig. Über Interaktionen mit YjcZ und der TM von DgcE vermittelt YjdA eine Komplexbildung. Die Interaktion von YjdA und DgcE sowie die Matrixproduktion hängen von der GTPase-Aktivität von YjdA ab. GTP wird daher als intrazelluläres Signal vorgeschlagen, das die Aktivierung von DgcE durch YjdA/YjcZ reguliert. Die MASE1-umfassende TM agiert als Zentrale der Signalintegration. Einerseits ist sie nötig für die DgcE-Aktivität und andererseits ist sie an einem massiven Abbau von DgcE beteiligt. Zudem wurden neu identifizierte Curli-regulierende Gene (rbsK, rbsR, ydcI, yieP, puuR) untersucht, wobei keines über das PdeR/DgcM/MlrA-Modul in die c-di-GMP-vermittelte CsgD-Expression eingreift. Flagellare Verknotungen in der unteren Schicht von E. coli Makrokolonien tragen zur Morphogenese dieser Makrokolonien bei. Diese Arbeit zeigt, dass Flagellenverknotungen zu einer verminderten Expression der Master-PDE PdeH beitragen, wodurch vermutlich die zelluläre c-di-GMP-Konzentration steigt und somit die Biofilmbildung begünstigt wird. / Biofilm formation of E. coli K12 requires the expression of the biofilm master regulator CsgD. This process depends on the signaling molecule c-di-GMP, which is synthesized by 12 diguanylate cyclases (DGCs with GGDEF domains) and degraded by 13 phosphodiesterases (PDEs with EAL domains). DgcE is the most complex DGC with a MASE1-containing transmembrane region (TM), three PAS, a GGDEF and a degenerate EAL domain, and it is essential for biofilm formation. This work shows that the regulation of the DgcE activity is highly complex. It was possible to assign activating and inhibitory roles to single domains of DgcE with regard to the expression of biofilm matrix components. C-di-GMP produced by DgcE is necessary for biofilm matrix production. The dimerization of DgcE is mainly mediated by the PAS region, whereas the EAL domain counteracts an active form of DgcE. DgcE-mediated matrix synthesis requires the activating signal input of the GTPase YjdA and its partner protein YjcZ. DgcE, YjdA and YjcZ form a protein complex in which YjdA directly interacts with YjcZ and the TM of DgcE. The interaction between DgcE and YjdA as well as the matrix expression depend on the GTPase activity of YjdA. Thus, it is proposed that GTP serves as an intracellular signal regulating the activation of DgcE by YjdA/YjcZ. The MASE1-containing TM proved to be a central hub for signal integration. It is both required for DgcE activity and for a massive degradation of DgcE. Furthermore, newly discovered curli-regulating genes (rbsK, rbsR, ydcI, yieP, puuR) have been analyzed. None of those gene products act on CsgD expression via the PdeR/DgcM/MlrA module. Flagellar entangling within the bottom layer of E. coli macrocolonies determines morphogenesis of macrocolonies. The data presented here suggest that the master PDE PdeH is somehow down-regulated by flagellar entangling, which probably results in a higher cellular c-di-GMP concentration, thereby promoting biofilm formation.

Der sekundäre Botenstoff c-di-GMP kommt in vielen Bakterienspezies vor und fördert dort die Bildung von Biofilmen. Für Abbau und Synthese von c-di-GMP in der Zelle sorgen Diguanylatzyklasen (DGCs) und Phosphodiesterasen (PDEs), an deren N-Terminus häufig Sensordomänen die enzymatische Aktivität steuern. In dieser Arbeit wurde die Regulation und Wirkungsweise einer Gruppe von PDEs mit einer neuartigen Sensordomäne, der CSS-Domäne, genauer charakterisiert. Die CSS-Domäne ist im Periplasma lokalisiert und besitzt zwei hochkonservierte Cysteine, von denen eines in dem namensgebenden CSS-Motiv arrangiert ist. Die Integration in die innere Membran erfolgt durch zwei Transmembrandomänen (TM1 und TM2), wobei TM2 die Verbindung zu der C-terminal gelagerten EAL-Domäne mit PDE-Aktivität herstellt. Die Analyse von PdeC als eine von fünf CSS-PDE in Escherichia coli K-12 zeigte, dass die Bildung einer Disulfidbrücke zwischen den konservierten Cysteinen vom oxidierende DsbA/DsbB-System katalysiert wird und zu einer Verminderung der enzymatischen Aktivität der EAL-Domäne führt. Im Gegensatz dazu führt die reduzierte freie Thiol-Form der CSS-Domäne zu einer stark erhöhten PDE-Aktivität verbunden mit der Dimerisierung über die TM2. Die Reduktion der CSS-Domäne führt außerdem zur Prozessierung durch die HtrA-Proteasen DegP und DegQ in ein membranständiges Fragment aus TM2+EAL-Domäne mit hoher enzymatischer Aktivität. Der Abbau durch DegP und DegQ im Periplasma ist sehr effizient. Auf der cytoplasmatischen Seite hingegen erfolgt die weitere Degradierung durch noch unbekannte Proteasen eher langsam, wodurch es unter bestimmten Bedingungen zur Akkumulierung der hochaktiven TM2+EAL-Form kommt. Durch das Zusammenspiel von redox-abhängiger Aktivitätskontrolle und Proteolyse der c-di-GMP-spezifischen PDE PdeC wird die Produktion der amyloiden Curli-Fasern und Cellulose reguliert, welche Hauptbestandteil der extrazellulären Matrix von Biofilmen sind. / The second messenger c-di-GMP promotes biofilm formation in many bacterial species. Phosphodiesterases (PDEs) and diguanylatcyclases (DGCs) - often controlled by various N-terminal sensor domains - degrade and synthesize c-di-GMP. The aim of this study was to characterize the mode of activation and physiological function of a new class of sensor domains, the CSS domain, which is coupled to an EAL domain with PDE activity and therefore potentially controlling c-di-GMP degradation. The CSS sensor domain contains two highly conserved cysteins in the periplasmic loop, of which one is arranged in the characteristic CSS motif. Integration into the inner membrane is ensured by two flanking transmembrane domains (TM1 and TM2), with TM2 providing a connection to the C-terminal EAL domain. Analysis of PdeC as one of 5 CSS-PDEs in Escherichia coli K-12 revealed a close linkage to the disulfide bond (DSB) system as well as important periplasmic proteases. Thus, formation of a DSB between the two conserved cysteins is promoted by the oxidizing DsbA/DsbB-system and reduces enzymatic activitiy of the EAL domain. In contrast, the free thiol form increases PDE activity and dimerizes via the TM2 domain. Moreover, reduction of the CSS domain results in degradation by the periplasmic HtrA proteases DegP and DegQ. These redundantly process PdeC to a shorter fragment containing the TM2 and EAL domain only, which shows dimerization as well and has high PDE activity. Degradation in the periplasm mediated by DegP and DegQ is very efficient. In contrast, further proteolysis in the cytoplasm by yet unidentified proteases is rather slow, allowing the accumulation of the highly active TM2+EAL form. Finally, the interplay of redox dependent activity control and proteolysis of the c-di-GMP specific PDE PdeC regulates production of curli and cellulose as major matrix components of the bacterial biofilm.