Browsing by Subject "Molekularbiologie"

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Evaluation and method development for the biosynthesis of microbial lipopeptides by bacillus species
(2023) Vahidinasab, Maliheh; Hausmann, Rudolf
Microbial lipopeptides are secondary metabolites produced by bacteria and single-celled microorganisms. They consist of a cyclic or linear peptide chain linked to a lipid residue. Due to their high-foaming biosurfactant properties, they have various industrial applications such as in detergents, food emulsifiers, bioremediation, and enhanced oil recovery. Additionally, they possess other functional properties such as antifungal activity, making them an environmentally friendly alternative to synthetic fertilizers and fungicides. Bacillus species produce cyclic lipopeptides known for their potent antifungal activity, which makes them a potential source of bio-fungicides in agriculture. However, the production titer of wild-type Bacillus species does not meet industrial needs. Thereby, genetic modification of producer strains and bioprocess engineering can help increase the production of lipopeptides. Nevertheless, the regulation and basis of biosynthesis for Bacillus lipopeptides are still not completely understood, and ongoing research aims to enhance their production. In general, three main lipopeptide families, including surfactins, iturins, and fengycins are produced by different Bacillus species. Among these, surfactin as the strong biosurfactant is the most extensively studied lipopeptide produced by Bacillus species. The focus of this doctoral thesis was mainly to evaluate the biosynthesis of iturin and fengycin families, which are strong antimicrobial lipopeptides produced by Bacillus subtilis and Bacillus velezensis. This involved developing strains through genetic engineering and enhancing the lipopeptide titer by evaluating the cultivation medium. Initially, the entire genome of the bacteria used in this thesis was examined in terms of lipopeptide biosynthesis, and the structure and yield of the different produced lipopeptides were analyzed. Regarding the lipopeptide producer derivatives of the domesticated laboratory model strain B. subtilis 168 and B. subtilis 3NA, a spore deficient strain appropriate for bioreactor cultivation, surfactin is the lipopeptide with the highest yield, while plipastatin which is a member of fengycin family, is produced in lower quantities. In the present thesis, the biosynthesis of plipastatin by B. subtilis BMV9 as the lipopeptide producer derivative of strain 3NA was evaluated. The study aimed to convert BMV9 to a constitutive plipastatin mono-producer strain. In this sense, overexpressing plipastatin biosynthesis operon using the stronger constitutive Pveg promoter led to a five-fold increase in plipastatin production. Interestingly, it was observed that deletion of srfAA-AD operon in BMV9 and the constructed constitutive plipastatin producer strain has not improved plipastatin production. Therefore, it can be stated that presumably the biosynthesis of plipastatin may be positively influenced in a post-transcriptional manner by the surfactin synthetase or some of its subunits. However, the regulatory mechanism behind this effect remained unknown and requires further research. Another attempt to enhance the plipastatin biosynthesis in strain BMV9 was repairing the degQ expression. One main genome characterization of strains with B. subtilis 168 and 3NA background is that the pleiotropic degQ gene expression, which is known to have a positive effect on plipastatin biosynthesis, is silenced due to a mutation in the promoter area. However, while repair of degQ expression in BMV9 increased the plipastatin production, combination of both repaired degQ expression and promoter exchange (Ppps::Pveg) has not significantly increased the plipastatin yield. To further evaluate the impact of degQ expression on surfactin and plipastatin biosynthesis, two strains of B. subtilis were selected: JABs24, a lipopeptide producer derived from the 168 strain, and DSM10T, the wild-type strain expressing native degQ. The findings demonstrated that surfactin biosynthesis is negatively affected by DegQ-associated DegU regulation, while increased plipastatin biosynthesis is achieved in the presence of native degQ expression. In addition to production of lipopeptides, the DegU regulatory system also plays a role in the formation of secretory proteases. A comparison of extracellular protease activities between JABs24 and DSM10T showed that degQ expression led to DSM10T having five times higher protease activity than JABs24. Interestingly, production of extracellular proteases has not affected the stability of both plipastatin and surfactin during cultivation, suggesting that lipopeptides are less targeted by extracellular proteases. The identification of proficient wild-type strains is critical to the advancement of bio-fungicide in agriculture. Therefore, the subsequent approach of this thesis centered on the production of microbial lipopeptide by wild-type B. velezensis strains. Here, the lipopeptide productivity and antifungal ability of B. velezensis UTB96 was higher than B. velezensis FZB42, as a well-established strain for biocontrol of plant pathogens in agriculture. Furthermore, addition of certain amino acids stimulated lipopeptide production, and using a bioreactor system resulted in enhancement of lipopeptide production, especially iturin A by UTB96. Overall, the doctoral thesis evaluates the biosynthesis of antimicrobial lipopeptides produced by B. subtilis and B. velezensis. The study involves genetic engineering such as promoter exchange, deletion of genes involved in competing biosynthetic pathways and cultivation medium development with amino acid supplementation to enhance the lipopeptide titer. The thesis also identifies B. velezensis UTB96 as a promising candidate for further research to be used as a wild-type antifungal agent in agriculture.
Microbial community structure and function is shaped by microhabitat characteristics in soil
(2016) Ditterich, Franziska; Kandeler, Ellen
Soil microorganisms play a key role in degradation processes in soil, such as organic matter decomposition and degradation of xenobiotics. Microbial growth and activity and therefore degradation processes are influenced by different ecological factors, such as substrate availability, pH and temperature. During soil development different microhabitats are formed which differ in their physiochemical properties. There is some evidence that mineral composition is a driver for specific microbial colonization. Thereby, the heterogeneity of soils with differences in mineral composition and substrate availability can lead to a spatial distribution of soil microorganisms. At the soil-litter interface, a biogeochemical hot spot in soil, the abundance and activity of soil microorganisms increases due to high substrate availability, and degradation processes such as pesticide degradation are enhanced. This thesis aimed to clarify the influence of habitat properties on the structure and function of the microbial community in soil. In particular, focus was on mineral-microbe interactions that result from the mineral composition and substrate availability in an artificial soils system. Furthermore this thesis was designed to increase our understanding of the bacterial and fungal roles in pesticide degradation at the soil-litter interface using 4-chloro-2-methylphenoxyacetic acid (MCPA) as a model xenobiotic. These two aspects of the thesis were examined in three studies. The first study focused on the succession of microbial communities and enzyme activities in an artificial soils system with varying mineral composition and substrate availability over a period of 18 months. In the second study a microcosm experiment was used to study the bacterial pathway of MCPA degradation at the soil-litter interface. Over a period of 27 days the succession of bacterial degraders was followed. The third study focused on the degradation of MCPA in soil by nonspecific fungal enzymes, through the addition of fungal laccases as well as litter during 42 days of incubation. Both studies indicated the involvement of fungi in MCPA degradation and the importance of the ecological behavior of different degraders as a function of substrate availability. Results of the first study indicated that the microbial community was affected by mineral properties under high substrate availability and by the availability of beneficial nutrients at the end of incubation when substrate had become limited. The measured enzyme activities provided clear evidence that microbial community structure was driven by nutrient limitation during incubation. In the presence of easily available organic substrates at the beginning of the experiment, the soil microbial community was dominated by copiotrophic bacteria (e.g. Betaproteobacteria), whereas under substrate limitation at the end of incubation, more recalcitrant compounds became important to oligotrophic bacteria (e.g. Acidobacteria), which then became dominant. The results of the second study indicated that the contribution of the potential degraders to degradation of MCPA differed, and this was also seen in the succession of specific bacterial MCPA degraders. Added litter stimulated MCPA degradation due to the availability of litter-derived carbon and induced a two-phase response of fungi. This was seen in the development of pioneer and late stage fungal communities. Both fungal communities were probably involved in MCPA degradation. Therefore, the third study focused on the fungal pathway. These results indicated that the fungal laccases used had no direct influence on degradation and were as efficient as litter in providing additional nutrient sources, increasing MCPA degradation by bacteria and fungi. The observed differences between litter and enzyme addition underscored the observation that the enzyme effect was short-lived and that substrate quality is an important factor in degradation processes. In conclusion, this thesis demonstrated that soil microbial communities and therefore degradation processes are driven by mineral composition as well as substrate availability and quality. In addition, this thesis extends our understanding of degradation processes such as the degradation of xenobiotics, with MCPA as model compound, in soil. The combined insights from all three studies suggest that the use of a simple system such as the artificial soil system can increase our understanding of complex mechanisms such as degradation of pesticides.
Microbial regulation of pesticide degradation coupled to carbon turnover in the detritusphere
(2015) Pagel, Holger; Streck, Thilo
Many soil functions, such as nutrient cycling or pesticide degradation, are controlled by microorganisms. Dynamics of microbial populations and biogeochemical cycling in soil are largely determined by the availability of carbon (C). The detritusphere is a microbial “hot spot” of C turnover. It is characterized by a concentration gradient of C from litter (high) into the adjacent soil (lower). Therefore, this microhabitat is very well suited to investigate the influence of C availability on microbial turnover. My thesis aimed at the improved understanding of biochemical interactions involved in the degradation of the herbicide 4-chloro-2-methylphenoxyacetic acid (MCPA) coupled to C turnover. In the detritusphere gradients of organic matter turnover from litter into the adjacent soil could be identified. Increased C availability, due to the transport of dissolved organic substances from litter into soil, resulted in the boost of microbial biomass and activity as well as in the acceleration of MCPA degradation. Fungi and bacterial MCPA-degraders benefited most from litter-C input. Accelerated MCPA degradation was accompanied by increased incorporation of MCPA-C into soil organic matter. The experimental results show that the transport of dissolved organic substances from litter regulates C availability, microbial activity and finally MCPA degradation in the detritusphere. In general, litter-derived organic compounds provide energy and resources for microorganisms. The following possible regulation mechanisms were identified: i) Litter might directly supply the co-substrate alpha-ketoglutarate (or surrogates) required for enzymatic oxidation of MCPA by bacterial MCPA degraders. Alternatively it might provide additional energy and resources for production and regeneration of the needed co-substrate. ii) Additional litter-C might alleviate substrate limitation of enzyme production by bacteria and bacterial consortia resulting in an increased activity of specific enzymes attacking MCPA. iii) Litter-derived organic substances might stimulate MCPA degradation via fungal co-metabolism by unspecific extracellular enzymes, either directly by inducing enzyme production, or by supplying primary substrates that provide the energy consumed by co-metabolic MCPA transformation. A new biogeochemical model abstracts these regulation mechanisms in such a way that C availability controls physiological activity, growth, death and maintenance of microbial pools. Based on a global sensitivity analysis, 41% (n=33) of all considered parameters and input values were classified as “very important” and “important”. These mainly include biokinetic parameters and initial values. The calibration of the model allowed to validate the implemented regulation mechanisms of accelerated MCPA degradation. The Pareto-analysis showed that the model structure was adequate and the identified parameter values were reasonable to reproduce the observed dynamics of C and MCPA. The model satisfactorily matched observed abundances of gene-markers of total bacteria and specific MCPA degraders. However, it underestimated the steep increase of fungal ITS fragments, most probably because this gene-marker is only inadequately suited as a measure of fungal biomass. The model simulations indicate that soil fungi primarily benefit from low-quality C, whereas bacterial MCPA-degraders preferentially use high-quality C. According to the simulations, MCPA was predominantly transformed via co-metabolism to high-quality C. Subsequently, this C was primarily assimilated by bacterial MCPA-degraders. The highest turnover of litter-derived C occurred by substrate uptake for microbial growth. Input and microbial turnover of litter-C stimulated MCPA degradation mainly in a soil layer at 0-3 mm distance to litter. As a consequence of this, a concentration gradient of MCPA formed, which triggered the diffusive upward transport of MCPA from deeper soil layers into the detritusphere. The results of the three studies suggest: The detritusphere is a biogeochemical hot spot where microbial dynamics control matter cycling. The integrated use of experiments and mathematical modelling gives detailed insight into matter cycling and dynamics of microorganisms in soil. Microbial communities need to be explicitly considered to understand the regulation of soil functions.
Modeling microbial regulation of pesticide turnover in soils
(2022) Chavez Rodriguez, Luciana; Streck, Thilo
Pesticides are widely used for pest control in agriculture. Besides their intended use, their long-term fate in real systems is not well understood. They may persist in soils, thereby altering ecosystem functioning and ultimately affecting human health. Pesticide fate is assessed through dissipation experiments in the laboratory or the field. While field experiments provide a close representation of real systems, they are often costly and can be influenced by many unknown or uncontrollable variables. Laboratory experiments, on the other hand, are cheaper and have good control over the governing variables, but due to simplification, extrapolation of the results to real systems can be limited. Mechanistic models are a powerful tool to connect lab and field data and help us to improve our process understanding. Therefore, I used mechanistic, process-based models to assess key microbial regulations of pesticide degradation. I tested my model hypotheses with two pesticide classes: i) chlorophenoxy herbicides (MCPA (2-methyl-4-chlorophenoxyacetic acid) and 2,4-D (2,4-Dichlorophenoxyacetic acid)), and ii) triazines (atrazine (AT)), in an ideal scenario, where bacterial degraders and pesticides are co-localized. This thesis explores some potential controls of pesticide degradation in soils: i) regulated gene expression, ii) mass-transfer process across the bacterial cell membranes, iii) bioenergetic constraints, and iv) environmental factors (soil temperature and moisture). The models presented in this thesis show that including microbial regulations improves predictions of pesticide degradation, compared to conventional models based on Monod kinetics. The gene-centric models achieved a better representation of microbial dynamics and enable us to explore the relationship between functional genes and process rates, and the models that used transition state theory to account for bioenergetic constraints improved the description of degradation at low concentrations. However, the lack of informative data for the validation of model processes hampered model development. Therefore, in the fourth part of this thesis, I used atrazine with its rather complex degradation pathway to apply a prospective optimal design method to find the optimal experimental designs to enable us identifying the degradation pathway present in a given environment. The optimal designs found suggest to prioritize determining metabolites and biomass of specific degraders, which are not typically measured in environmental fate studies. These data will lead to more robust model formulations for risk assessment and decision-making. With this thesis, I revealed important regulations of pesticide degradation in soils that help to improve process understanding and model predictions. I provided simple model formulations, for example the Hill function for gene expression and transition state theory for bioenergetic growth constraints, which can easily be integrated into biogeochemical models. My thesis covers initial but essential steps towards a predictive pesticide degradation model usable for risk assessment and decision-making. I also discuss implication for further research, in particular how mechanistic process-based modeling could be combined with new technologies like omics and machine learning.
Plasmopara viticola, the downy mildew of grapevine : phenotypic and molecular characterization of single sporangium strains infecting hosts with different resistance levels
(2015) Gómez Zeledón, José Javier; Spring, Otmar
The downy mildew of grapevine, Plasmopara viticola, is one of the most important pathogens in viticulture. Its genetic diversity had been assessed in some previous studies using molecular markers, but the diversity of the infection behavior has not yet been addressed adequately. Therefore, the development of a fast, reliable and uncomplicated assay to screen for pathogen phenotypes on host with different resistance levels was a major task of this work. A leaf disc test was proposed, evaluating sporulation and necrosis produced by the pathogen on Vitis plants with different susceptibility. Using this bioassay, interesting strains were assessed and kept for future studies. The urgent need to work with genetic homogeneous inoculum was shown, because the assays revealed a high phenotypic diversity in isolates collected from the field as a bulk sample. Hence, a cloning technique to obtain single sporangium strains was found useful to avoid working with mixed genotypes. The leaf disc bioassay also allowed screening for fungicide resistance in P. viticola populations. Isolates resistant to dimethomorph and metalaxyl, two important fungicides for oomycetes control, were detected. Higher resistance was associated with fields were the fungicide application was high as well. Some strains were even resistant to doses where the fungicide exhibits phytotoxic activity to grapevine. The approach of characterizing P. viticola pathotypes on different host plants of Vitis vinifera cultivars and Vitis species from North America and Asia revealed a broad spectrum of fully susceptible to completely resistant reactions. This information is of direct practical value in future plant breeding programs, but also provides the chance to select specific host-pathogen combinations to study the mechanisms of resistance or susceptibility. Fluorescence microscopy revealed how the infection progress of highly and lowly virulent strains advance in tolerant and susceptible hosts, and which points of the infection are interesting for future studies. On the molecular level, effectors were investigated to trace their possible involvement in the infection process. It was found that RXLR 1, NLP 1, Elicitin like 2, Glucanase inhibitor 2 and 4 , and 1,3-ß Glucanase 2 are candidates which are upregulated in the earliest infection stages. Following the here established methodology and suggested strategy it should be possible in the future to get a better insight in the mechanisms of infection and resistance of grapevine downy mildew.