Browsing by Subject "Pseudomonas"

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Applied molecular bioprocess control using RNA thermometers : exploiting temperature responsive elements for rhamnolipid production
(2022) Noll, Philipp; Hausmann, Rudolf
The highest titer reported for heterologous Rhamnolipid (RL) production is 14.9 g/L. However, biomass generation, as a large carbon sink, was a significant drawback in this process with roughly 50 more biomass than product produced. This problem is addressed in this thesis leveraging temperature as control variable and a molecular temperature sensor, an RNA thermometer (RNAT). RNAT generally refers to secondary loop structures, in the 5’ untranslated region of the mRNA, that form at certain temperatures and therefore regulate translation in dependence of temperature. The ROSE (repression of heat shock gene expression) RNAT evaluated in the first original research article in the heterologous system P. putida KT2440 pSynpro8oT_rhlAB originates from P. aeruginosa. The ROSE element regulates, in dependence of ambient temperature, the translation of rhlA and via a polar effect also the translation of rhlB therefore indirectly RL synthesis. It was found that in the ROSE RNAT-controlled system, the RL production rate was 60% higher at cultivations of 37°C than at 30°C. However, besides the regulatory effect of the RNAT, as revealed by control experiments, multiple unspecific metabolic effects may be equally responsible for the increase in production rate. After screening for even more efficient regulatory structures, a fourU RNAT was identified. Natively, this fourU RNAT regulates the expression of the heat shock gene agsA of Salmonella enterica and its regulatory capability can easily be modified by site-directed mutagenesis. The experimental data collected in the second original research article confirms the functionality of the fourU RNAT in the heterologous RL production system. The data suggested improved regulatory capabilities of the fourU RNAT compared to the ROSE element and a major effect of temperature on RL production rates and yields. The average RL production rate increased by a factor of 11 between 25°C and 38°C. Control experiments confirmed that a major part of this increase originates from the regulatory effect of the fourU RNAT rather than from an unspecific metabolic effect. With this system YP/X values well above 1 (about 1.4 gRL/gBM) could be achieved mitigating the problem of high biomass formation compared to product synthesis. Also, YP/S values of about 0.2 gRL/gGlc at elevated temperatures of 37-38°C were reached in shake flasks. The system was subsequently tested in a proof-of-concept bioreactor process involving a temperature switch. With this simple batch experiment and a temperature switch from 25°C to 38°C not only a partial decoupling of biomass formation from product synthesis was achieved but also an around 25% higher average specific rhamnolipid production rate reached compared to the so far best performing heterologous RL production process reported in literature (average specific production rate: 24 mg/(g h) vs. 32 mg/(g h)). However, to achieve higher titers while reducing side product formation a suitable feeding strategy and more complex temperature profiles may be required. Temperature variations in turn cause several metabolic changes, many of which are complex and interdependent. Models that describe biological processes as a function of temperature are thus essential for improved process understanding. The goal of the peer reviewed review article “Modeling and Exploiting Microbial Temperature Response”, shown in this thesis, was to present an overview of various temperature models, aid comprehension of model intent and to facilitate selection and application. Since not all metabolic interdependencies and mechanisms during temperature variation are known for the reasonable connection of input-output relationships, a suitable modeling approach seemed to be neural networks. Neural networks as black box models do not require mechanistic a priori knowledge but representative historic datasets. To collect training data, different temperature profiles or constant temperatures for a bioreactor process with P. putida KT2440 pSynpro8oT_rhlAB were applied and concentration curves for biomass, glucose and RL recorded. Subsequently, the data was fed into the neural network to compute RL titer as output. An exponential temperature profile yielded at the highest RL value of approx. 9 g (around 13 g/L) less biomass (around 12 g/L) than product. These values were reached after only 30 h consuming just 45 g of glucose. Hence, at this timepoint 36 weight-% of the consumed glucose could be assigned to mono-RL (YP/S = 0.19 gRL/gGlc) and biomass (YX/S = 0.17 gBM/gGlc. The so far best performing heterologous RL production process, yielded 23.2 g (14.9 g/L) mono-RL from >250 g of consumed glucose (YP/S = 0.10 gRL/gGlc) in >70 h using the same strain and medium but a constant temperature of 30°C.
Beneficial microbial consortium improves winter rye performance by modulating bacterial communities in the rhizosphere and enhancing plant nutrient acquisition
(2023) Behr, Jan Helge; Kampouris, Ioannis D.; Babin, Doreen; Sommermann, Loreen; Francioli, Davide; Kuhl-Nagel, Theresa; Chowdhury, Soumitra Paul; Geistlinger, Joerg; Smalla, Kornelia; Neumann, Günter; Grosch, Rita
The beneficial effect of microbial consortium application on plants is strongly affected by soil conditions, which are influenced by farming practices. The establishment of microbial inoculants in the rhizosphere is a prerequisite for successful plant-microorganism interactions. This study investigated whether a consortium of beneficial microorganisms establishes in the rhizosphere of a winter crop during the vegetation period, including the winter growing season. In addition, we aimed for a better understanding of its effect on plant performance under different farming practices. Winter rye plants grown in a long-time field trial under conventional or organic farming practices were inoculated after plant emergence in autumn with a microbial consortium containing Pseudomonas sp. (RU47), Bacillus atrophaeus (ABi03) and Trichoderma harzianum (OMG16). The density of the microbial inoculants in the rhizosphere and root-associated soil was quantified in autumn and the following spring. Furthermore, the influence of the consortium on plant performance and on the rhizosphere bacterial community assembly was investigated using a multidisciplinary approach. Selective plating showed a high colonization density of individual microorganisms of the consortium in the rhizosphere and root-associated soil of winter rye throughout its early growth cycle. 16S rRNA gene amplicon sequencing showed that the farming practice affected mainly the rhizosphere bacterial communities in autumn and spring. However, the microbial consortium inoculated altered also the bacterial community composition at each sampling time point, especially at the beginning of the new growing season in spring. Inoculation of winter rye with the microbial consortium significantly improved the plant nutrient status and performance especially under organic farming. In summary, the microbial consortium showed sufficient efficacy throughout vegetation dormancy when inoculated in autumn and contributed to better plant performance, indicating the potential of microbe-based solutions in organic farming where nutrient availability is limited.
Neu auftretende bakterielle Blattfleckenerreger an Radies und Entwicklung eines Resistenztests als Grundlage für die Züchtung resistenter Sorten
(2014) Scholze, Inka S.; Vögele, Ralf
Radish is one of the most important vegetable crops in Rhineland Palatinate. During the last decades, an increase in bacterial leaf spots on radish has been observed. Although the bulb is unaffected, leaf spot symptoms lead to a decline in sales and profits as German consumers prefer marketable red radish bunches including the freshness-indicating foliage. So far, preventive methods for the control of bacterial pathogens on radish were limited to irrigation strategies (for example drip irrigation) or field hygiene. These methods are, however, often difficult to implement and their effect predominantly insufficient. Resistant or tolerant breeds would provide a solution, however, breeding companies have been lacking the necessary information regarding the causative agents and their biology and epidemiology needed for the development of such breeds. Previous examinations of infested plants and seed lots suggest different pseudomonads and Xanthomonas campestris as possible agents. The main objective of this study was therefore to identify the relevant pathogens causing leaf-spot symptoms on radish. Furthermore, another objective was to clarify the infection and growth requirements of bacterial pathogens on hosts for the development of a screening method for resistance to leaf spot pathogens on radish. On account of increasing compensation claims from breeding companies by farmers, the role of seed transmissibility was also to be determined. during a three year observation, bacterial pathogens from plants and seed lots were isolated and characterized. The characterization of unknown bacterial strains was performed by a combination of physiological and molecular methods. Molecular characterization methods such as 16s rDNA sequence analysis and MLST (multilocus sequence typing) were needed to complete the designation of P. syringae pathovars. In addition, virulence assays clarified the importance of single pathovars. Hence bacterial leaf spot pathogens could be determined as P. syringae pv. maculicola, P. viridiflava, P. cannabina pv. alisalensis and X. campestris whereas the former two species were the most abundant in Rhineland Palatinate. Infection trials on radish plants concluded that the different pathogens induced different typical symptom characteristics. To evaluate the influence of environmental factors such as humidity and temperature, it was demonstrated that inoculations with pseudomonads and a leaf spot inducing X. campestris strain caused successful infection and symptom development under a wide temperature range. Humidity was shown to be the most influential factor limiting infestation intensity with P. viridiflava displaying a higher demand for humid conditions than P. syringae pv. maculicola and X. campestris. All three types induced a higher disease infestation on radish plants in high humidity conditions (~94% rH) contrary to a lower infestation in dry conditions (~63% rH). In a first test series, plants inoculated with P. viridiflava were almost symptom free under dry conditions, whereas P. syringae and X. campestris were still able to induce leaf spots. Based on the results of infections assays, it was possible to develop a screening method on radish plants for resistance to the leaf spot inducing bacterial pathogen P. syringae pv. maculicola. The screening method was based on the spray-inoculation of radish leaves fixed in water filled orchid tubes, incubated under high humidity in a climate chamber at 24 °C day/10 °C night. Determination of resistance was performed 7 to 10 days past inoculation by rating disease intensity of infested leaves. Whereas tests could be performed by inoculations with P. viridiflava and P. cannabina as well as P. syringae, the latter proved to be most suitable for the screening method.
Quantitative Proteomanalyse von Pseudomonaden zur Aufklärung biotechnologisch relevanter Stoffwechselwege
(2013) Simon, Oliver; Huber, Armin
The main focus of this work was a quantitative proteome analysis of a variety of Pseudomonas strains with respect to the biotechnological synthesis of the base chemicals glyoxylic acid, butanol and vanillin. In addition, effects of the terpene citronellol on the proteome of P. aeruginosa were investigated. A second key aspect of this work involved the establishment of proteomics methods for the analysis of complex samples, especially for the analysis of membrane proteins. Using carbonate extraction followed by label-free MS-based quantification allowed the identification and quantification of a significant number of hydrophobic proteins which were not covered by the 2D-DIGE approach. In addition, the GeLCMSMS workflow was found to be a simple and efficient method for the analysis of total bacterial lysates. Using this method, about 30% of all proteins encoded by the P. putida KT2440 genome could be identified and quantified. In conclusion, this work demonstrated that different proteomics methods can substantially contribute to biotechnological strain development and the understanding of cellular networks.
The functional role of phosphorus-mobilizing bacteria in the rhizosphere of tomato and maize
(2017) Nassal, Dinah; Kandeler, Ellen
Phosphorus (P) is an essential plant nutrient. However, global P reserves are being increasingly exploited and surplus P applied by P fertilization is steadily accumulating in the form of plant-unavailable P compounds in arable soils. Future plant production will therefore require a more effective and sustainable P fertilization regime. One promising approach is the use of phosphorus-mobilizing bacteria (PMB), which are able to mobilize P in soil through mineralization or solubilization so effectively that plant P supply is improved. Increases in plant growth and P uptake by the addition of PMB have been reported several times, but PMB’s functional mechanisms in soils and plants are still poorly understood. However, an understanding of PMB’s functional mechanisms is necessary to evaluate both the potential and limitations of their use as well as to develop practical application recommendations. This thesis aimed to provide a better understanding of PMB’s functional mechanisms in soil; the foci here were mechanisms and interactions of P mineralization with indigenous soil microorganisms. We aimed to identify P mineralization-dependent and -independent as well as direct and indirect mechanisms of PMB on soil and plants. To this end, three rhizobox experiments were performed in the greenhouse using tomato and maize as the test plants and Pseudomonas sp. RU47 (RU47) as the PMB. To identify effective P mineralization beyond the level of endogenous microbial activity, a treatment using unselectively cultivated soil bacteria for inoculation was included. Furthermore, the addition of devitalized RU47 cells provided the opportunity to identify indirect mechanisms. In all three rhizobox experiments the activities of acid and alkaline phosphomonoesterases in rhizosphere and bulk soil were determined, as the latter could be clearly identified as being of microbial origin. Effects on microbial community structure in soil were estimated by denaturing gradient gel electrophoresis (DGGE) and/or phospholipid fatty acid analysis. For deeper investigations of potential effects on microbial population composition and possible dependencies on soil conditions, a fourth experiment was performed using maize, three different Pseudomonas strains possessing PMB abilities, and three different soils varying in parameters which included organic C, pH, and P content. Microbiome shifts in soil were quantitatively determined via quantitative PCR using domain- (bacteria, archaea, fungi) and six bacterial phylum-specific primers. Our experiments showed that tomato plants grown under low P availability soil conditions improved in both growth and P uptake when viable RU47 cells were added. This effect was accompanied by increased alkaline phosphatase activity (PA) in the rhizosphere. We also observed plant growth-promotion effects and a trend of increased PA by the addition of dead RU47 cells. Based on DGGE results, which indicated the promotion of indigenous rhizobacteria, we assume a priming effect induced by the addition of C sources in the form of bacterial residues (dead RU47), which resulted in increased indigenous microbial activity in the rhizosphere. In each rhizobox experiment viable RU47 cells were able to colonize the rhizosphere at high abundances, persisting up to 50 days after sowing. We found indications of phytohormonal influences with the addition of both viable and dead RU47 cells, but this was more pronounced in dead than in viable RU47 treatments. Increasing P availability in soil by mineral P fertilization seemed to improve RU47’s ability to colonize and persist, which was shown by an increased RU47 abundance in both rhizosphere and bulk soils. However, despite an observable slight tendency, strengthened plant growth-promotion that positively correlated with improved RU47 abundance in the rhizosphere could not be detected. In general, colonization by viable RU47 cells did not significantly affect microbial community structure, either in the rhizosphere or in bulk soil. Using three different PMB strains, including RU47, in three contrasting soils, inoculation effects on the microbial community occurred heterogeneously, differing between the strains, soils, and time. Changes at the domain level were due primarily to nutrient availability in the soil, which differed between the soils and over time. Individual shifts in microbial community structure occurred more frequently in the rhizosphere than in bulk soil, but colonizing PMB neither increased bacterial abundance in rhizosphere bacteria, nor displaced copiotrophic rhizobacteria (indicative of C competition).