| Zusammenfassung: | Burkholderia plantarii PG1 (formerly glumae) is a model organism known as causal agent of bacterial panicle blight in rice plants and has become one of the most common seed-borne bacterial rice diseases in the world. Nevertheless, this organism has biological interest. First, B. plantarii PG1 is the producer of the extracellular lipase LipA to produce enantiopure pharmaceuticals. In addition, B. p... Burkholderia plantarii PG1 (formerly glumae) is a model organism known as causal agent of bacterial panicle blight in rice plants and has become one of the most common seed-borne bacterial rice diseases in the world. Nevertheless, this organism has biological interest. First, B. plantarii PG1 is the producer of the extracellular lipase LipA to produce enantiopure pharmaceuticals. In addition, B. plantarii PG1 is a native rhamnolipid producer which is used in pharmaceutical, chemical, food and cosmetic industries. Furthermore, it is distinguished by a unique Quorum sensing system in Burkholderia and possesses more scientific interesting properties which were investigated in this work. The genome of B. plantarii PG1 contains a hydroxyethylthiazole kinase ThiM, which is part of a salvage pathway in the thiamine metabolism conceivably involved in the regulation of the endogenous CRISPR/CAS I-F system due to its unique genomic position upstream of the endonuclease Cas1. For further insights, a comparative transcriptome analysis was performed to elucidate possible effects of a ThiM deletion mutant. Different growth phases and media were investigated, resulting in significant transcriptional changes. In the first analysis of the transcription levels, the absence of the kinase ThiM increased the transcription of csy1-3 in LB medium and caused a decrease of csy1-3 in M9 minimal medium without thiamine. However, a comparative analysis demonstrated that the medium effect caused the transcriptional changes. Although this medium effect was present, an influence of the kinase ThiM on the regulation of the CRISPR/CAS I-F system could not be completely excluded. For further advances, a comparative transcriptome profiling should provide insights by identifying expression patterns and analysing metabolic networks under different conditions. The kinase ThiM is part of the salvage pathway in thiamine metabolism to produce thiamine pyrophosphate (TPP), the active form of thiamine. TPP is an essential cofactor in several metabolic pathways. The influence of different media of LB and M9 medium without thiamine and M9 medium with supplemented thiamine were investigated at the late exponential and the late stationary growth phase in B. plantarii PG1 and a ThiM deletion mutant. In this analysis, the interplay of metabolic processes related to the thiamine metabolism was revealed. The reduced presence of TPP resulted in a diminished activity of the metabolic pathways of glycolysis, the citrate cycle, oxidative phosphorylation, leucine degradation, arginine biosynthesis, histidine metabolism, thiamine metabolism, nitrogen metabolism and fatty acid metabolism. Finally, it is the most comprehensive transcriptome dataset of B. plantarii PG1 providing further insights for investigations.
The regulation of the CRISPR/CAS system was further investigated. Here three putative promoter sites were identified, and their activity experimentally evaluated by constructed reporter strains. Thus, the activity of the promoter sites was proven. The regulation of the CRISPR/CAS system in B. plantarii PG1 is still unknown. Therefore, other possible regulators were investigated and two were identified here. Experimental analysis of the putative regulators investigated in the promoter reporter strains revealed a repression of the activity of the promoter sites of the CRISPR/CAS system. The possible repressors are two cyclic AMP receptor proteins named CRP1 and CRP3 in this study which are dependent on the glucose metabolism. Comparative transcriptome analyses were applied to confirm the observed experimental results. Further experiments are required to verify the regulatory processes and the role of the possible regulators here. As in literature described the CRISPR/CAS system is regulated by the Quorum sensing system in some species and first insights of an influence on the CRISPR/CAS system in B. plantarii PG1 were shown in the study of Rong Gao in 2015. Therefore, Rong Gao constructed and investigated three QS deletion mutants (B. plantarii PG2-PG4, formerly glumae) in B. plantarii PG1. Here in this study, the CRISPR/CAS promoter fusions were investigated in the three QS deletion mutants of B. plantarii PG2-4 and were exposed to environmental stress conditions via different salt and glucose concentrations. Thereby, effects in the different QS deletion mutant strains as well as in changing salt concentrations were observed. Altered salt concentrations increased the activity of the promoter sites of the CRISPR/CAS system in the wildtype B. plantarii PG1 and in the QS deletion mutants of PG3 and PG4, demonstrating the effect of the QS system and additionally of environmental changes on the CRISPR/CAS system. |
