Charakterisierung einer Doppelmutante alternativer ECF-ähnlicher RNA-Polymerase Sigma Faktoren des Stickstoff-fixierenden bakteriellen Leguminosen-Symbionten Sinorhizobium meliloti
Zusammenfassung der Projektergebnisse
The title of my initially proposed research project for the DFG research fellowship was “Characterization of an alternative ECF-like RNA polymerase sigma factor double mutant of the legume-interacting nitrogen-fixing bacterial symbiont Sinorhizobium meliloti”. The respective bacterial double mutant for the ECF sigma factors RpoE6 and RpoE8, and results of preliminary phenotypic analyses were available when I joined the Long lab at Stanford. After a few months and several detailed complementation and reconstruction experiments it was clear, that the symbiotic defect of this strain was caused by a secondary mutation, namely an insertion of the S. meliloti insertion element ISRm1 in nodF, a symbiosis-essential gene. Furthermore, even a bacterial strain lacking all of the 11 ECF sigma factors and the corresponding anti-sigma factors were indistinguishable from wild type in symbiotic behavior. Thus, this project was not pursued any longer. However, a second project that I started during my first year in the lab turned out to be successful, resulting in the publication of a research article in the Journal of Bacteriology. This work was on relA and dksA genes, major players in the stringent response, which is a transcriptional regulatory mechanism in bacteria triggered by limiting nutrient conditions. I investigated the role of two DksA-like proteins from S. meliloti in culture and in interaction with its host plant M. sativa. Deletion mutants were analyzed using growth tests on different media, nodulation assays on plants, enzyme activity assays, reporter gene studies, expression profiling, complementation experiments, and other techniques. The results showed, that only one DksA-like paralog is functional, and the corresponding mutant displays free-living as well as symbiotic phenotypes. Modifications of essential motifs in the other paralog failed to establish DksA function. A relA mutant showed severe pleiotropic phenotypes in all conditions tested. I will continue working on ongoing projects. I will investigate the role of relA and dksA during symbiosis in more detail. This includes observation of infection processes, global transcription profiling, imaging of reporter strains and plant marker lines, and suppressor mutant analyses. As the central objective of my future research proposals I want to focus on the characterization of M. truncatula mutants defective in symbiosis.
