Mechanismus und Funktion des Programmierten Zelltodes in Caulobacter crescentus
Zusammenfassung der Projektergebnisse
Multi-cellular organisms rely on programmed cell death (PCD) to eliminate excess or potentially harmful cells from the system. More recently, PCD has also been studied in unicellular bacteria where this genetic program selectively triggers the suicide of individual cells. Bacterial PCD is observed in various species as a response to environmental stresses including nutrient starvation or DNA damage. While cell death under these conditions has in some cases been attributed to the induction of toxins or bacterial viruses harboured by the bacterial chromosome, the mechanisms of inducing PCD are mostly unknown. Furthermore, previous studies have not demonstrated general benefits for bacterial PCD, such that the function of PCD in these unicellular organisms has remained mysterious. The goal of the proposed research was to characterize PCD mediated by the DNA damage-induced endonuclease BapE in the alpha-proteobacterium Caulobacter crescentus. In the course of the SOS response, BapE non-specifically fragments the bacterial chromosome, eventually causing cell death. When analysing the regulation of BapE I determined that its transcriptional start site was positioned within the binding site of LexA, the master regulator of the SOS response. I furthermore used a fluorescent reporter to exclude that additional stress conditions could trigger BapE expression. The selective expression in response to DNA damage and tight transcriptional control by LexA suggested that BapE was likely a dedicated factor of the SOS response in Caulobacter. To investigate the potential benefit of BapE-mediated cell death for a bacterial community experiencing DNA damage, I analysed the transcriptomes of cells during the SOS response. As revealed by fluorescence microscopy of Caulobacter carrying a reporter to monitor BapE expression, induction of the endonuclease - and thus of cell death - was highly heterogeneous. Employing the reporter signal as a determinant of the cellular status, i.e. to define the bacteria undergoing PCD, I specifically separated the different populations and will be able to compare their transcriptomic profiles. In addition, RNA seq of unsorted samples prior to and after induction of the SOS response allowed for a detailed analysis of the transcriptional profile of the Caulobacter stress response to DNA damage. Having served mainly as a model organism of bacterial cell biology, gene expression regulation, e.g. in response to diverse environmental stresses has received less attention in research of Caulobacter. Especially the post-transcriptional control, i.e. gene expression regulation on the RNA level, has not been addressed in detail in Caulobacter. My transcriptome analysis suggests a rich landscape of non-coding RNAs present under normal growth conditions, but also specifically induced upon DNA damage. Small regulatory RNAs (sRNAs) are non-coding transcripts that act as versatile modulators of gene expression in bacteria via direct interactions with target messenger RNAs. An unexpected route of the project was the identification and characterization of a novel sRNA induced in response to DNA damage, and during growth in minimal medium. The twocomponent system ChvIG was discovered in a genome-wide transposon screen as an activator of this riboregulator, which I named ChvR. Pulse-overexpression of ChvR followed by a global transcriptome analysis on microarrays identified a TonB-dependent receptor as the sole target of ChvR under the tested conditions. Employing a combination of genetic and biochemical assays I showed that ChvR represses CC_3013 at the post-transcriptional level through direct basepairing. Fine-mapping of the interaction between ChvR and its binding partner revealed that the sRNA employs two independent sequence elements to recognize the target mRNA. A manuscript summarizing my results is currently under preparation. Genome-wide bioinformatic-based or experimental screens have identified hundreds of sRNAs candidates in different bacterial species. However, the bulk of studies investigating the function and mechanisms of sRNAs have been focusing on gamma-proteobacteria like Escherichia coli. From my initial experiments I already identified several unique features of regulation via non-coding RNAs in Caulobacter, which may lead to the establishment of new concepts of RNA-based control of gene expression in bacteria.
Projektbezogene Publikationen (Auswahl)
- Post-transcriptional gene regulation by an Hfq-independent small RNA in Caulobacter crescentus. Nucleic Acids Research, gky765, Aug 2018
Fröhlich KS, Förstner KU, Gitai Z
(Siehe online unter https://doi.org/10.1093/nar/gky765)
