PhD Project "A novel DNA-binding regulator coordinates riboflavin and roseoflavin metabolism in Streptomyces davaonensis" (MM09) - Heidelberg University

Date: 01/09/2023

Application Deadline: 30/06/2023

The objective of the present project is to make a first step towards the elucidation of mechanisms employed to regulate expression of the roseoflavin and riboflavin genes.

In one of our recent publications on the characterization of roseoflavin and riboflavin biosynthetic genes in S. davaonensis we reported on the role of the putative regulator gene marR located immediately downstream of the gene rosB encoding the key enzyme of roseoflavin biosynthesis (Kissling et al., 2020). S. davaonensis MarR contains helix-turn-helix motifs typical for DNA binding regulators and it is very likely that MarR stimulates transcription by binding RNApolymerase. First, this gene (marR) is strongly expressed in the roseoflavin production phase but is transcribed from the opposite direction. Second, when marR was deleted from the chromosome of S. davaonensis three out of four of the resulting strains showed a reduction of roseoflavin levels in the supernatant. Third, the gene product of marR is similar to the response regulator DegU of Bacillus subtilis being part of the two-component signal transduction system DegS-DegU. All these findings indicate that marR may be involved in regulating expression of rosB i.e. roseoflavin biosynthesis. Moreover, we found in our recent publication that promoter regions upstream of some S. davaonensis roseoflavin and riboflavin biosynthetic genes regulate differently depending on the strain background (Kissling et al., 2020). When coupled to a reporter gene and integrated into the chromosome of Streptomyces coelicolor, the promoter regions did not stimulate gene expression, neither in the exponential nor in the stationary phase. However, when the same constructs were introduced into the chromosome of S. davaonensis reporter gene expression was strongly enhanced showing that a species specific regulation occurs. 

The objective of the present project is to make a first step towards the elucidation of mechanisms employed to regulate expression of the roseoflavin and riboflavin genes. These genes are scattered around the chromosome and do not form operons. Our working hypothesis is that MarR is activated by a yet unknown protein and, in an activated form, binds to the promoter region upstream of rosB. Binding of activated MarR stimulates binding of RNA polymerase which leads to enhanced gene expression of rosB.

Planned experiments in the PhD project:
The first experiment will be to characterize a S. davaonensis marR deletion strain. This strain already has been constructed and we have to validate by growth experiments whether roseoflavin synthesis is affected (first experiments showed that less roseoflavin is synthesized). Second, we have to show that complementation with marR restores the phenotype. The corresponding plasmid has also been constructed and is ready for conjugation. In the following work the DNA binding activity of MarR shall be validated by biochemical studies and the yet unknown interaction partner of MarR (the sensor histidine kinase) shall be identified employing a novel (tool proximity‐dependent biotin identification; BioID; Fig. 2), which we will setup in close cooperation with the group of Oliver Valerius from the University of Göttingen. 
The following questions shall be answered: Is MarR the only regulator? Is there a superordinate regulator? Does the availability of nitrogen affect expression of the roseoflavin and/or riboflavin biosynthetic genes? Which extracellular signals affect expression of the roseoflavin and/or riboflavin biosynthetic genes i.e. which signal triggers roseoflavin production?

Personal qualifications: A person interested in antibiotics, antivitamins and microbiomes. Moreover, since we use molecular biology tools, the candidate also should be experienced with regard to state-of-the-art cloning techniques

Please submit your application exclusively through the HBIGS application system: http://www.hbigs.uni-heidelberg.de/main_application.html

Please read more under "Open PhD Positions" on: http://www.hbigs.uni-heidelberg.de