Investigating the molecular basis of the bacterial endocytosis-like protein uptake

• Applicants: Dr. Damien Devos; Professor Dr. Christian Jogler
• Subject Area: Metabolism, Biochemistry and Genetics of Microorganisms ; Microbial Ecology and Applied Microbiology
• Term: from 2013 to 2018
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 239740592

Background:Unique in the bacterial domain, the planctomycetes bacteria Gemmata obscuriglobus display vesicle mediated endocytosis-like phenomenon. Bacterial membrane coat proteins (MCs), structurally similar to eukaryotic clathrin, are involved in vesicle formation. Since little is known about the molecular basis of this trait, including MC interaction partners, it remains enigmatic whether the bacterial endocytosis phenomenon is homolog or analog to the hallmark trait of eukaryogenesis.Hypothesis:Besides MCs, true vesicle formation will require additional proteins, such as motor proteins. We hypothesise that MCs engage partners to mediate endocytosis and that several bacteria of the PVC-superphylum share this endocytosis-like phenomenon. MCs are essential for vesicle formation and thus for the endocytosis-like phenomenon. Consequently PVC bacteria encoding MCs are expected to possess this trait.Objectives:To verify our hypothesis we will follow three major lines of research that will bring our complementary skills together.A: Development of genetic tools for Gemmata obscuriglobus.The analysis of the molecular background of the endocytosis like phenomenon was hampered by the lack of genetic tool. Recently we developed such tools for Planctomyces limnophilus that will be transferred to G. obscuriglobus to allow for in vivo MC-protein tagging employing fluorescent markers and the generation of mutants. B: Analysis of endocytosis like uptake of proteins in selected PVC bacteria.Among the PVC superphylum, currently genetic tools are exclusively available for P. limnophilus and Verrucomicrobium spinosum. Both species possess MCs, but were not analyzed regarding endocytosis. To verify our hypothesis we will study the capability of P. limnophilus and V. spinosum to perform endocytosis-like protein uptake. We will employ genetics to determine the molecular basis of this trait in the genomic background of P. limnophilus, V. spinosum and G. obscuriglobus. While the comparative analysis in different species is key to elucidate minor differences, this provides us with a reasonable backup as well. To demonstrate MCs being involved in endocytosis like uptake employing GFP or dtTomato labeling and (superresolution) time lapse fluorescent microscopy in wild type and mutant background in one model organism would be sufficient to proof our hypothesis. C: Biochemical characterization of the endocytosis-like phenomenon in various PVC members.We will determine whether other proteins are involved in the endocytosis-like phenomenon using bioinformatics and biochemical approaches. This include yeast-2-hybrid approaches and a new co-immuno precipitation logic (with Mike Rout, NY USA). Identified proteins will be subject to GFP labeling and (super resolution) fluorescent microscopic analysis. In addition, mutants will be constructed to determine if the identified proteins are essential for the endocytosis like phenomenon.

• DFG Programme: Research Grants
• International Connection: Spain