Assembly and dynamics of a bacterial DNA uptake machinery at the single molecule level

• Applicant: Professor Dr. Peter Graumann
• Subject Area: Metabolism, Biochemistry and Genetics of Microorganisms ; Microbial Ecology and Applied Microbiology
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 549961924

Natural competence describes the ability of some bacteria to actively take up DNA from the environment and incorporate it into their genome. Competence is a major source of DNA exchange between bacteria, and leads to spreading of antibiotic resistances. Thus, the process is of high relevance for evolution, relevant for human health, and is also highly interesting in terms of its mechanism that employs homologous recombination (HR) via widely conserved RecA protein. We have recently found that the model organism Bacillus subtilis expresses a competence-specific pilus to take up DNA all over its surface. Taken up DNA can equilibrate within the periplasm via DNA-binding protein ComEA, and is then converted into ssDNA and moved into the cytosol by a protein complex found at one cell pole. Thus, DNA uptake is a two-tier process, uncoupling transport through the cell wall from polar uptake into the cytosol. While the picture of the path of DNA is slowly taking shape, some major questions still remain. It has been shown that competence pili in some bacteria can bind to dsDNA, but it is not clear how dsDNA would fit through a narrow channel, as its bending stiffness would not allow pulling in dsDNA bound in the middle of the long molecule. It is also unclear how ssDNA that is transported into the cytosol is stably introduced into the genome. We have recently found that major pilin ComGC can bind to dsDNA as well as to ssDNA. ssDNA binding would explain how DNA could be taken up, because ssDNA parts of long dsDNA molecules could be easily bent and taken through a pilus channel. We plan to prove this point by characterizing ssDNA and dsDNA binding of purified pilins and identify their binding surfaces. ssDNA binding will be tested in vivo to prove our model. We will attempt to solve 3D structures of all pilins. The structure of competence pili will be analyzed by atomic force microscopy and electron microscopy, providing insight into their molecular mode of action. We hypothesize that ssDNA taken up into the cytosol is made available for RecA binding via two proteins, DprA and ComFC, so RecA can efficiently form protein/ssDNA filaments that integrate incoming ssDNA into the chromosome. Our recently analyses show that putative DNA endonuclease CoiA plays an important role in transformation in B. subtilis and could be the missing crossover resolvase. CoiA localizes to the cell poles and moves onto the nucleoids upon addition of DNA, where it could act as last step of HR. By a combined biochemical/cell biological/genetic approach, we will clarify the pathway of ssDNA into the chromosome. CoiA bears resemblance to proteins having unstructured N- and C-termini, and could thus represent a candidate for a phase separation process during DNA recombination, which we will test. The planned experiments will answer two important questions at the very beginning and end of cellular transformation, completing the picture of natural transformation.

• DFG Programme: Research Grants