Study on late competence proteins involved in natural transformation of Bacillus subtilis
The following study comprises in vivo and in vitro data on several of the so-called late competence proteins, which are involved in natural transformation of Bacillus subtilis. The gram-positive bacterium B. subtilis belongs to those bacteria, who are able to take up DNA from their environment and i...
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|The following study comprises in vivo and in vitro data on several of the so-called late competence proteins, which are involved in natural transformation of Bacillus subtilis. The gram-positive bacterium B. subtilis belongs to those bacteria, who are able to take up DNA from their environment and incorporate the foreign DNA by homologous recombination into their own chromosome; a feature named competence. This fascinating ability is carried out by only a portion of the bacterial culture, expressing specific proteins, encoded by the late competence operons. If exogenous double-stranded DNA is about to be taken up from the environment, it needs to first cross the thick cell wall of B. subtilis, with a width of ~40 nm. In case of B. subtilis, this first border is crossed by a putative pseudopilus who transfers the DNA inside of the cell. The energy for this particular process is probably provided by the assembly/disassembly ATPase ComGA. The taken-up DNA is then further transferred into the cytosol by the so-called competence complex or competence machinery. The complex consists out of specific competence proteins, which assemble at the membrane, including a DNA-binding transmembrane protein (ComEA) and an aqueous channel protein (ComEC).
In the following thesis, the unknown role of the protein ComEB has been further elucidated in the context of competence, and its enzymatic function was analysed in vitro. It was found that the protein carries out deaminase activity, which is not essential for transformation. In case of ComEC, an amino acid, D573, has been identified as essential for transformation. Truncations of the protein, supposed to carry out an exonuclease function, were heterologously expressed and purified as GST-tag fusions, but, probably due to aggregations of the proteins, no enzymatic activity was detected. The intracellular diffusion of fluorophore fusions of several competence proteins, namely ComEB-mV, ComGA-mV, ComEC-mV and mV-ComEA was analysed via single-molecule tracking, in the presence and absence of exogeneous DNA. In case of ComGA, a C-terminal fusion to mVenus was analysed and it was found that the protein becomes more dynamic in the presence of DNA. Further, the localization and diffusion of a fluorescently labeled PCR product inside of competent Bacillus cells was analysed for the first time. The diffusive behaviour and localization of the stained DNA resembles the diffusion of mV-ComEA. This led to the hypothesis that ComEA serves as a reservoir for taken-up DNA, similar to what is already known for orthologues of ComEA from other naturally competent bacteria.