Bacterial mating, or conjugation, is the transfer of DNA from a donor bacterial cell to a recipient cell. Mating contributes to the spread of antibiotic resistance as well as to bacterial evolution. During conjugation, DNA is transferred through a Type 4 Secretion System (T4SS) that is made up of multiple proteins embedded in the donor cell membrane. We are interested in characterizing the function and localization of the T4SS of the integrative and conjugative element ICEBs1 of Bacillus subtilis. Previously, we discovered that ConE (VirB4) and ConB (VirB8) are both critical components of the ICEBs1 T4SS. ConE-GFP localizes to the membrane of the donor cell, predominantly at the poles of the cell. We hypothesize that ConB will localize similarly to ConE as several lines of evidence indicate that the two proteins interact with each other. Using isothermal assembly, we constructed fusions between the conB gene and the genes for three different fluorescent proteins or tags: monomeric superfolder green fluorescent protein (msfGFP), mCHERRY, and HaloTag. Three different tags were chosen to increase our chances of obtaining a fluorescent fusion that is also functional for mating and to enable us to do dual color co-localization of ConB-mCHERRY and ConE-GFP. The constructs were transformed into Escherichia coli and their sequences verified. We have transformed the plasmids into B. subtilis, and recombined the fusions onto the chromosome. Next, the location of ConB will be visualized using fluorescence microscopy. Our study will provide a greater understanding of the localization of the T4SS of B. subtilis.

Fluorescence localization of the conjugation machinery of Bacillus subtilis

2019 Spring National Meeting

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