Abstract 1533 Investigation of the Membrane Topology of the Bacterial Conjugation proteins ConC and ConG

Bacterial conjugation, also known as mating, is a major form of horizontal gene transfer. During conjugation, a donor bacterium passes genetic material through a translocation channel to a recipient cell. This process allows for the evolution of bacterial populations and the spread of antibiotic resistance genes. Our research focuses on studying the integrative and conjugative element ICEBs1 of Bacillus subtilis, a Gram-positive bacterium. We studied the membrane topology of ConC and the VirB6-like protein ConG, which are two of the multi-pass membrane proteins that comprise the ICEBs1 conjugation channel. The location of their C-termini was determined using a plate-based blue/white screening assay. The genes encoding LacZ and PhoA were fused to the ends of our genes of interest and transformed into B. subtilis. Strains were then streaked onto indicator plates and incubated for color development. LacZ encodes for the enzyme β-galactosidase, which can cleave the substrate 5-bromo-4-chloro-3-indolyl-beta-D-galacto-pyranoside (X-gal), forming a blue pigment if localized in the cytoplasm, but not if it is located outside the cell. PhoA encodes for the enzyme alkaline phosphatase, which can cleave the substrate 5-Bromo-4-chloro-3-indolyl phosphate (X-P), producing a blue pigment if localized extracellularly but is inactive if cytoplasmic. Previously, we had predicted that ConC's and ConG's C-termini would be extracellular. We discovered that ConC's C-terminus was cytoplasmic. The strain expressing ConC-LacZ turned blue, while the strain expressing ConC-PhoA remained beige. The membrane topology of ConG was studied using a mutant of ConG that removed its large C-terminal domain as our results with the full-length protein were inconclusive. We found that the C-terminus of the ConG mutant was cytoplasmic. Overall, our results shed new light on the proposed model of the ICEBs1 conjugation channel. This research was funded by Suffolk University.