Conjugation, or mating, is the transfer of DNA from a bacterial donor cell to a recipient cell. Conjugation contributes to the movement of genes involved in antibiotic resistance and pathogenesis. ICEBs1 is an integrative and conjugative element of Bacillus subtilis. ICEBs1 encodes for conjugation machinery, classified as a Type IV Secretion System (T4SS), that consists of multiple interacting proteins including ConB, ConC, ConD, ConE, ConG, and CwlT. Our focus is on the bitopic transmembrane mating protein ConB. ConB has four domains: a cytoplasmic N-terminal domain (NTD), a transmembrane segment (TMS), and extracellular central and C-terminal NTF2 domains. Previous research showed that deletion of any of these four domains results in significantly lower mating frequencies, suggesting that each domain is critical for function. Here, we analyze how deletions of ConB’s domains affect ConB protein levels in B. subtilis using quantitative infrared fluorescence western blotting. conB domain deletion mutants were fused to the mCherry gene to allow for detection by an anti-mCherry antibody. We found that the mCherry antibody quantitatively detects ConB-mCherry in B. subtilis. Site-directed mutagenesis was used to separately delete each of the four domains encoded by conB. So far, we have discovered that separate deletion of three of the four ConB’s domains does not alter ConB proteins levels greatly. We conclude that the defects for at least three of the four mutants in mating were due to defects in protein function, and not due to low protein levels. Our results provide a new understanding on the domain structure of ConB in B. subtilis.
Support or Funding Information
This research was funded by a National Science Foundation Research at Undergraduate Institutions grant.
