This is interesting (yet perplexing) because it has been proposed that the specialized secretory apparatus ESX-1 of M. smegmatis that lacks an EssB/YukC/TraF homologue carries out DNA transfer . By raising a polyclonal antibody against EssB, we find that the protein sediments
with S. aureus membranes in a manner similar to SrtA, a well-characterized membrane embedded protein . Residues 229–251 roughly define a hydrophobic sequence reminiscent of a transmembrane spanning segment (PTMD). Interestingly, recombinant EssB behaves as a soluble oligomer in E. coli with a rod-shaped like structure and the PTMD sequence appears to be necessary and sufficient for this oligomerization process. Obviously, this conformation may simply represent an energetically this website favorable state for an otherwise membrane-spanning.
Nonetheless, recombinant EssBNM and EssBMC are more prone to multimerization than intact EssB suggesting that the full-length sequence limits or selleck chemical regulates the oligomerization of the protein. Protein translocators of other secretion systems such as the Tat or holin pathways undergo regulated multimerization to facilitate pore function in the membrane [30, 31]. In S.aureus , the presence of the PTMD targets EssBNM and EssBMC to the membrane. This targeting appears to affect the function of endogenous EssB in wild-type staphylococci. On the contrary, EssBΔM (lacking PTMD) is soluble. It is unable to complement the essB mutant and it displays no dominance over wild-type for EsxA secretion. As such, none of the truncated EssB variant could complement wild-type EssB for secretion. Further studies are needed to determine whether the PTMD sequence serves as an autonomous membrane-spanning domain or whether it provides a mean to associate
with another integral membrane protein encoded within the ESS cluster. Deletion of essB in strain USA300 leads to loss of EsxA secretion and EsxA remains in the cell. Because overproduction of EssB is not toxic in E. coli , we do not believe that this protein alone is capable of forming a pore for the passage of secreted substrates. Interestingly, Avelestat (AZD9668) two other proteins EsaB and EsaD also AG-014699 order accumulate in the essB mutant. While the exact role of EsaB is still unknown, it does not appear to be a secreted substrate , and thus the reason for this increase is unclear but it points to additional biochemical interactions within proteins of the ESS cluster. Recent evidence suggests that EsaD is a membrane protein also required for EsxA secretion . Perhaps EssB interacts physically with EsaD to either complete or regulate formation of the translocon. Future studies are needed to address this possibility and determine whether EssB is an integral or peripheral element of the ESS translocon. Conclusions The ESS pathway is an alternate and conserved secretion system of several Gram-positive bacteria. Here, we show that EssB is found in the membrane of S.