Bacteriocin sublancin-168

Bacillus subtilis strain 168 produces the extremely stable lantibiotic sublancin 168, which has a broad spectrum of bactericidal activity. Both sublancin 168 production and producer immunity are determined by the SPbeta prophage. While the sunA and sunT genes for sublancin 168 production have been known for several years, the genetic basis for sublancin 168 producer immunity has remained elusive. Therefore, the present studies were aimed at identifying an SPbeta gene(s) for sublancin 168 immunity. By systematic deletion analysis, we were able to pinpoint one gene, named yolF, as the sublancin 168 producer immunity gene. Growth inhibition assays performed using plates and liquid cultures revealed that YolF is both required and sufficient for sublancin 168 immunity even when heterologously produced in the sublancin-sensitive bacterium Staphylococcus aureus. Accordingly, we propose to rename yolF to sunI (for sublancin immunity). Subcellular localization studies indicate that the SunI protein is anchored to the membrane with a single N-terminal membrane-spanning domain that has an N(out)-C(in) topology. Thus, the bulk of the protein faces the cytoplasm of B. subtilis. This topology has not yet been reported for known bacteriocin producer immunity proteins, which implies that SunI belongs to a novel class of bacteriocin antagonists.

The synthesis of sublancin 168, a unique S-glucosylated bacteriocin antibiotic, is described. The natural product and two S-glycosylated variants were successfully prepared via native chemical ligation followed by folding. The synthetic glycopeptides were shown to possess primarily an α-helical secondary structure by CD and NMR studies.

Bacillus subtilis strain 168 produces the extremely stable and broad-spectrum lantibiotic sublancin 168. Known sublancin 168-susceptible organisms include important pathogens, such as Staphylococcus aureus. Nevertheless, since its discovery, the mode of action of sublancin 168 has remained elusive. The present studies were, therefore, aimed at the identification of cellular determinants for bacterial susceptibility toward sublancin 168. Growth inhibition and competition assays on plates and in liquid cultures revealed that sublancin 168-mediated growth inhibition of susceptible B. subtilis and S. aureus cells is affected by the NaCl concentration in the growth medium. Added NaCl did not influence the production, activity, or stability of sublancin 168 but, instead, lowered the susceptibility of sensitive cells toward this lantibiotic. Importantly, the susceptibility of B. subtilis and S. aureus cells toward sublancin 168 was shown to depend on the presence of the large mechanosensitive channel of conductance MscL. In contrast, MscL was not involved in susceptibility toward the bacteriocin nisin or Pep5. Taken together, our unprecedented results demonstrate that MscL is a critical and specific determinant in bacterial sublancin 168 susceptibility that may serve either as a direct target for this lantibiotic or as a gate of entry to the cytoplasm.