Sara Lundström:
Characterization of a Bacillus licheniformis gene cluster required for functional expression of a bacteriocin

Date: 31-05-2012    Supervisor: Michael Askvad Sørensen

The aim of this PhD project was to characterize the gene cluster required for functional expression of a bacteriocin (UniProt ordered locus name BL00275) from Bacillus licheniformis ATCC 14580. This bacteriocin, with the proposed name formosin (ForD) is encoded in a gene located in the chromosome of B. licheniformis ATCC 14580 with three adjacent genes (UniProt ordered locus names BL00274-BL00272), proposed to be named forE, forF, and forG, respectively. Genetic in silico analysis showed that these four genes are arranged in an operon situated in a genomic island with host defensive properties. Structural and functional studies demonstrated that ForE and ForG constitute an ABC transporter required in both secretion of and immunity to formosin. ForF is an accessory protein to the ForEG ABC transporter containing an N-terminal transmembrane domain. No function could be linked to ForF. Secretion analysis revealed formosin to have two secretion signals; one N-terminal sec-dependent signal peptide and one C-terminal ABC transporter signal. However, only when secreted through the ForEG ABC transporter could formosin be detected in the medium. Characterization of formosin showed that it is a 9.6kDa heat-labile bacteriocin belonging to the lactococcin 972 family with an observed bacteriolytic effect on Bacillus subtilis. ForG is a structural homolog of a previously described “immunity” protein associated with this protein family. Investigation of lactococcin 972-like proteins and adjacently encoded genes, in addition to the results obtained within this project, concluded that the members of the lactococcin 972 family are associated with ABC transporters and not transmembrane immunity proteins as previously predicted.