Construction of a Nisin-inducible host-vector system for foreign gene expression in Gram-positive bacteria

Several strains of Lactococcus lactis produce the lantibiotic peptide nisin. Nisin biosynthesis is autoregulated by a two-component regulatory system NisK-NisR. In this thesis, a transferable nisin inducible host-vector expression system was developed in S.pneumoniae and transferred to a variety of bacterial species, including Streptococcus gordonii, Streptococcus pyogenes, Streptococcus agalactiae and Enterococcus faecalis. Pneumococcal recombinant strain TP15B13, displaying on surface the HA1 antigenic domain of influenza hemagglutinin using as fusion partner the S. pyogenes surface protein M6, was constructed. M6-based HA1 fusion protein, upon nisin induction, is targeted to the membrane, exported and anchored on the surface by sequences of the M6 protein as indicated by dot blot and flow cytometry analysis on whole bacterial cells using specific M6 polyclonal and HA1 monoclonal antibodies. The synthetic construct MBT6, constituted by: (i) the m6-ha1 fusion gene under the control of the nisA promoter, (ii) the nisin two-component system nisK-nisR, (iii) the gene ermB conferring erythromycin resistance, was developed and cloned. The resulting insertion vector pMBT6 was constructed and transformed in an S. pneumoniae recipient strain carrying the integrative and conjugative element Tn5253, where the MBT6 construct integrates. Then recombinant Tn5253::MBT6 was transferred by conjugation from S. pneumoniae to different transformable and non-transformable bacterial species, where M6HA1 expression upon nisin induction was assayed.