Segregational instability was observed following the transformation of lactococcal vectors into a range of strains. Loss of resident plasmids was seen when using pGKV11 but not with pNZ18 or pIL252. A recombinant plasmid carrying the gene for phospho-beta-galactosidase was unstable in some background strains and this may involve IS element activity. The role of IS elements in genetic instability was investigated and a wide distribution of IS904 and ISSIS in lactococcal strains was discovered.
The mobilisation of lactococcal vectors by natural gene transfer processes was studied. The conjugative plasmid pBU1-7 mobilised numerous nonconjugative plasmids. In the widely used L.lactis strains 712 and Bu-2 plasmid vectors were conjugally transferred even in the absence of a conjugative plasmid. This may involve a known chromosomally located sex factor at least in the case of L.lactis 712. Lysates of temperate bacteriophages were found to transduce plasmid vectors even though they were very much smaller than the bacteriophage genome. These observations suggest that commonly used lactococcal strains have the capacity to horizontally transfer plasmid vectors by both conjugation and transduction. The frequencies observed under perfect laboratory conditions were low but even this might be eliminated by the removal of temperate bacteriophages and sex factors from genetically engineered microorganisms (GEM).
Structural genes for the bacteriophage lysins of bacteriophages 0vML3 and US3 were characterised and isolated such that their expression under the control of an inducible lactococcal promoter could be investigated. For the ovML3 lysin a complex organisation with 2 overlapping and in frame ORFs was discovered.
Promoter probe strategies were developed using promoterless lux genes. The promoter and repressor of the inducible lactose operon from plasmid pLP712 were coloned, sequenced and characterised.
Expression of the US3 lysin gene in L. lactis under control of th e lactose promoter was achieved and lactose caused growth inhibition of the strain. The ovML3 lysin was expressed in a variety of lactococcal strains under the control of a bacteriophage promoter. Without osmotic buffering these constructs grew normally through log phase but lysed once stationary phase was reached. This was probably because lysin was only active from outside of the cell and limited spontaneous lysis at stationary phase initiated a cascade of lysis due to released lysin. These observations demonstrate that biologically contained lactococci can be produced by engineering lethal genes.