Background and objectives

Lactoccoci are extensively used by agro-food industries in food fermentation, for example for making various dairy products. The advances in genetic technology now make strain improvement by genetic modification possible. The BAP, BEP and BRIDGE programmes of the European Community have been major contributors to this progress. However, practical exploitation of this potential requires that genetically modified micro-organisms be stable and accepted by the public. For genetically modified organisms to become accepted, they must, among other issues, be demonstrated to be safe. A particular concern is the spread of genetic material from the modified strain to other strains or even other species (called horizontal spread). Another major issue is the survival and environmental impact of genetically modified micro-organisms. This project aimed to investigate the stability of genetically modified lactococci, and to study the horizontal spread of genes from such strains. The second aim was to develop genetically contained lactococci, that is, strains which are not viable outside their industrial workplace.

Approach and methodology

The approach involved analysing the stability of genetically modified lactococci, including the segregational and structural instability of plasmid vectors. There are various natural gene transfer processes, particularly conjugation and transduction, and the contribution that these processes may make to horizontal gene transfer from genetically modified lactococci was investigated. To develop systems for biological containment of genetically modified strains, various lethal gene systems were developed and assessed. Appropriate inducible gene expression systems were then designed for the lethal genes, and these different elements combined so create genetically modified lactococci with in-built containment systems. Finally, the performance and characteristics of these containment systems, and lactococci carrying them, were evaluated.

Main findings and outcome

Commonly used lactococcal vectors were mobilised by natural bacterial mating processes (conjugation) and by bacteriophage-mediated transduction. The frequencies of transfer were low. Furthermore, these experiments indicated that improved containment of genetically modified lactococci could be further improved by elimination of temperate bacteriophages (which promote transduction) and sex factors (which promote conjugation). Vector stability was determined and the significance of transposable elements as sources of instability was established. Two lysin genes were characterised. Systems were developed to isolate controllable promoters that may be suitable for use as genetic switches. This included exploitation of light-emitting lux genes and the development of a model controlled gene expression system based on the lactose repressor and operon. Lysin genes were successfully expressed in lactococci using these new systems. Lactose-induced growth inhibition was demonstrated with the bacteriophage US3 lysin, and an autolytic strain was created using the bacteriophage VML3 lysin. Without osmotic protection, this autolytic strain lysed spontaneously as it entered stationary phase proving that the lethal gene concept for containment is effective.

Conclusions

The potential of natural gene transfer processes to mediate horizontal spread of genetic material was low, but nevertheless present. The elimination of such potential from genetically modified micro-organisms would improve their containment. A lethal gene concept was developed and shown to be feasible using bacteriophage lysin genes and inducible gene expression signals. The approach was successfully validated in genetically modified lactococci.