Background and objectives

Chemical pesticides are generally cost-effective in controlling pests and diseases, and have in consequence become an integral part of modern agriculture. However, these same chemical inputs are also implicated in ecological, environmental and human health problems, so the need to find effective alternative approaches with minimal deleterious effects is obvious. Natural and genetically modified organisms (GMOs) provide an alternative to chemical controls, however this alternative also has biological risks associated. In particular, the implications of the biosafety risks of using GMOs need to be carefully considered before they are implemented. Ideally, a bacterium would persist for long enough in the environment to achieve control of the pest (i.e. so it is effective), but no longer (i.e. without ecological risk).

The specific aim of this project was two-fold. Firstly, to improve the use of ‘self-destruct’ systems to limit bacterial persistence, and secondly, to construct a bacterial strain with enhanced desiccation tolerance. The self-destruct system and the enhanced desiccation resistance will be combined in a single strain, and its persistence will be tested in greenhouse trials.

Approach and methodology

Two control systems were developed for ‘killing’ genes in the plant rhizosphere (the environment surrounding the plant root). This involved construction of a prolonged persistence bacterial strain with a functional ‘self-destruct’ system. One system uses a cold-shock inducible promoter isolated from Pseudomonas putida, which is being tested for functionality in available suicide cassettes. The second system is based on a range of lux genes inserted into the P. putida chromosome, which can be screened on the basis of light emission from the bacteria in response to corn root exudates. In addition, new broad host range toxins for self-destructing bacteria were developed to circumvent the possibility that low level read-through of the killing genes would lead to the selection of mutants with non-functional lethal toxins. Two strategies were employed. Firstly, mutations that rendered the host cells resistant to the lethal action of the toxins were constructed and analysed. An in vitro system was established for analysing the toxin activity.

Colonisation assays are being conducted in various crops including corn, spinach and beans in pot assays in greenhouses to monitor the population dynamics in the rhizosphere over a period of up to 12 weeks. P. putida clones overproducing the osmoprotectant sugar trehalose, are being characterised for desiccation, (using trehalose-accumulation, osmotic tolerance and other growth characteristics of the genetically engineered bacteria). Colonisation by the wild-type and the trehalose-overproducing clones will be tested. The compatibility of the control systems for both desiccation tolerance and self-destruct genes, as well as desiccation tolerance testing in micro-organisms containing both genetic modifications, is under development. Optimisation of drying protocols for Pseudomonas with inducible trehalose synthase systems, formulation of desiccation-tolerant bacteria for seed coating, and testing of the resistance of genetically engineered bacteria to repeated cycles of desiccation and rehydration, are all being developed.
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Main findings and outcome

P. putida KT2442 is a root-colonising strain which can use proline, one of the major components in root exudates, as its sole carbon and nitrogen source. A P. putida mutant, unable to grow with proline as the sole carbon and nitrogen source, was isolated after random mini-Tn5-Km mutagenesis. The mini-Tn5 insertion was located at the putA gene, which is adjacent to and divergent from the putP gene. The putA gene codes for a protein of 1315 amino acid residues which is homologous to the PutA protein of Escherichia coli, Salmonella enterica serovar Typhimurium, Rhodobacter capsulatus, and several Rhizobium strains. Regions of the P. putida PutA protein showed homology to the proline dehydrogenase of Saccharomyces cerevisiae and Drosophila melanogaster, as well as to the pyrroline-5-carboxylate dehydrogenase of S. cerevisiae and a number of aldehyde dehydrogenases. This suggests that in P. putida, both enzymatic steps for proline conversion to glutamic acid are catalysed by a single polypeptide. The putP gene was homologous to the putP genes of several prokaryotic micro-organisms, and its gene product is an integral inner-membrane protein involved in proline uptake. The expression of both genes was induced by the addition of proline to the culture medium and was regulated by PutA. In a P. putida putA-deficient background, expression of both putA and putP genes was maximal and proline independent.
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Conclusions

P. putida KT2442 responds to root exudates, allowing gene expression to be turned on or off depending on the niche being colonised. This strain synthesises trehalose and an increase in the production of this carbohydrate can increase persistence under desiccation conditions. Regulatory circuits responding to plant exudates, and killing genes are being combined to control, at will, the survival of the strain under environmental conditions.