International Symposium on Nitrogen Fixation with Non-Legumes edition:9 location:Leuven, Belgium date:1-5 September 2002
Rice (Oryza sativa L.) is the staple in the diet of over 40% of the world’s population making it the most important food crop currently produced. A bacterium repeatedly isolated from rice in China is Pseudomonas stutzeri A15 (Vermeiren et al., 1999). This nitrogen-fixing endophyte may provide the rice plant with fixed nitrogen and hence promote plant growth. At present, the mechanisms which enable strain A15 to colonize and infect rice roots and survive within rice plants, are not known. To identify bacterial promoters specifically induced during the interaction with the host plant, the ‘in vivo expression technology’ (IVET) is used (Rainey, 1999; Rainey and Preston, 2000).
IVET is a promoter trapping technique based on the complementation of a mutation in an essential biosynthetic gene (Mahan et al., 1993). The prerequisite for applying this technique under these circumstances is that the mutation cannot be complemented by production of plant metabolites. Therefore a P. stutzeri A15 dapB mutant was constructed. The dapB gene encodes a dihydrodipicolinate reductase and is involved in diaminopimelic acid biosynthesis. Diaminopimelic acid is an essential component of peptidoglycan and the precursor of lysine.
A genome library was constructed by inserting DNA fragments in front of a promoterless dapB gene with the P. stutzeri A15 dapB mutant as the host strain. The screening of this library for specifically in vivo expressed promoters is in progress. Already, a number of genes with specific in vivo expression could be identified. These genes seem to be involved in stress response, nutrient acquisition, adaptation to different environments or regulation. In addition to these genes, some genes without significant homology to genes in the database or genes with unknown function were isolated as well.