Aspects of marker/reporter stability and selectivity in soil microbiology

Based on several experiences of microbial release using genetically modified Rhizobium leguminosarum, we have highlighted a number of aspects related to the suitability of introduced markers such as resistance to mercury and b-galactosidase activity, the latter serving the function of high-expression level reporter gene obtained by the introduction of a synthetic promoter conferring strong inducible expression in Gram-negative bacteria. In vitro expression and in vivo performances of the chosen examples have been followed in model strains comparing gene dosage and expression levels. The technical possibility of unambiguously monitoring the marked GMM has been evaluated in medium- and long-term experiments carried out both in microcosms and soil, also including the presence of the plant symbiotic host. Marker stability, regardless the nature of the gene, was shown to be dependent on the location of the genetic modification and on its degree of gene expression regulation. Reporter strength was found to be an advantage allowing the distinction of marker-bearing bacteria while negatively affecting their genetic stability. Plasmid-borne regulated reporters were found to be stable up to the stages of rhizosphere colonization, but were more critically selected against upon symbiotic host invasion.