Mini-Tn7 vectors for stable expression of diguanylate cyclase PleD∗in Gram-negative bacteria

Abstract

Background: The cyclic diguanylate (c-di-GMP) is currently considered an ubiquitous second messenger in bacteria that influences a wide range of cellular processes. One of the methodological approaches to unravel c-di-GMP regulatory networks involves raising the c-di-GMP intracellular levels, e.g. by expressing a diguanylate cyclase (DGC), to provoke phenotypic changes. Results: We have constructed mini-Tn7 delivery vectors for the integration and stable expression of the pleD∗gene encoding a highly active DGC, which can be used to artificially increase the intracellular levels of c-di-GMP in Gram negative bacteria. The functionality of these new vectors has been validated in several plant-interacting α- and γ-proteobacteria. Similarly to vector plasmid-borne pleD∗, the genome-borne mini-Tn7pleD∗constructs provide significant increases in intracellular c-di-GMP, provoking expected phenotypic changes such as enhanced polysaccharide production, biofilm formation and reduced motility. However, the mini-Tn7pleD∗constructs resulted far more stable in the absence of antibiotics than the plasmid-based pleD∗constructs. Furthermore, we have also implemented an inducible system to modulate pleD∗expression and intracellular c-di-GMP rises "on demand". Conclusions: mini-Tn7pleD∗constructs are very stable and are maintained during bacterial free-living growth as well as during interaction with eukaryotic hosts, in the absence of selective pressure. This high stability ensures experimental homogeneity in time and space with regard to enhancing c-di-GMP intracellular levels in bacteria of interest.