Early transcriptomic response of Alnus glutinosa to Frankia alni symbiont, an upregulated nsLTP (non-specific Lipid Transfer Protein) is implicated in early and late stages of symbiosis

The response of Alnus glutinosa to Frankia alni is complex with several sequential physiological modifications that include calcium spiking, root hair deformation, penetration, induction of primordium, formation and growth of nodule. A transcriptomic study of seedlings in hydroponics after early contact (2.5 days) with Frankia alni, either with a culture supernatant or with living cells separated from the roots by a dialysis membrane, permitted to identify plant genes which expression level was modified upon early contact with Frankia. Forty-two genes were significantly up-regulated in both experiments, most of them encoding biological processes such as oxidative stress or response to stimuli. Among them, the most upregulated gene was a non-specific lipid transfer protein encoding gene with a fold change of 141. This nsLTP was found to increase Frankia nitrogen fixation at sub-lethal concentration. Interestingly, it was immunolocalized to a region of the deformed root hair at an early infection stage and later in nodules, it was localized around bacterial vesicles suggesting a role in early and late stages of symbiosis.

A Promiscuous Halogenase for the Derivatization of Flavonoids

Halogenation often improves the bioactive properties of natural products and is used in pharmaceutical research for the generation of new potential drug leads. High regio- and stereospecificity, simple reaction conditions and straightforward downstream processing are the main advantages of halogenation using enzymatic biocatalysts compared to chemical synthetic approaches. The identification of new promiscuous halogenases for the modification of various natural products is of great interest in modern drug discovery. In this paper, we report the identification of a new promiscuous FAD-dependent halogenase, DklH, from Frankia alni ACN14a. The identified halogenase readily modifies various flavonoid compounds, including those with well-studied biological activities. This halogenase has been demonstrated to modify not only flavones and isoflavones, but also flavonols, flavanones and flavanonols. The structural requirements for DklH substrate recognition were determined using a feeding approach. The homology model of DklH and the mechanism of substrate recognition are also proposed in this paper.

Stable Genetic Transformation and Heterologous Expression in the Nitrogen-fixing Plant Endosymbiont Frankia alni ACN14a

Genus Frankia is comprised primarily of nitrogen-fixing actinobacteria that form root nodule symbioses with a group of hosts known as the actinorhizal plants. These plants are evolutionarily closely related to the legumes, which are nodulated by the rhizobia. Both host groups utilize homologs of nodulation genes for root-nodule symbiosis, derived from common plant ancestors. However the corresponding endosymbionts, Frankia and the rhizobia, are distantly related groups of bacteria, leading to questions of their symbiotic mechanisms and evolutionary history. To date, a stable system of genetic transformation has been lacking in Frankia. Here, we report the successful electrotransformation of Frankia alni ACN14a, by means of replicating plasmids expressing chloramphenicol-resistance for selection, and the use of GFP as a marker of gene expression. We have identified type IV methyl-directed restriction systems, highly-expressed in a range of actinobacteria, as a likely barrier to Frankia transformation and circumvented this barrier by using unmethylated plasmids, which allowed the transformation of F. alni as well as the maintenance of the plasmid. During nitrogen limitation, Frankia differentiates into two cell types: the vegetative hyphae and nitrogen-fixing vesicles. When the plasmid transformation system was used with expression of egfp under the control of the nif gene cluster promoter, it was possible to demonstrate by fluorescence imaging the expression of nitrogen fixation in vesicles but not hyphae in nitrogen-limited culture.