Characterization of Diverse Mechanisms of Salicin Degradation in Populus Microbiome Isolates

Investigations of metabolic interactions between plant and microbes are vital to designing approaches to promote plant growth and health. Populus, a potential biofuel crop, interacts with its microbiome through numerous metabolites, but research on phenolic glycosides (PGs) is still lacking despite the fact that Populus allocates significant amount of carbon to PGs. Therefore, this study asked the following questions: what is the prevalence of microbiome isolates that utilize PGs as growth substrates? what is the diversity in the mechanisms of PG utilization? For this study, salicin was used as the model PG substrate. Four naturally occurring salicin degradation systems designated – Bgl, Sal, Lac and Cbg that have been described in literature were investigated in 407 Populus bacterial isolates with available genome sequences. These four systems appear to be present in phylogenetically diverse microbial associates suggesting independent evolution of these mechanisms. Following this, four bacterial strains – Caulobacter sp. AP07, Citrobacter sp. YR018, Agrobacterium rhizogenes OK036 and Rahnella sp. OV744 were selected for further study. Caulobacter sp. AP07 was demonstrated to possess two different salicin systems – Lac and Sal that were both found to be active during growth on salicin minimal media. Citrobacter sp. YR018, on the other hand, utilized salicin most likely by gaining mutations. A few possible loci that could have been mutated are proposed in this study. Agrobacterium rhizogenes OK036 was investigated for its unique ability to degrade both the glucosyl and salicyl moieties of salicin unlike other organisms in this study that only utilize glucosyl group. Using proteomic, metabolomic and other experimental assays, Agrobacterium was demonstrated to utilize salicin by first cleaving glucose off of salicyl alcohol, which is then channeled into gentisate pathway. A few unique features of this novel pathway were explored further using bioinformatic approaches.Finally, salicin co-metabolism by co-culture of Rahnella sp. OV744 and Pseudomonas sp. GM16 was elucidated. Using proteomic and metabolomic along with other quantitative assays OV744 and GM16 were demonstrated to be involved in a unidirectional cross-feeding interaction during growth on salicin minimal media. The co-culture study illustrates a possible mechanism Populus might utilize to fine-tune its microbial colonization process