The novel Beta-rhizobia Cupriavidus sp. UYMMa02A strain employs nod-independent strategies to interact with Mimosa sp. host plants

Abstract

UYMMa02A is a Beta-rhizobia strain of the Cupriavidus genus isolated from nodules of Mimosa magentea in Uruguay and able to form effective nodules with several Mimosa species, including its original host. Phylogenetic analyses indicated that UYMMa02A belongs to a novel Cupriavidus species, different to the previously characterized C. taiwanensis and C. necator rhizobial strains. However, the genome of UYMMa02A revealed the presence of nod, nif and fix genes highly syntenic to the model strain C. taiwanensis LMG19424, suggesting the presence of common mechanisms implicated in the symbiotic establishment with plant hosts. The objective of this work was to analyze the importance of nod genes during the Cupriavidus-Mimosa symbiotic interaction. For that, UYMMa02A was cultivated in the presence of different flavonoids and Mimosa pudica host plant, while the bacterial response was assessed using a combination of genomic and proteomic approaches. In order to determine the expression levels of the UYMMa02A nod operon, we constructed reporter plasmids containing transcriptional fusions between the promoter region of nodB, the first gene of the operon, and the gfp reporter gene. When cultures of UYMMa02A containing the pnodB-gfp were incubated with luteolin, apigenin or M. pudica root exudates, we could not detect any fluorescence, suggesting that UYMMa02A nod genes are not induced in these conditions. To further characterize the role of these genes, we constructed an UYMMa02A nodD- mutant and analyzed its symbiotic phenotype. Surprisingly, the nodD minus mutant strain was able to form symbiotically effective nodules on M. pudica plants, indicating the existence of a nodD independent infection mechanism. Finally, using a proteomic approach, we identified a total of 28 differentially expressed proteins in the presence of pure flavonoids or the host plant, M.pudica. The characterization of the identified proteins will certainly help to identify alternative mechanisms involved in the Cupriavidus-mimosa symbiosis.