Role of erythritol catabolism by Rhizobium johnstonii in the rhizospheres of lentil, pea, and vetch

Abstract

Legumes are known for their high protein content and ability to improve soil fertility. They form a symbiotic relationship with rhizobia through exchange of signaling molecules and plant exudates, such as carbon sources, which, in turn, enable biological nitrogen fixation through the formation of nodules. In the rhizosphere, the environment around the roots, bacteria compete against each other for root colonization and nodulation. Rhizobium johnstonii is capable of catabolizing erythritol, a known pea root exudate. Previous deletion mutation of the catabolic genes (Yost et al., 2006) created strains unable or with reduced ability to grow on media containing erythritol as a sole carbon source. Subsequently, the mutant strains were impaired in their ability to compete against the wild type to colonize pea root cells. In this study, I expanded the investigation to include lentils and vetch, alongside pea, to assess competition between wild type and mutant strains in different legume plants. It was determined that erythritol catabolism is important for Rhizobium for successful colonization and nodulation of all three legume species. In this research, I demonstrate that the capacity to catabolize erythritol is an important trait that is required across all these three plants. Previous deletion mutations in the transporter genes resulted in strains with reduced growth rates compared to the wild type when grown on minimal medium with erythritol as the sole carbon source. Non-specific transporters can sometimes facilitate the uptake of similar molecules, even if they are not the primary substrate, although their involvement in erythritol uptake remains unclear. To further explore this, I employed an IN-seq library and TRANSIT analysis, but these approaches did not reveal any additional specific transporters responsible for erythritol uptake in Rhizobium johnstonii bv. viciae VF39sm mutants. The IN-Seq approach may not be effective for identifying erythritol transporters due to the redundancy of alternative transporter systems. Additionally, metabolic flexibility and compensatory mechanisms might explain the observed growth on erythritol, as bacteria are able to adapt by upregulating various transporter pathways under selective pressure. In 2022, Mullock observed the upregulation of rhi genes (rhizosphere induced genes involved in a quorum sensing system) in Rhizobium johnstonii bv. viciae 3841 when growing on erythritol and compared the results with growth of this strain on mannitol, suggesting that erythritol may act as a signaling molecule in the rhizosphere. Now the key question is whether erythritol functions as a signaling molecule in the rhizospheres of lentil, pea, and vetch plants, in addition to serving as a carbon source. To investigate the role of erythritol as a signaling molecule rather than solely a carbon source, I integrated rhiA promoter-gusA fusions into wild type, catabolic and transporter mutant strains to measure gene expression through the GUS assay. rhiA is part of the rhiABC operon, which is involved in the quorum sensing system of Rhizobium and plays a role in the regulation of genes associated with rhizosphere-induced proteins. While β-glucuronidase activity was detected, it did not show consistent trends in expression patterns across strains. Therefore, further investigation is suggested to characterize the role of erythritol as a signaling molecule for regulation of genes not involved in erythritol catabolism.