Biology subjects

Oresnik, I. J.

Publications and source records attributed to Oresnik, I. J..

3 recordsLinked to original sources

bioRxiv2026

Sinorhizobium meliloti possesses a complete Embden-Meyerhoff-Parnas pathway that is indispensable for symbiotic nitrogen fixation

Rhizobia are an agronomically valuable group of bacteria capable of entering into endosymbiotic relationships with leguminous plants, during which they fix atmospheric nitrogen using energy derived from the metabolism of plant-provided dicarboxylic acids. It is generally assumed that the gluconeogenic catabolism of dicarboxylic acids proceeds via the Embden-Meyerhoff-Parnas pathway in rhizobia. However, rhizobia are classically thought to lack the phosphofructokinase enzyme required for conversion of fructose-1,6-bisphosphate to fructore-6-phosphate as part of this pathway. Here, we demonstrate that a model rhizobium, Sinorhizobium meliloti, encodes a phosphofructokinase, completing the Embden-Meyerhoff-Parnas pathway of this organism. Biochemical characterization of the S. meliloti phosphofructokinase demonstrates that it can catalyze the reversible phosphorylation of fructose-6-phosphate under in vitro conditions in a pyrophosphate-dependent, rather than ATP-dependent, manner. We further show that S. meliloti also encodes a distinct fructose-1,6-bisphosphatase that can phenotypically complement the loss of the phosphofructokinase enzyme. Loss of both enzymes results in a block of the gluconeogenic pathway in S. meliloti and results in S. meliloti being unable to fix nitrogen in symbiosis with alfalfa (Medicago sativa). Phylogenetic analyses and complementation studies demonstrate that PPi-dependent phosphofructokinases are broadly distributed across the phylum Pseudomonadota (syn. Proteobacteria), including most rhizobial species of the class Alphaproteobacteria, suggesting both that PPi-dependent phosphofructokinases are likely more broadly distributed than is generally recognized, and that the catabolism of dicarboxylic acids in most rhizobia proceeds via a PPi-dependent phosphofructokinase. SIGNIFIGANCECentral carbon metabolism is an important biochemical network that bridges the gap between substrate catabolism and biosynthetic reactions in all living organisms. However, much of what we know about metabolism comes from the study of a few model organisms such as the bacterium Escherichia coli. Here, we identified the enzyme catalyzing a key step of central carbon metabolism in rhizobia (nitrogen-fixing bacterial symbionts of legumes), which until now had remained undetected. We show that this enzyme is dependent on pyrophosphate, which is different than the situation in E. coli, helping to explain why previous studies failed to identify this enzyme in rhizobia and highlighting the limitations associated with generalizing our understanding of metabolism from a limited subset of organisms.

microbiology↗

bioRxiv2026

Isolation of rhizobia from Ontario soils that are effective at fixing nitrogen with common bean (Phaseolus vulgaris)

Common bean (Phaseolus vulgaris) is an important crop in Canada and globally. Like other legumes, common bean (Phaseolus vulgaris) establishes symbiotic interactions with nitrogen fixing bacteria called rhizobia. However, nitrogen fixation by rhizobia in association with common bean is often suboptimal, constraining its productivity and necessitating the application of nitrogen fertilizer. To support the development of high-performing, locally adapted rhizobial inoculants for Ontario common bean growers, we isolated 216 common bean-nodulating rhizobia from southern Ontario soils using a nodule trapping approach with four common bean cultivars. Whole genome sequencing followed by phylogenomic analyses of the 216 rhizobial isolates revealed substantial diversity, assigning them to 11 Rhizobium species, including two novel species. Nearly all isolates belong to the symbiovar phaseoli, spanning the nodC {gamma}-a, {gamma}-b, and alleles, with four isolates belonging to the symbiovar gallica. Soil origin had a significant impact on the species-level community composition recovered during the nodule trapping experiments, indicative of biogeographical structuring of common bean-nodulating rhizobia across southern Ontario. In contrast, host trapping cultivar had only a minor influence of the recovered Rhizobium population diversity. Greenhouse assays demonstrated that one of the novel Rhizobium species exhibited the highest average symbiotic effectiveness, although high-quality isolates were found across multiple species. Together, these results revealed a diverse and genomically variable Rhizobium community capable of forming effective symbioses with common bean in southern Ontario soils. Importantly, our genome-sequenced Rhizobium collection will serve as a valuable resource for identifying competitive and high-quality strains for the development of inoculants tailored to Ontario common bean production. IMPORTANCECommon bean is a globally important food crop, yet its productivity is often limited by suboptimal nitrogen fixation, forcing growers to rely on synthetic fertilizers. Consequently, identifying high-performing, locally adapted inoculant strains is essential for reducing dependence on synthetic nitrogen fertilizers and improving the sustainability of temperate agroecosystems. Our study provides a genome-sequenced collection of common bean-nodulating Rhizobium from southern Ontario, revealing substantial species and genomic diversity across sampling locations. Greenhouse studies allowed us to identify multiple isolates, including isolates from a novel Rhizobium species, that consistently fix nitrogen with, and enhance the growth of, common bean plants. Our findings highlight strong biogeographical structuring of rhizobial communities and demonstrate that Ontario soils already harbour strains with high symbiotic potential. In addition, our Rhizobium collection represents a foundational resource to support future inoculant development and enables future work on the ecology, evolution, and applied optimization of legume-rhizobium symbioses.

microbiology↗

bioRxiv2019

Characterization of Sorbitol Dehydrogenase SmoS from Sinorhizobium meliloti 1021

Sinorhizobium meliloti 1021 is a Gram-negative alphaproteobacterium with a robust capacity for carbohydrate metabolism. The enzymes that facilitate these reactions assist in the survival of the bacterium across a range of environmental niches, and they may also be suitable for use in industrial processes. SmoS is a dehydrogenase that catalyzes the oxidation of the commonly occurring sugar alcohols sorbitol and galactitol into fructose and tagatose respectively using NAD+ as a cofactor. The main objective of this study is to evaluate SmoS using biochemical techniques. The nucleotide sequence was codon optimized for heterologous expression in E. coli BL21 (DE3) GOLD cells, the protein was subsequently overexpressed and purified. Size exclusion chromatography and X-ray diffraction experiments suggest that SmoS is a tetrameric peptide. SmoS was crystallized to 2.1 [A] in the absence of substrate and 2.0 [A] in complex with sorbitol. SmoS was characterized kinetically and shown to have a preference for sorbitol despite a higher affinity for galactitol. Computational ligand docking experiments suggest that galactitol oxidation proceeds slowly because tagatose binds the protein in a more energetically favorable complex than fructose, and is retained in the active site for a longer time frame following oxidation which reduces the rate of the reaction. These results supplement the inventory of biomolecules with the potential for industrial applications and enhance our understanding of metabolism in the model organism S. meliloti.

biochemistry↗