Search bioRxiv⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2025.11.03.686230

Engineering and evaluation of Sinorhizobium meliloti nodulation (nod) gene reporter systems in rhizobia and non-rhizobia

Luu, C. X.·Geddes, B. A.
View original source ↗Export metadata ↓

Abstract

Developing N{square}-fixing partnerships between diazotrophic microbes and non-legumes can enhance soil fertility and reduce dependence on synthetic fertilizers. Unlike legumes, non-legumes lack the genetic ability to form root nodule symbiosis with rhizobia but can form facultative associations with free-living diazotrophs. Engineering root nodule symbiosis in non-legumes remains a central aim in synthetic biology to enhance biological nitrogen fixation in cereals. Such a symbiosis will require specific symbiotic signaling that mimics the rhizobium-legume interaction. However, lack of effective tools for identifying compatible and engineerable microbial partners is a key challenge. To address this, we have developed inducible nodulation (nod) gene reporters to screen both rhizobial and non-rhizobial strains capable of expressing Sinorhizobium meliloti nod genes, which encode bacterial signals initiating nodule formation in legumes. The reporters include a superfolder GFP reporter controlled by the inducible nod box promoter (PnodA), plant signal-dependent activators nodD1 and nodD2, and a constitutively mScarlet-I marker, named nodD1-PnodA and nodD2-PnodA. Their functionality was validated in various S. meliloti backgrounds using in vitro induction and two in planta induction approaches. These advancements facilitated the identification of both rhizobia and non-rhizobia capable of expressing S. meliloti nod genes, thereby supporting the development of synthetic N{square}-fixing symbioses in cereals.

Source connections

Explore related subjects

microbiologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Luu, C. X., Geddes, B. A.. 2025-11-03. Engineering and evaluation of Sinorhizobium meliloti nodulation (nod) gene reporter systems in rhizobia and non-rhizobia. https://doi.org/10.1101/2025.11.03.686230

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

A population-scale landscape of the subgingival microbiome reveals divergent routes to periodontal dysbiosis

Periodontitis is an archetypical mucosal inflammatory disease in which microbiome dysbiosis at the tooth-epithelial interface interacts with host genetic and behavioral risk factors to drive immune-mediated tissue destruction. Although subgingival microbiome compositional shifts are thought to parallel disease severity, microbiome variation at the population-level and its relationship to periodontal clinical phenotypes and disease-modifying factors remain poorly defined. Here, we use unsupervised manifold learning to map the compositional landscape of the subgingival microbiome in 1,355 adults spanning periodontal health to severe periodontitis. We identified eight latent microbiome states organized along a branching continuum from eubiosis to dysbiosis. An intermediate microbial configuration marked ecological destabilization and bifurcation into two distinct periodontitis-associated dysbiotic trajectories, distinguished by links to gingival inflammation and smoking. Although the microbiome trajectories broadly tracked periodontal destruction, a minority of individuals showed discordant microbiome-clinical phenotypes, with some individuals with periodontitis retaining otherwise eubiotic microbiomes enriched for low-abundance pathobionts, while some cases of health or mild disease had highly dysbiotic communities, suggesting distinct host susceptibility. Together, these findings define a population-scale ecological landscape of the subgingival microbiome, reveal divergent trajectories to periodontal dysbiosis, and highlight heterogeneity in the relationship between microbial community structure and clinical disease expression.

microbiology↗
bioRxiv2026

Beta-lactam enhancement against methicillin-resistant Staphylococcus aureus by cell wall blockade is autolysis-dependent: a butyrolactone derivative as case in point

Methicillin-resistant Staphylococcus aureus (MRSA) is non-susceptible to beta-lactams. Blockade of cell wall biosynthesis is a potential target for beta-lactam enhancement but requires further investigation. A butyrolactone derivative enhanced beta-lactams against MRSA strains by reducing the availability of D-Ala-D-Ala. Unlike D-cycloserine, it did not inhibit D-Ala-D-Ala ligase (Ddl). Nor did it show an additive or synergistic effect when combined with cycloserine, indicating a unique mechanism for blocking cell wall precursor production that does not involve the traditional Lipid II pathway. Notably, beta-lactam potentiation by our chemical or D-cycloserine was highly dependent on the intrinsic autolytic ability of the tested MRSA strains. Strains that resisted lysis upon Triton X-100 exposure showed a minimal increase in beta-lactam susceptibility, whereas highly autolytic strains showed significant changes in their beta-lactam MICs. We have thus identified autolytic ability as the Achilles Heel in the strategy of targeting cell wall biosynthesis for beta-lactam potentiation.

microbiology↗
bioRxiv2026

Rapid and largely reversible shifts in the canine fecal metabolome during dietary change

Diet can rapidly change the fecal metabolome, but less is known about recovery after the original diet is restored. We used untargeted UPLC-MS metabolomics to analyze 72 fecal samples from nine Pumi dogs during an owner-managed switch from dry food to raw food and back to dry food. Diet phase accounted for a large proportion of variation in both ionization modes. More than 13,000 LC-MS features changed at the first sampling point after the switch to raw food, with a similarly large response after return to dry food. Among features significant in both comparisons, more than 99% changed in opposite directions. At the final sampling point, no positive-mode (ESI+) features and only 13 negative-mode (ESI-) features differed from the second dry-food baseline under the same threshold. BARF-associated patterns persisted in analyses excluding individual dogs and in pedigree-adjusted candidate models, although individual feature effects depended on normalization. Putative metabolites from several biochemical classes differed in their response and recovery. The fecal metabolome therefore changed rapidly and returned largely toward baseline, with differences among dogs.

microbiology↗