Background Nitrogen-fixing bacteria (NFBs) play a critical role in biological nitrogen fixation for supplying essential nitrogen nutrients to plants in agriculture and natural ecosystems. Especially, these bacteria and Leguminosae plants form symbiosis to improve plant growth and soil fertility. Theoretically, the inoculation of NFBs into soils increases biological nitrogen fixation, but the efficiency of NFBs is frequently compromised by the low capacity of NFB root colonization. In this study, we introduced the synthetic bacterium EcCMC, which was genetically engineered to express the surface-displayed artificial polysaccharide (PS)-recognizing protein Cmc, to test if it can improve NFBs root colonization in representative Leguminosae plants, including Astragalus sinicus and Medicago sativa. Rhizosphere microbiomes, biochemical indicators, and plant yields were evaluated after 28 days in the three treatments, i.e., the control group without addition of any exogenous bacterium, the NFBs plus EcM (bacteria only expressing mCherry rather than Cmc) group, and the NFBs plus EcCMC group (n = 3). Results Owing to its polysaccharide-binding capacity, EcCMC strongly bound to the surface of A. sinicus roots. This binding was followed by the increased recruitment of the exogenous NFBs, Sinorhizobium meliloti and Sphingomonas endophytica, on the roots. As revealed by amplicon sequencing of the 16S rRNA gene, a combined inoculation of EcCMC and the NFBs increased the relative abundance of both Rhizobiales and Sphingomonadales, two important bacterial groups involved in nitrogen fixation. Consistently, metabolomic analysis showed that the metabolites involved in nitrogen fixation remarkably accumulated in the rhizosphere soils inoculated with NFBs plus EcCMC. Moreover, inoculation of NFBs plus EcCMC increased the activity of nitrogenase from 10.8 ~ 11.3 to 16.2 nmol/min/g (significant difference, p < 0.05, t-test), together with the total soil nitrogen levels from 217 ~ 258 to 414 mg/kg (significant difference, p < 0.05), and the soil organic matter levels from 19.5 ~ 20.8 to 23.6 mg/kg (significant difference, p < 0.05). Consequently, the yield of A. sinicus was remarkably improved by the inoculation of NFBs plus EcCMC. Similar results were observed in the experiments using Medicago sativa. Conclusions This study sheds a novel light on a synthetic biology-assisted regulation of rhizosphere microbiomes for enhanced nitrogen fixation and soil fertility in Leguminous plants. The designed polysaccharide-binding protein may be used as a universal tool to promote plant growth and enhance crop resilience in the future. <MediaObject ID="MOESM2"> <VideoObject FileRef="MediaObjects/40168_2025_2189_MOESM2_ESM.mp4" VideoID="8Y6FY-UFBRawQ3zVkR4S4y"> <Caption Language="En" xml:lang="en"> <CaptionContent> Video Abstract </CaptionContent> </Caption> </VideoObject> </MediaObject> Graphical Abstract

Synthetic bacterium-facilitated colonization of nitrogen-fixing bacteria for remodeling the rhizosphere microbiome and improving plant yield

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2025年11月29日

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Shuo Liu,
Zirun Zhao,
Yuxin Ji,
Hangqi Zhu,
Yingfeng Sun,
Mingchun Li,
Qilin Yu

摘要

Background

Nitrogen-fixing bacteria (NFBs) play a critical role in biological nitrogen fixation for supplying essential nitrogen nutrients to plants in agriculture and natural ecosystems. Especially, these bacteria and Leguminosae plants form symbiosis to improve plant growth and soil fertility. Theoretically, the inoculation of NFBs into soils increases biological nitrogen fixation, but the efficiency of NFBs is frequently compromised by the low capacity of NFB root colonization. In this study, we introduced the synthetic bacterium EcCMC, which was genetically engineered to express the surface-displayed artificial polysaccharide (PS)-recognizing protein Cmc, to test if it can improve NFBs root colonization in representative Leguminosae plants, including Astragalus sinicus and Medicago sativa. Rhizosphere microbiomes, biochemical indicators, and plant yields were evaluated after 28 days in the three treatments, i.e., the control group without addition of any exogenous bacterium, the NFBs plus EcM (bacteria only expressing mCherry rather than Cmc) group, and the NFBs plus EcCMC group (n = 3).

Results

Owing to its polysaccharide-binding capacity, EcCMC strongly bound to the surface of A. sinicus roots. This binding was followed by the increased recruitment of the exogenous NFBs, Sinorhizobium meliloti and Sphingomonas endophytica, on the roots. As revealed by amplicon sequencing of the 16S rRNA gene, a combined inoculation of EcCMC and the NFBs increased the relative abundance of both Rhizobiales and Sphingomonadales, two important bacterial groups involved in nitrogen fixation. Consistently, metabolomic analysis showed that the metabolites involved in nitrogen fixation remarkably accumulated in the rhizosphere soils inoculated with NFBs plus EcCMC. Moreover, inoculation of NFBs plus EcCMC increased the activity of nitrogenase from 10.8 ~ 11.3 to 16.2 nmol/min/g (significant difference, p < 0.05, t-test), together with the total soil nitrogen levels from 217 ~ 258 to 414 mg/kg (significant difference, p < 0.05), and the soil organic matter levels from 19.5 ~ 20.8 to 23.6 mg/kg (significant difference, p < 0.05). Consequently, the yield of A. sinicus was remarkably improved by the inoculation of NFBs plus EcCMC. Similar results were observed in the experiments using Medicago sativa.

Conclusions

This study sheds a novel light on a synthetic biology-assisted regulation of rhizosphere microbiomes for enhanced nitrogen fixation and soil fertility in Leguminous plants. The designed polysaccharide-binding protein may be used as a universal tool to promote plant growth and enhance crop resilience in the future.

Video Abstract

Graphical Abstract