A Novel Nitrogen-Fixing Bacterium Raoultella electrica Isolated from the Midgut of the Leafhopper Recilia dorsalis

Insects · 5 authors, 1 centre

AI SUMMARY

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POPULATIONRaoultella electrica strain Rd210413 isolated from the digestive tract of adult leafhopper Recilia dorsalis

INTERVENTIONGrowth on nitrogen-free Ashby medium and acetylene reduction assay to measure nitrogenase activity

COMPARISONComparison with Bradyrhizobium japonicum (positive control) and Escherichia coli DH5α (negative control) for growth and nitrogenase activity

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TL;DR

This study isolated a novel nitrogen-fixing bacterium, *Raoultella electrica* strain Rd210413, from the digestive tract of the leafhopper *Recilia dorsalis*, a rice pest. Genome sequencing revealed that *R. electrica* possesses all genes required for nitrogen fixation (nifHDK, nifAL, nifJ, nifBENQSUX), and fluorescence in situ hybridization confirmed its colonization in the midgut and filter chamber of the leafhopper. The bacterium demonstrated nitrogenase activity of 43.60 nmol·mL⁻¹·h⁻¹, suggesting a potential role in providing nitrogen nutrition to its insect host, though further studies are needed to confirm this symbiotic relationship.

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{"Background": "Herbivorous insects, including leafhoppers, face nitrogen nutrition deficiencies due to the high C:N ratio of their diets (e.g., bark, leaves, or sap). Symbiotic microorganisms can provide nitrogen nutrition through nitrogen recycling, fixation, or upgrading. While nitrogen fixation by symbiotic microorganisms has been extensively demonstrated in termites, evidence in Hemiptera remains limited. The leafhopper *Recilia dorsalis* (Cicadellidae: Deltocephalinae) is a major rice pest in Asia that transmits viral pathogens including Rice dwarf virus, Rice stripe mosaic virus, and Rice tungro virus. Leafhoppers harbor the obligate bacterium 'Candidatus Sulcia muelleri' and a partner \u03b2-proteobacterium that provide 10 essential amino acids, but no nitrogen-fixing symbionts have been previously identified in leafhoppers. The *Raoultella* genus (Enterobacteriaceae, Proteobacteria) includes four known species\u2014*R. ornithinolytica*, *R. planticola*, *R. terrigena*, and *R. electrica*\u2014several of which have demonstrated nitrogen fixation capacity in other contexts.", "Methods": "Fifty adult *R. dorsalis* were collected from a rice field in Jiaxing, Zhejiang Province, China, and maintained at 26 \u00b1 1\u00b0C with a 16:8 h light-to-dark cycle. Whole digestive tracts were dissected under sterile conditions, ground in PBS, diluted 1000-fold, and plated on LB agar under aerobic and anaerobic conditions at 28\u00b0C for 48 h. Pure colonies were screened on Ashby nitrogen-free medium to identify nitrogen-fixing isolates. The bacterial strain was identified via 16S rRNA gene sequencing using primers 27F and 1492R, with phylogenetic analysis performed using MEGA 7 (Maximum Likelihood method, Hasegawa\u2013Kishino\u2013Yano model, 1000 bootstrap replicates). Genome sequencing was performed using Illumina HiseqX-Ten and PacBio RSII platforms, with assembly via Falcon and circularization via Circulator. Gene prediction used prodigal, RNAmmer, RNAscan-SE, Rfam, and RepeatMasker, with annotation against COG, GO, KEGG, NR, and Swiss-Prot databases. Fluorescence in situ hybridization (FISH) was performed on dissected digestive tracts from 30 leafhoppers using a Cy3-labeled probe targeting *R. electrica* 16S rRNA. Transmission electron microscopy used negative staining with 2% sodium phosphotungstate. Nitrogen fixation capacity was assessed by growth on LB and Ashby nitrogen-free media, with *Bradyrhizobium japonicum* and *Escherichia coli DH5\u03b1* as positive and negative controls. Growth curves were recorded using Bioscreen C at 28\u00b0C with OD600 measured every 30 min. Acetylene reduction assay (ARA) was performed in sealed 60 mL bottles with He:C\u2082H\u2082 (90:10, v/v) gas mixture; ethylene concentration was measured by gas chromatography (GC-2014, Shimadzu) with a flame-ionization detector. nifH gene expression was measured by RT-qPCR using the \u0394\u0394CT method with 16S rRNA as internal control.", "Key Results": "A nitrogen-fixing bacterial strain was isolated and identified as *Raoultella electrica* (strain Rd210413, GenBank OQ595258). Transmission electron microscopy revealed rod-shaped cells approximately 3.2 \u03bcm in length and 1.1 \u03bcm in diameter with flagella but no capsule or spores. FISH showed *R. electrica* distributed throughout the midgut and filter chamber of *R. dorsalis*, primarily localized in the gut lumen near the epithelium. The genome comprised a single circular chromosome of 5,282,709 base pairs with 55.58% G+C content, encoding 4,890 protein-coding sequences (86.68% of genome), 86 tRNA genes, 25 rRNA genes (8 for 16S rRNA, 8 for 23S rRNA, 9 for 5S rRNA), and 121 sRNA genes. All nitrogen fixation genes were located in a 29.4 kbp region (nifQ to nifJ), including nifHDK (structural protein), nifAL (transcriptional regulator), nifJ (FeMo co-factor synthesis), and nifBENQSUX (electron transfer). Phylogenetic analysis of nifH showed 98.98% homology with *Klebsiella michiganensis* and *R. planticola*, and 98.63% with *R. terrigena*. Growth assays showed no significant differences among *R. electrica*, *E. coli*, and *B. japonicum* on LB medium, but on Ashby nitrogen-free medium only *R. electrica* and *B. japonicum* grew, with *B. japonicum* growing significantly faster. RT-qPCR showed nifH expression was significantly higher on Ashby medium than on LB medium. Acetylene reduction assay demonstrated nitrogenase activity of 43.60 nmol\u00b7mL\u207b\u00b9\u00b7h\u207b\u00b9 for *R. electrica*, compared to 325.34 nmol\u00b7mL\u207b\u00b9\u00b7h\u207b\u00b9 for *B. japonicum*.", "Clinical Implications": "This study identifies the first nitrogen-fixing bacterium isolated from a leafhopper species, expanding the known range of nitrogen-fixing symbionts in Hemiptera. The finding that *R. electrica* colonizes the midgut and filter chamber of *R. dorsalis* and possesses all genetic machinery for nitrogen fixation suggests a potential nutritional symbiotic role, though direct evidence of nitrogen transfer to the host remains to be established. The relatively low nitrogenase activity (43.60 nmol\u00b7mL\u207b\u00b9\u00b7h\u207b\u00b9) compared to plant-associated nitrogen-fixing bacteria is consistent with patterns observed in other insect gut symbionts. The presence of additional nitrogen metabolism genes (assimilatory nitrate reduction, dissimilatory nitrate reduction, denitrification) suggests *R. electrica* may have versatile nitrogen utilization capabilities. These findings provide a foundation for understanding how gut microbes contribute to nitrogen nutrition in herbivorous insects and may inform future strategies for pest management or symbiotic manipulation in rice agroecosystems."}

PICO

PPOPULATION

Raoultella electrica strain Rd210413 isolated from the digestive tract of adult leafhopper Recilia dorsalis

IINTERVENTION

Growth on nitrogen-free Ashby medium and acetylene reduction assay to measure nitrogenase activity

OOUTCOME

R. electrica grew on nitrogen-free medium, showed nifH gene upregulation under nitrogen-depleted conditions, and exhibited nitrogenase activity of 43.60 nmol·mL⁻¹·h⁻¹

STUDY TYPE

other

SPECIALTY

entomology

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ORIGINAL PAPER

PMC10231068

A Novel Nitrogen-Fixing Bacterium Raoultella electrica Isolated from the Midgut of the Leafhopper Recilia dorsalis

Insects

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ABSTRACT

Simple Summary Nitrogen is a vital element that all living organisms require for growth and development. Although the atmosphere has an abundant supply of gaseous nitrogen, unfortunately, most organisms are unable to utilize it. Some microorganisms are capable of converting nitrogen gas to ammonia, which serves as a nitrogen source available to most organisms. Ammonia is subsequently utilized by organisms to synthesize a range of nitrogen-containing compounds that are necessary for life activities. For example, nitrogen-fixing microorganisms aid leguminous plants in obtaining the required nitrogen. Herbivorous insects face significant growth and development restrictions due to low total nitrogen content in their food. In this study, we isolated a nitrogen-fixing bacterium, Raoultella electrica , from the digestive tract of the leafhopper, Recilia dorsalis . R. electrica has all the nitrogen fixation genes and colonizes the gut lumen of leafhoppers. We further evaluated the growth rate of R. electrica in nitrogen-containing and nitrogen-free media and measured its nitrogenase activity through an acetylene reduction assay. These findings may be useful in studying the biological nitrogen fixation by gut microbes in host insects. Abstract Nitrogen is a crucial element for the growth and development of insects, but herbivorous insects often suffer from nitrogen nutrition deficiencies in their diets. Some symbiotic microorganisms can provide insect hosts with nitrogen nutrition through nitrogen fixation. Extensive research has clearly demonstrated the process of nitrogen fixation by symbiotic microorganisms in termites, while evidence supporting the occurrence and significance of nitrogen fixation in the diets of the Hemiptera is less conclusive. In this study, we isolated a strain of R. electrica from the digestive tract of a leafhopper, R. dorsalis , and found that it had nitrogen-fixing capabilities. Fluorescence in situ hybridization results showed that it was located in the gut of the leafhopper. Genome sequencing revealed that R. electrica possessed all the genes required for nitrogen fixation. We further evaluated the growth rate of R. electrica in nitrogen-containing and nitrogen-free media and measured its nitrogenase activity through an acetylene reduction assay. The findings of these studies could shed light on how gut microbes contribute to our understanding of nitrogen fixation.

CITATION EXPORT

Qiuyan Huang, Yilu Feng, Hong-Wei Shan et al.. A Novel Nitrogen-Fixing Bacterium Raoultella electrica Isolated from the Midgut of the Leafhopper Recilia dorsalis. Insects. (2023). https://doi.org/10.3390/insects14050431

AUTHORS

5 · 1 CENTRES

Qiuyan Huang, Yilu Feng, Hong-Wei Shan, Jian-Ping Chen, Wei Wu

State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-Products, Key Laboratory of Biotechnology in Plant Protection of Ministry of Agriculture and Zhejiang Province, Institute of Plant Virology, Ningbo University, Ningbo 315211, China; [email protected] (Q.H.);