Genes · 5 authors, 5 centres
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This study sequenced the genome of Rhizobium sp. '10ap3', a nitrogen-fixing bacterium isolated from pigeonpea root nodules. The genome lacks common nodulation (nodABC) and nitrogen fixation (nif) genes, suggesting an alternative purine-based pathway may initiate symbiosis with pigeonpea. The strain's genome also contains genes for stress tolerance and iron acquisition, making it a candidate for commercial bio-inoculant development.
**Background:** Pigeonpea (Cajanus cajan) is a drought-tolerant grain legume that improves soil fertility through symbiotic nitrogen fixation with soil rhizobia. While pigeonpea associates with diverse rhizobial strains, significant variation in nitrogen fixation efficiency exists among compatible strains, and the genetic basis for this variation remains unclear. The symbiotic process involves key bacterial genes (nod, nif, fix) that control nodulation and nitrogen fixation. This study aimed to sequence the genome of a pigeonpea-compatible rhizobial strain, determine its genome size, and identify key functional genes.
**Methods:** The rhizobial strain Rhizobium sp. '10ap3', previously isolated from pigeonpea root nodules in South Africa and shown to have superior symbiotic efficiency, was revived on yeast mannitol agar. DNA was extracted using the Wizard genomic DNA extraction kit. Sequencing was performed on the Illumina MiSeq platform using a 2 × 300 bp paired-end strategy with the MiSeq Reagent Kit v3. Adapter sequences and low-quality bases were trimmed, and overlapping pairs were merged using CLC Genomic Workbench version 8.5.1, resulting in 8,265,062 reads for de novo assembly. Gene annotations were performed with the NCBI Prokaryotic Genome Annotation Pipeline.
**Key Results:** Sequencing generated 10,364,436 raw sequence reads, yielding 8,265,062 trimmed reads. The genome of strain '10ap3' is a large circular chromosome of 6,297,373 bp with a G+C content of 60.0%. The assembly consisted of 36 contigs (including scaffolds), with a maximum scaffold size of 876,087 bp. The genome contains 6,013 total genes, of which 5,961 are coding sequences (99.13%), and 5,833 protein-coding genes could be assigned specific functions. The genome includes 3 ribosomal RNA operons, 45 tRNAs, 4 ncRNAs, and 128 pseudogenes. No plasmids were detected. Notably, the genome lacks common nodulation genes (nodABC) and nitrogen fixation genes (nif). Only one nodulation-related gene, nolR (a DNA-binding transcription factor), was identified on contig 12. Genes for purine biosynthesis precursors—inosine-5-monophosphate, adenosine monophosphate nuclease, and adenylosuccinate—were present on the chromosome. Additional genes identified include those involved in nitrogen metabolism, stress response, phosphorus metabolism, iron acquisition, siderophore aerobactin synthesis, and auxin biosynthesis (tryptophan synthase α and β chains).
**Clinical Implications:** This study provides genomic evidence that the common nodABC genes are not universally required for successful symbiosis, as strain '10ap3' demonstrates symbiotic efficiency with pigeonpea despite lacking these genes. The presence of purine biosynthesis genes suggests an alternative nodulation pathway using purine derivatives to trigger nodule formation. The strain's genome also contains genes for abiotic stress tolerance and iron acquisition, indicating potential for survival in challenging soil conditions. These findings support the development of '10ap3' as a commercial bio-inoculant for pigeonpea, particularly in iron-deficient or stress-prone soils. Future research should evaluate symbiotic efficiency under various soil moisture stress conditions and compare genome composition across distinct nitrogen-fixing genera associated with pigeonpea.