Transfer of Rhizobium leguminosarum nodD1 and nodG, and Azotobacter chroococcum nifH1 and nifV, into Bacillus megaterium using physicochemical and molecular techniques.

Talabani, Shahen Kamil

doi:10.58928/ku25.16211

	Transfer of Rhizobium leguminosarum nodD1 and nodG, and Azotobacter chroococcum nifH1 and nifV, into Bacillus megaterium using physicochemical and molecular techniques.
Kirkuk University Journal for Agricultural Sciences (KUJAS)
Volume 16, Issue 2, June 2025, Pages 79-89 PDF (1.21 M)
Document Type: Research Paper
DOI: 10.58928/ku25.16211
Author
Shahen Kamil Talabani^*
High Education- University of Sulaimani- Engineering Sciences College
Abstract
This study involved two techniques for transferring important genes related to nitrogen fixation into Bacillus megaterium. Initially, morphological characteristics and molecular properties were analyzed to identify isolated bacteria. This process led to the isolation of Bacillus megaterium, Azotobacter chroococcum, and Rhizobium leguminosarum using a specific growth medium. Morphologically, the results showed that all the bacterial colonies were circular, convex, and smooth. A. chroococcum was creamy color, while B. megaterium and R. leguminosarum appeared white. The application of molecular approaches relied on identifying chromosomal DNA, specifically the nifH1 and nifV genes of A. chroococcum, and the plasmid DNA including the nodD1 and nodG genes of R. leguminosarum. Additionally, genomic DNA and two primers of B. megaterium were included. The nodD1 and nodG genes were successfully transferred from R. leguminosarum to B. megaterium, resulting in 46 colonies on agar plates of Sperber medium that contained tetracycline and ampicillin as genetic markers. Furthermore, the nifH1 and nifV genes were transferred into the transformant B. megaterium from A. chroococcum via a conjugation mechanism, leading to the growth of 170 colonies on plates of Sperber's agar medium containing erythromycin and ampicillin as genetic markers. To confirm the success of these transfers, molecular analyses were conducted to detect the nodD1 and nodG genes in the transformant B. megaterium, as well as the nifH1 and nifV genes in the conjugant B. megaterium. This was achieved using appropriate primers and PCR conditions for all the genes, followed by gel electrophoresis, which demonstrated the successful completion of both techniques.
Keywords
Nitrogen fixation genes; Bacterial transformation; Bacterial conjugation; PCR; Gel electrophoresis

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