Abstract
Sinorhizobium fredii is well known for its ability to establish symbiosis with diverse legumes such as Glycine max (soybean, determinate nodules) and Cajanus cajan (pigeon pea, indeterminate nodules). In order to make screening of S. fredii genes related to symbiosis cost-effective, we constructed a large Tn5 insertion mutant library of S. fredii CCBAU45436 using the signature-tagged mutagenesis (STM) technique. This STM library contains a total of 25,500 independent mutants distributed in 17 sublibraries tagged by corresponding distinct DNA bar-code sequences. After the pilot screening of 255 mutants in 15 batches, Tag85-4, Tag4-17, Tag4-11 and Tag10-13 were found to have attenuated competitiveness (0–30 % in nodule occupation) compared to the wild-type strain when inoculated on soybean. Further characterization of these mutants suggests that Tag4-11 (a pyrC mutant) and Tag10-13 (a nrdJ mutant) are defective in establishing symbiosis with soybean. The pyrC mutant induced uninfected pseudonodules while the nrdJ mutant formed significantly more nodules containing bacteroids with poor persistence ability. When these two mutants were tested on pigeon pea, host-specific symbiotic defects were found. These results demonstrated the STM library as a valuable resource for identifying S. fredii genes relevant to symbiosis.
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Acknowledgments
We thank Dr. Anke Becker and Dr. Hisayuki Mitsui for providing pG18Mob2 and pTnMod-OΩ, respectively. We also thank Dr. J. Peter W. Young for polishing the language. This work was supported by National Basic Research Program of China (973 Program 2015CB158300), Innovative Project of State Key Laboratory of Agrobiotechnology (2014SKLAB4-1), and Chinese Universities Scientific Fund for graduate students (2012YJ035).
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Communicated by Shuang-Jiang Liu.
Dan Wang and Yuan Chun Wang have contributed equally to this work.
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Wang, D., Wang, Y.C., Wu, L.J. et al. Construction and pilot screening of a signature-tagged mutant library of Sinorhizobium fredii . Arch Microbiol 198, 91–99 (2016). https://doi.org/10.1007/s00203-015-1161-9
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DOI: https://doi.org/10.1007/s00203-015-1161-9