Abstract
Conventional systems used to tag and transfer symbiotic plasmids (pSyms) of rhizobial strains are based in mutagenesis with transposons. In those processes, numerous clones must be analyzed to find one of them with the transposon inserted in the pSym. Following this strategy, the insertion might interrupt a gene that can affect the symbiotic phenotype of the bacteria tagged. Here, we have developed a new system based in homologous recombination that generates Sinorhizobium fredii strains with pSyms tagged by the insertion of a suicide vector which harbor a truncated copy of S. fredii HH103 nodZ gene, a mob site, and a kanamycin-resistant gene. When it is introduced by conjugation in a S. fredii strain, the vector integrates in pSym by only one recombination event. This pSym tagged can be transferred in matting experiments to other strains in the presence of a helper plasmid. Following this method, we have tagged several strains and transferred their pSyms to a recipient strain demonstrating the potential of this new system.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Oldroyd GE (2013) Speak, friend, and enter: signaling systems that promote beneficial symbiotic associations in plants. Nat Rev Microbiol 11:252–263
Hoykaas PJJ, van Brussel AAN, den Dulk-Ras H et al (1981) Sym plasmid of Rhizobium trifolii expressed in differentrhizobial species and Agrobacterium tumefaciens. Nature 291:351–353
De Jong TM, Brewin NJ, Phillips DA (1981) Effects of plasmid content in rhizobium leguminosarum on pea nodule activity and plant growth. J Gen Microbiol 124:1–7
Brewin NJ, Wood EA, Young JPW (1983) Contribution of the symbiotic plasmid to the competitiveness of Rhizobium leguminosarum. J Gen Microbiol 129:2973–2977
Djordjevic MA, Zurkowski W, Shine J et al (1983) Sym plasmid transfer to various symbiotic mutants of Rhizobium trifolii, R. leguminosarum and R. meliloti. J Bacteriol 156:1035–1045
Simon R, Priefer U, Pühler A (1983) Vector plasmids for in vivo and in vitro manipulations of Gram-negative bacteria. In: Pühler A (ed) Molecular genetics of the bacteria-plant interaction. Springer, Berlin, p 98
Simon R (1984) High frequency mobilization of gram-negative bacterial replicons by the in vivo constructed Tn5-Mob transposon. Mol Gen Genet 196:413–420
Simon R, Quandt J, Klipp W (1989) New derivatives of transposon Tn5 suitable for mobilization of replicons, generation of operon fusions and induction of genes in Gram- negative bacteria. Gene 80:161–169
Hynes MF, Quandt J, O’Connell MP et al (1989) Direct selection for curing and deletion of rhizobium plasmids using transposons carrying the Bacillus subtilis sacB gene. Gene 78:111–120
Schäfer A, Tauch A, Jäger W et al (1994) Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18 and pK19: selection of defined deletions in the chromosome of Corynebacterium glutamicum. Gene 145:69–73
Espuny MR, Ollero FJ, Bellogín RA et al (1987) Transfer of the Rhizobium leguminosarum biovar trifolii plasmid pRtr5a to a strain of Rhizobium sp. that nodulates on Hedysarumcoronarium. J Appl Bacteriol 63:13–20
Simon R, Priefer U, Puhler A (1983) A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram negative bacteria. Biotechnol 1:784–791
Figurski DH, Helinski DR (1979) Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci U S A 76:1648–1652
Noreen S, Schlaman HRM, Bellogín RA et al (2003) Alfalfa nodulation by Sinorhizobium fredii does not require sulfated nod-factors. Func Plant Biol 30:1219–1232
Buendía-Clavería AM, Chamber M, Ruiz-Sainz JE (1989) A comparative study of the physiological characteristics, plasmid content and symbiotic properties of different Rhizobium frediistrains. Syst Appl Microbiol 12:203–209
Keyser HH, Bohlool BB, Hu TS et al (1982) Fast-growing rhizobia isolated from root nodules of soybean. Science 215:1631–1632
Yang SS, Bellogín RA, Buendía A et al (2001) Effect of pH and soybean cultivars on the quantitative analyses of soybean rhizobia populations. J Biotechnol 91:243–255
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor
Beringer JE (1974) R factor transfer in Rhizobium leguminosarum. J Gen Microbiol 84:188–198
Morrison NA, Hau CY, Trinick MJ (1983) Heat curing of a Sym plasmid in a fast-growing Rhizobium sp. that is able to nodulate legumes and the nonlegume Parasponia sp. J Bacteriol 153:527–531
Eckardt T (1978) A rapid method for the identification of plasmid deoxyribonucleic acid in bacteria. Plasmid 1:584–588
Acknowledgments
This work was funded by the Spanish Ministry of Science and Innovation, grant number PID2019-107634RB-I00, and supported by FEDER funds, grant number FEDER-US 1259948.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2024 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Cutiño, A.M., del Carmen Sánchez-Aguilar, M., Ruiz-Sáinz, J.E., del Rosario Espuny, M., Ollero, F.J., Medina, C. (2024). A Novel System to Selective Tagging of Sinorhizobium fredii Symbiotic Plasmids. In: Medina, C., López-Baena, F.J. (eds) Host-Pathogen Interactions. Methods in Molecular Biology, vol 2751. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3617-6_17
Download citation
DOI: https://doi.org/10.1007/978-1-0716-3617-6_17
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-3616-9
Online ISBN: 978-1-0716-3617-6
eBook Packages: Springer Protocols