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The Rhizobium leucaenae CFN 299 pSym plasmid contains genes expressed in free life and symbiosis, as well as two replication systems

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Abstract

The alpha-protobacterium Rhizobium leucaenae CFN 299 is able to nodulate and fix nitrogen in symbiosis with a wide range of legumes, including Phaseolus vulgaris (common bean). Strain CFN 299 contains a 500-kb symbiotic plasmid (pSym) that encodes genes required for nodulation and nitrogen fixation as well as many genes whose function is unknown. In this work, we characterized the transcriptional expression of 16 pSym genes in common bean nodules and in free-living cells grown in culture. A functionally diverse group of genes were expressed during discrete stages of the symbiosis or in free-living cells. These included genes whose products are involved in nodulation and nitrogen fixation, carbon metabolism, vitamin synthesis, sulfur utilization, conjugation, transposition and DNA replication. We also examined the functionality of two replication systems encoded on pSym and found that repABC, but not repC2, is required for pSym replication.

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Acknowledgments

We thank M.A. Rogel-Hernández, M. F Mora, F. J. Santana, P. Gaytan, E. Bustos, S. Becerra, J. Yañez, M. L. Zavala, L. Medina-Aparicio, A. Vazquez and J. E. Rebollar-Flores for technical help. This work was supported by a grant from the Dirección General de Asuntos del Personal Académico, DGAPA/UNAM IN203215, to I.H.L.

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Authors and Affiliations

  1. Departamento de Microbiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Av. Universidad 2001, Cuernavaca, Morelos, 62210, Mexico

    Aurora Gamez-Reyes, Noé Becerra-Lobato & Ismael Hernández-Lucas

  2. Laboratorio de Fisiología Molecular de Plantas, Centro de Investigación en Biotecnología, Universidad Autónoma del Estado de Morelos, Av. Universidad 2001, Cuernavaca, Morelos, 62209, Mexico

    José Augusto Ramírez-Trujillo

  3. Centro de Ciencias Genómicas, Universidad Nacional Autónoma de México, Cuernavaca, Morelos, 62210, Mexico

    Esperanza Martínez-Romero & Michael F. Dunn

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  1. Aurora Gamez-Reyes
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  2. Noé Becerra-Lobato
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  3. José Augusto Ramírez-Trujillo
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  4. Esperanza Martínez-Romero
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  6. Ismael Hernández-Lucas
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Correspondence to Ismael Hernández-Lucas.

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Aurora Gamez-Reyes, Noé Becerra-Lobato and José Augusto Ramírez-Trujillo contributed equally to this work.

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Fig. S1

Representative ethidium bromide-stained Eckhardt gels showing Rhizobium leucaenae plasmid profile. R. leucaenae CFN 299, containing 1.5 MB megaplasmid, the 500 kb symbiotic (pSym) plasmid, plasmid B of 240 kb and plasmid A 185 kb. CFN 299-10 lacking pB and 300 kb fragment of pSym. The plasmid profile of CFN 299 derivatives generated in this work is also shown (DOCX 257 kb)

Fig. S2

Autoradiogram of Southern blot of HindIII digested genomic DNA hybridized with pVO155 probe. CFN 299 and CFN 299-10 shows no hybridization signal. The CFN 299 derivatives generated in this work only present a single hybridization band (DOCX 464 kb)

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Gamez-Reyes, A., Becerra-Lobato, N., Ramírez-Trujillo, J.A. et al. The Rhizobium leucaenae CFN 299 pSym plasmid contains genes expressed in free life and symbiosis, as well as two replication systems. Ann Microbiol 67, 263–273 (2017). https://doi.org/10.1007/s13213-017-1257-3

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