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Applied and Environmental Microbiology, January 2005, p. 460-466, Vol. 71, No. 1
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.1.460-466.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Non-Frankia Actinomycetes Isolated from Surface-Sterilized Roots of Casuarina equisetifolia Fix Nitrogen{dagger}

María Valdés,1 Néstor-Octavio Pérez,1,{ddagger} Paulina Estrada-de los Santos,2 Jesús Caballero-Mellado,3 Juan José Peña-Cabriales,2 Philippe Normand,4 and Ann M. Hirsch5*

Departamento de Microbiología, Escuela Nacional de Ciencias Biológicas, I. P. N., Plan De Ayala y Carpio México, D.F.,1 Centro de Investigación sobre Fijación de Nitrógeno, UNAM, Cuernavaca,3 Departamento de Biotecnología y Bioquímica, CINVESTAV, Irapuato, México,2 Ecologie Microbienne UMR CNRS 5557, Université de Lyon, Villeurbanne, France,4 Department of Molecular, Cell, and Developmental Biology and Molecular Biology Institute, University of California, Los Angeles, California5

Received 29 April 2004/ Accepted 18 August 2004

Based on partial 16S sequences, we previously described a novel group of nonsymbiotic, acetylene reduction activity-positive actinomycetes which were isolated from surface-sterilized roots of Casuarina equisetifolia growing in Mexico. An amplified rRNA restriction analysis confirmed that these actinomycetes are distinct from Frankia, a finding substantiated by a 16S rRNA gene phylogenetic analysis of two of the Mexican isolates. Further support for these actinomycetes being separate from Frankia comes from the very low DNA-DNA homology that was found. Nevertheless, the Mexican isolates may be diazotrophs based not only on their ability to grow in N-free medium and reduce acetylene to ethylene but also on the results from 15N isotope dilution analysis and the finding that a nifH gene was PCR amplified. A comparison of the nifH sequences from the various isolates showed that they are closely related to nifH from Frankia; the similarity was 84 to 98% depending on the host specificity group. An analysis of complete 16S rRNA gene sequences demonstrated that the two strains analyzed in detail are most closely related to actinobacteria in the Thermomonosporaceae and the Micromonosporaceae.

* Corresponding author. Mailing address: Department of Molecular, Cell, and Developmental Biology, University of California, 405 Hilgard Ave., Los Angeles, CA 90095-1606. Phone: (310) 206-8673. Fax: (310) 206-5413. E-mail: ahirsch{at}ucla.edu.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

{ddagger} Present address: Centers for Disease Control and Prevention, Atlanta, GA 30333.

Applied and Environmental Microbiology, January 2005, p. 460-466, Vol. 71, No. 1
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.1.460-466.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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