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Applied and Environmental Microbiology, February 2001, p. 725-732, Vol. 67, No. 2
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.2.725-732.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Nitrogen Fixation Genes in an Endosymbiotic Burkholderia Strain

Daniela Minerdi,1 Renato Fani,2 Romina Gallo,2 Alessandra Boarino,1 and Paola Bonfante1,*

Dipartimento di Biologia Vegetale dell'Università di Torino and Centro di Studio sulla Micologia del Terreno del Consiglio Nazionale delle Ricerche, 10125 Turin,1 and Dipartimento di Biologia Animale e Genetica, Università degli Studi di Firenze, 50125 Florence,2 Italy

Received 26 July 2000/Accepted 4 December 2000

In this paper we report the identification and characterization of a DNA region containing putative nif genes and belonging to a Burkholderia endosymbiont of the arbuscular mycorrhizal fungus Gigaspora margarita. A genomic library of total DNA extracted from the fungal spores was also representative of the bacterial genome and was used to investigate the prokaryotic genome. Screening of the library with Azospirillum brasilense nifHDK genes as the prokaryotic probes led to the identification of a 6,413-bp region. Analysis revealed three open reading frames encoding putative proteins with a very high degree of sequence similarity with the two subunits (NifD and NifK) of the component I and with component II (NifH) of nitrogenase from different diazotrophs. The three genes were arranged in an operon similar to that shown by most archaeal and bacterial diazotrophs. PCR experiments with primers designed on the Burkholderia nifHDK genes and Southern blot analysis demonstrate that they actually belong to the genome of the G. margarita endosymbiont. They offer, therefore, the first sequence for the nif operon described for Burkholderia. Reverse transcriptase PCR experiments with primers designed on the Burkholderia nifH and nifD genes and performed on total RNA extracted from spores demonstrate that the gene expression was limited to the germination phase. A phylogenetic analysis performed on the available nifK sequences placed the endosymbiotic Burkholderia close to A. brasilense.

* Corresponding author. Mailing address: Dipartimento di Biologia Vegetale dell'Università di Torino, Viale P. A. Mattioli 25, 10125 Turin, Italy. Phone: 390116502927. Fax: 390116707459. E-mail: p.bonfante{at}csmt.to.cnr.it.

Applied and Environmental Microbiology, February 2001, p. 725-732, Vol. 67, No. 2
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.2.725-732.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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