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Phase Variation and Genomic Architecture Changes in Azospirillum

Ludovic Vial, Céline Lavire, Patrick Mavingui, Didier Blaha, Jacqueline Haurat, Yvan Moënne-Loccoz, René Bally, and Florence Wisniewski-Dyé^*

UMR CNRS 5557 Ecologie Microbienne, IFR 41 Bio-Environnement et Santé, Université Lyon 1, 43 Boulevard du 11 Novembre 1918, 69622 Villeurbanne Cedex, France

Received 12 April 2006/ Accepted 25 May 2006

The plant growth-promoting rhizobacterium Azospirillum lipoferum 4B generates in vitro at high frequency a stable nonswimming phase variant designated 4V_I, which is distinguishable from the wild type by the differential absorption of dyes. The frequency of variants generated by a recA mutant of A. lipoferum 4B was increased up to 10-fold. The pleiotropic modifications characteristic of the phase variant are well documented, but the molecular processes involved are unknown. Here, the objective was to assess whether genomic rearrangements take place during phase variation of strain 4B. The random amplified polymorphic DNA (RAPD) profiles of strains 4B and 4V_I differed. RAPD fragments observed only with the wild type were cloned, and three cosmids carrying the corresponding fragments were isolated. The three cosmids hybridized with a 750-kb plasmid and pulse-field gel electrophoresis analysis revealed that this replicon was missing in the 4V_I genome. The same rearrangements took place during phase variation of 4BrecA. Large-scale genomic rearrangements during phase variation were demonstrated for two additional strains. In Azospirillum brasilense WN1, generation of stable variants was correlated with the disappearance of a replicon of 260 kb. For Azospirillum irakense KBC1, the variant was not stable and coincided with the formation of a new replicon, whereas the revertant recovered the parental genomic architecture. This study shows large-scale genomic rearrangements in Azospirillum strains and correlates them with phase variation.

Present address: UMR CNRS INSA 5122 Microbiologie et Génétique, IFR 41 Bio-Environnement et Santé, Université Lyon 1, 43 Boulevard du 11 Novembre 1918, 69622 Villeurbanne Cedex, France.

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