Phase Variation and Genomic Architecture Changes in Azospirillum

Azospirillum is a plant growth-promoting rhizobacterium associated with roots of monocots, including important crops, such as wheat, corn, and rice. Both in greenhouse and in field trials, Azospirillum was shown to exert beneficial effects on plant growth and crop yields, under various soil and climatic conditions (15). The actual benefit from biological nitrogen fixation has been questioned, and plant growth promotion by Azospirillum seems to be due mainly to production of phytohormones (9, 15). The most abundant phytohormone produced is indole-3-acetic acid, allowing an increase in the number of lateral roots and root hairs; this results in a higher absorption of water and minerals from the soil (9). Bacterial populations, especially in soil or in the rhizosphere, have to endure fluctuating environmental conditions. Bacteria have evolved different strategies to adapt to these environments. Phase variation is one adaptive process by which bacteria undergo frequent, usually reversible phenotypic changes resulting from genetic or epigenetic alterations at specific genetic loci (29). This process is used by several bacterial species to generate intrapopulation diversity that increases bacterial fitness and is important in niche adaptation or to escape host defenses (reviewed in references 46 and 55). In contrast to spontaneous mutations, which occur at a frequency of approximately 10−8 to 10−6 mutations per growing cell per generation, phase variation occurs at frequencies higher than 10−5 switches per cell per generation (29). Various mechanisms control phase variation. These include DNA inversion or duplication, deletion, transposition, homologous recombination, slipped-strand mispairing, and differential methylation (reviewed in reference 55). Phase variation in pathogenic bacteria—for example, switching of type IV pili in Neisseria gonorrhoeae (27), differential expression of surface layer proteins in Campylobacter fetus (16), and loss of virulence in the phytopathogen Ralstonia solanacearum (43)—has been extensively studied. However, phase variation is not restricted to pathogenic bacteria. Indeed, it also occurs during rhizosphere colonization of various plants by several strains of beneficial plant-associated Pseudomonas (1, 48). For instance, the regulation of biocontrol traits (production of antifungal metabolites, chitinases, and biosurfactants) by phase variation was reported for Pseudomonas spp. strains (54). Azospirillum lipoferum 4B, a strain isolated from a rice rhizosphere, generates in vitro at high frequencies (10−4 to 10−3 per cell per generation) a stable phase variant named 4VI (3, 30). Variant colonies are readily distinguishable from wild-type colonies by the differential absorption of dyes incorporated into the growth medium. The 4VI variant exhibits pleiotropic modifications; it gained assimilation of certain sugars but lost the ability to assimilate other sugars (3), to reduce triphenyl tetrazolium chloride, to bind some dyes, to swim (4), and to reduce nitrous oxide (our unpublished results). A. lipoferum 4T, a nonswimming strain displaying all of the features of the 4VI variant, and strain A. lipoferum 4B have been isolated simultaneously from rice rhizosphere at the same frequency (8). A. lipoferum 4T retains the ability to efficiently colonize rice roots (2). Like 4VI, A. lipoferum 4T was found to be genetically very close to A. lipoferum 4B (3, 8, 28), suggesting that A. lipoferum 4T could in fact be a 4VI variant of strain 4B generated within the soil ecosystem. We recently showed that inactivating recA in strain 4B resulted in a higher frequency of generation of variants (57), contrasting with many studies of other bacteria showing either no effect or the abolition of phase variation process in recA mutants. This finding suggests the possibility that phase variation is accompanied by genetic rearrangements. As of now, the molecular mechanism underlying these nonreversible changes in A. lipoferum 4B remains to be determined. In this study, the objective was to determine whether genomic rearrangements take place during phase variation of strain 4B and related Azospirillum strains. The current work shows that genomic rearrangements are concomitant with phase variation in three species of Azospirillum.