Abstract
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.
Highlights
Azospirillum is a plant growth-promoting rhizobacterium associated with roots of monocots, including important crops, such as wheat, corn, and rice
Since phase variation on strain A. lipoferum 4B has been extensively studied and the frequency of variants generated by a 4BrecA mutant was increased up to 10-fold, we focused first on genome rearrangements taking place when 4B switches to 4VI
Phase variation and DNA rearrangement phenomena in Azospirillum strains were investigated by using media containing dyes, plasmid and pulsed-field gel electrophoresis (PFGE) profiles, and random amplified polymorphic DNA (RAPD) and Southern hybridization of genomic DNA patterns
Summary
Azospirillum is a plant growth-promoting rhizobacterium associated with roots of monocots, including important crops, such as wheat, corn, and rice. 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). Phase variation is not restricted to pathogenic bacteria It occurs during rhizosphere colonization of various plants by several strains of beneficial plant-associated Pseudomonas [1, 48]. Like 4VI, A. lipoferum 4T was found to be genetically very close to A. lipoferum 4B [3, 8, 28], suggesting
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