R. Carreño-López 1,2, L. Pereg-Gerk1, B.E. Baca2, C. Elmerich1
Microbiologic et Environnement, Instituí Pasteur, 25-28 rue Dr. Roux, 75724 Paris Cedex, France
2Centro de Microbiología, Universidad Autónoma de Puebla, Apdo. Postal 1622, C.P. 72000, Puebla, Pue. México
Azospirillum bacteria live in close association with plants roots. The attachment of the bacterium to the root surface is essential for the establishment of an efficient colonization. Bacteria are highly motile in liquid media by way of a single polar flagellum responsible for swimming. Motility by swarming on semisolid media is made possible by numerous lateral flagella (Michiels et al. 1991). The aim of this work was to characterize a locus affecting swarming motility of strain Sp7S, a derivative of A. brasilense Sp7, Sp7S which was also shown to carry a mutation in capsular polysaccharide production (gene flcA) (Pereg-Gerk et al. 1998) and was impaired in surface root colonization. A gene bank of the strain Sp7 was introduced by conjugation into Sp7-S and a plasmid clone termed pAB7115 that restored swarming was isolated. The physical map of the plasmid was established and the locus responsible for the complementation was identified after mutagenesis with Tn5. Determination of the nucleotide sequence of the complementing region resulted in the identification of two genes.
The deduced translation product of the first one was highly similar to a phosphoribosyl-aminoimidazol carboxylase, encoded by purK, a gene involved in purine biosynthesis. Inactivation of purK did not lead to auxotrophy as described in Escherichia coli and Salmonella typhimurium (Tiedeman et al. 1989). The second one (ORF1) showed similarity with proteins of unknown function described in E. coli, Aqualifex aeolicus and Synechocystis spp., and a low similarity was found with a diguanylate cyclase motif from Acetobacter xylinus (24%). Disruption of both genes, in wild type Sp7, led to prototrophic mutant strains with decreased swarming properties, although both mutants showed lateral flagella. It is hypothesized that ORF1 and purK genes play a role in controlling the swarming motility possibly in the functioning of the lateral flagella. Root surface colonization of wheat was studied. No major difference was found between the wild type and the two mutants, although an effect on long-term colonization cannot be excluded, because lateral flagella enable bacterial movement along the root surface.
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