INSTITUTO DE BIOTECNOLOGIA Y BIOLOGIA MOLECULAR
Unidad Ejecutora - UE
congresos y reuniones científicas
In Azospirillum brasilense Cd a mutation in fmlB results in a truncated polar flagellum, lack of swimming, and altered attachment to maize roots
FERNANDO ROSSI; WALTER FERRARI; MARINA CASTRO; SONIA FISCHER; ANALIA PRINCIPE; MARIANO PISTORIO; EDGARDO JOFRE
Congreso; XXV Reunión Latinoamericana de Rizobiologia (XXV RELAR).; 2011
Azospirillum brasilense is a motile Gram-negative bacterium that can adapt its flagellation to different environments. A mixed flagellation system allows movement of bacteria in liquid media (swimming) by a polar flagellum, present in all growth conditions, while the synthesis of lateral flagella is induced to allow the movement on solid surfaces (swarmming). The structural component of the lateral flagella in A. brasilense Sp7, the flagellin Laf1, has been previously characterized. A laf1 mutant completely loses its capability to swarm on solid media but the absence of lateral flagella does not seem to influence bacterial adhesion to wheat roots. In contrast, the structural gene of the polar flagellum has not yet been identified. The polar flagellin protein, however, was shown to play a role in the adhesion of Azospirillum to plant roots. Although previous studies have confirmed that the polar flagellin is glycosylated in A. brasilense, the genes required for such glycosylation as well as the role of this modification, have not been described. The best-characterized example is the flagellin of Camplylobacter spp. which is decorated with as many as 19 O-linked glycans contributing near of 10% to the weight of flagellin. Most strains appear to carry the genes for synthesis of two distinct nine-carbon sugars that decorate flagellin: pseudaminic acid (PseAc) and an acetamidino form of legionaminic acid (LegAm). The PseB/PseC proteins, encoded by Cj1293 and Cj1294, respectively that function as dehydratase/aminotransferase have been shown essential enzymes for the synthesis of PseAc in Campylobacter jejuni 81-176. Mutations in either Cj1293 or Cj1294 resulted in a non-motile phenotype with accumulation of intracellular flagellin, impaired autoagglutination, and changes in attachment and/or invasion of intestinal ephytelium. In this study we generated a Tn5 mutant of A. brasilense Cd severely impaired in swimming. Genetic characterization allowed to the identification of the flmB gene whose deduced amino acid sequence showed high similarity and identity to a pyridoxal phosphate-depend aminotransferase of Caulobacter crescentus. The flmB gene is part of an operon with flmA which encodes a dehydratase/reductase that uses NAD+ as cofactor and UDP-glucose as specific substrate. The putative role of both proteins is related to changes of the flagellin before subunit assembly within the flagellar filament. Analysis by transmission electron microscopy revealed that the flmB mutant strain produced a truncated polar flagellum compared to the wild-type strain. Moreover, the attachment of the flmB mutant strain to maize roots was significantly lower than the wild-type strain in competition assays suggesting the participation of the polar flagellum during the establishment of the plant-bacteria interaction. Complementation assays of mutant flmB with the wild-type flmB allele fully restored the wild-type phenotype indicating the absence of polar effects of the Tn5 insertion on flmB downstream genes.