FlmA and FlmB influence polar flagellum structure, flagellin glycosylation and surface polysaccharides of Azospirillum brasilense Cd

ROSSI, F; FERRARI, W; MEDEOT, D; LIAUDAT, J.P; MORALES, G; FISCHER, S; PISTORIO, M; JOFRÉ, E

La Falda

Workshop; II Workshop Latinoamericano sobre PGPR; 2014

Bacteria belonging to genus Azospirillum, a model for studying associative plant-microbe interactions, have one polar flagellum - made up of repeating flagellin subunits (FlaA)- when grown in liquid media and use a distinct lateral flagella system (Laf) for swarming motility, over solid surfaces. FlaA is a glycoprotein which O-linked polysaccharide has the same monosaccharide residues than O-LPS. The genes required for such glycosylation, as well as, the role of this modification in A. brasilense, have not been described yet. Here, we demonstrated the involvement of the locus flmAB in FlaA O-glycosylation, polymerization and assembly of the polar flagellum as well as in polysaccharide biosynthesis. Motility test: 2 μl of bacterial culture at OD600nm= 1.0 was applied to the center of motility agar (0,3%) or swarm agar (0,6%) and incubated for 48 h. AFM Imaging: Bacteria were harvested by centrifugation, cells were suspended in 100 μl dH2O and then deposited on a mica surface. Samples were dried 1 h at 30 oC before imaging with an atomic force microscope Agilent 5550, equipped with a 90 μm multipurpose scanner. Mass Spectrometry: Flagellins were purified and separated by 2D-PAGE. Protein bands were excised, in-gel digested using trypsin and analyzed by MALDI TOF/TOF. LPS/EPS isolation and analysis: EPSs were precipitated from 2 days-old culture supernatants with three volumes of cold ethanol and quantified by the anthrone method. LPS were isolated using the classic phenol-water method and were separated by SDS-PAGE. In A. brasilense, mutations in either flmA (dehydratase) or flmB (aminotransferase) resulted in a severely impaired swimming motility. The phenotype of swarming motility was also affected in the flmA and flmB mutant strains. Swarming motility was induced in both mutant strains whereas in the wild type strain this motility phenotype was not detected under the tested conditions. AFM analysis of cells from mutant strains, grown in liquid medium, evidenced an extremely short polar flagelum in contrast to the observed for the wild type strain. Furthermore, mass spectrometry analysis of purified flagellin from the wild type strain showed two O-glycosylated variants FLaA1 and FlaA2. In contrast, in the mutant strains no glycosylation signals were detected suggesting that the products of these genes are required for flagellin glycosylation and polar flagellum assembly. Analysis of surface polysaccharides from flmA and flmB mutant strains showed alterations in the LPS profile and overproduction of EPS compared to the wild type strain, suggesting that the products of flmA and flmB are also involved in the biosynthetic pathways of these polysaccharides. Based on our findings, we conclude that the products of flmA and flmB from A. brasilense Cd are required for flagellin glycosylation, polar flagellum assembly, and are also involved in surface polysaccharides biosynthesis. Probably, the induction of swarming motility observed in the flmA and flmB mutant strains be the consequence of the lack of polar flagellum.