Lipopolysaccharide Precursor Mutants Disrupt Unipolar Polysaccharide Adhesin Synthesis and Cell Surface Functions in Agrobacterium tumefaciens

Agrobacterium tumefaciens is a facultative phytopathogen and causative agent of crown gall disease, capable of attaching to surfaces via a polarly localized unipolar polysaccharide (UPP) adhesin. The UPP is composed of two distinct forms, one characterized by N -acetylglucosamine residues (UPP _GlcN ) and the other by N -acetylgalactosamine residues (UPP _GalN ). Two genes with presumptive roles in dTDP-L-rhamnose biosynthesis, rfbA and rfbD, were identified in a transposon screen to be involved specifically in UPP _GalN biosynthesis. Examination of independent rfbA and rfbD mutations validates the transposon mutant phenotypes, with UPP _GalN specific defects observed as well as general attachment defects and UPP-mediated cellular aggregation across all rfb mutants. Additionally, despite retaining flagella, mutations in the rfb genes impart flagellar motility defects. Suppressor mutations that rescue the non-motile Δ rfbD phenotype disrupt the remaining rfb genes ( rfbA , rfbB, and rfbC ), the phosphoglucomutase exoC and a putative glycosyl transferase ATU-RS21610. Single deletion mutants in the dTDP-L-rhamnose pathway also have hallmarks of compromised outer membrane integrity, such as increased sensitivity to high-molecular weight antibiotics and increased expression of target genes for the widely conserved ChvG-ChvI two component system outer membrane stress response. Thus, defects in the dTDP-L-rhamnose pathway cause multiple cell surface deficiencies, resulting in outer membrane stress, impaired flagellar motility, defective UPP production, and irregular adhesion.