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 ΔrfbD 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.IMPORTANCEBiofilms are clinically and industrially relevant in many different contexts, with substantial health impacts and monetary costs. Surface attachment and motility are essential components necessary for bacterial biofilm formation. Many microbes utilize specific surface appendages or structures to facilitate attachment and motility. This study probes the process of surface attachment and subsequent biofilm formation in Agrobacterium tumefaciens and has revealed connections between the creation of polysaccharide precursors required for biofilm formation and outer membrane functions such as motility and membrane integrity. These findings broaden our understanding of the complex interactions between outer membrane surface functions and how disruption of relevant precursor pools can have multifaceted impacts that may represent useful targets for new antimicrobial approaches.