Many early molecular events in symbiotic infection are documented, yet factors enabling Rhizobium to progress within the plant-derived infection thread, penetrate the root cortical cells, and ultimately survive within the intra-cellular symbiosome compartment as mature nitrogen-fixing bacteroids are poorly understood. Rhizobial surface polysaccharides (SPS), including the capsular polysaccharides (K-antigens), exist in close proximity to plant-derived membranes throughout the infection process. SPSs are essential for bacterial survival, adaptation, and as potential determinants of nodulation and/or host specificity, yet relatively few studies have examined the role of K-antigens in these events. We constructed a mutant that lacks genes essential for the production of the K-antigen strain-specific sugar precursor, pseudaminic acid in the exceptionally broad host range Rhizobium sp. NGR234. The complete structure of the K-antigen of strain NGR234 was established, and it consists of disaccharide repeating units of glucuronic and pseudaminic acid having the structure ®4)--D-glucuronic acid-(1®4)--5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-nonulosonic acid-(2®. Deletion of three genes located in the rkp-3 gene cluster, rkpM, rkpN and part of rkpO, abolished pseudaminic acid synthesis, yielding a mutant in which the strain-specific K-antigen was totally absent: other surface glycoconjugates, including the lipopolysaccharides, exopolysaccharides, and flagellin glycoprotein appeared unaffected by the deletion. The NGRrkpMNO mutant was symbiotically defective, showing reduced nodulation efficiency on several legumes. Interestingly, K-antigen production in the parent strain was found to decline after rhizobia were exposed to plant flavonoids, and the decrease coincided with flavonoid-induction of a symbiotically active (bacteroid-specific) rhamnan-LPS, suggesting an exchange of SPS occurs during bacterial differentiation in the developing nodule.