In natural and artificial aquatic environments, population structures and dynamics of photosynthetic microbes are heavily influenced by the grazing activity of protistan predators. Understanding the molecular factors that affect predation is critical for controlling toxic cyanobacterial blooms and maintaining cyanobacterial biomass production ponds for generating biofuels and other bioproducts. We previously demonstrated that impairment of the synthesis or transport of the O-antigen component of lipopolysaccharide (LPS) enables resistance to amoebal grazing in the model predator-prey system consisting of the heterolobosean amoeba HGG1 and the cyanobacterium Synechococcus elongatus PCC 7942 (R. S. Simkovsky et al., Proc Natl Acad Sci U S A 109:16678 -16683, 2012, http://dx.doi.org/10.1073/pnas.1214904109). In this study, we used this model system to identify additional gene products involved in the synthesis of O antigen, the ligation of O antigen to the lipid A-core conjugated molecule (including a novel ligase gene), the generation of GDP-fucose, and the incorporation of sugars into the lipid A core oligosaccharide of S. elongatus. Knockout of any of these genes enables resistance to HGG1, and of these, only disruption of the genes involved in synthesis or incorporation of GDP-fucose into the lipid A-core molecule impairs growth. Because these LPS synthesis genes are well conserved across the diverse range of cyanobacteria, they enable a broader understanding of the structure and synthesis of cyanobacterial LPS and represent mutational targets for generating resistance to amoebal grazers in novel biomass production strains. L ipopolysaccharide (LPS) serves as a critical interface between aGram-negative bacterium's internal physiology and its abiotic and biotic environments, protecting the cell from specific antimicrobials, evading the innate immune and complement systems, enabling symbiosis with eukaryotic hosts, acting as a recognition factor for phage infection, and protecting bacteria against digestion by predatory amoebae (1-5). Alterations to any of the three primary components of the LPS structure (the endotoxin lipid A, the core oligosaccharide, or the O-antigen polysaccharide), through changes in sugar content or sequence, acylation, phosphorylation, or other molecular modifications, can alter these interactions, modulate the toxicity of the endotoxin, or prevent cellular behaviors such as motility (6, 7).While much is known about the structure and synthesis of LPS in enteric and pathogenic proteobacteria, little is known about either aspect of LPS in photosynthetic cyanobacteria and how the cyanobacterial LPS affects interactions with the environment (8-11). To date, only three cyanobacterial LPS structures have been reported: the partial structural determination of the filamentous freshwater cyanobacterium Oscillatoria planktothrix LPS (12) and the complete structural determination of the LPSs of the unicellular marine Synechococcus strains WH8102 and CC9311 (9). Beyond these structures, the remaining...