Cengic I, Uhlén M, Hudson EP
J. Bacteriol. - (-) - [2018-05-29; online 2018-05-29]
Functional surface display of small affinity proteins, namely Affibodies (6.5 kDa), was evaluated for the model cyanobacterium Synechocystis sp. PCC 6803, through anchoring to native surface structures. These structures included confirmed or putative subunits of the type IV pili, the S-layer protein, and the heterologous Escherichia coli autotransporter antigen 43 system. The most stable display system was determined to be through C-terminal fusion to PilA1, the major type IV pili subunit in Synechocystis, in a strain unable to retract these pili (ΔpilT1). Type IV pili synthesis was upheld, albeit reduced, when fusion proteins were incorporated. However, pili-mediated functions such as motility and transformational competency were negatively affected. Display of Affibodies on Synechocystis and the complementary anti-idiotypic Affibodies on E. coli or Staphylococcus carnosus was able to mediate interspecies cell-cell binding by Affibody complex formation. The same strategy was however not able to drive cell-cell binding and aggregation of Synechocystis-only mixtures. Successful Affibody-tagging of the putative minor pilin PilA4 showed that it locates to the type IV pili in Synechocystis, and that its extracellular availability depends on PilA1. In addition, Affibody-tagging of the S-layer protein indicated that the domains responsible for anchoring and secretion of this protein are located at the N- and C-terminus, respectively. This study can serve as a basis for future surface display of proteins on Synechocystis for biotechnological applications.Importance Cyanobacteria are gaining interest for their potential as autotrophic cell factories. Development of efficient surface display strategies could improve their suitability for large-scale applications by providing options for designed microbial consortia, cell immobilization, and biomass harvesting. Here, surface display of small affinity proteins was realized by fusing them to the major subunit of the native type IV pili in Synechocystis sp. PCC 6803. Display of complementary affinity proteins allowed specific cell-cell binding between Synechocystis and Escherichia coli or Staphylococcus carnosus Additionally, successful tagging of the putative pilin PilA4, helped determine its localization to the type IV pili. Analogous tagging of the S-layer protein shed light on the regions involved in its secretion and surface anchoring.
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