Surface Display of Small Affinity Proteins on Synechocystis sp. Strain PCC 6803 Mediated by Fusion to the Major Type IV Pilin PilA1

Abstract

Functional surface display of small affinity proteins, namely Affibodies (6.5 kDa), was evaluated for the model cyanobacterium 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 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 , in a strain unable to retract these pili (Δ). 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 and the complementary anti-idiotypic Affibodies on or 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-only mixtures. Successful Affibody-tagging of the putative minor pilin PilA4 showed that it locates to the type IV pili in , 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 for biotechnological applications. 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 sp. PCC 6803. Display of complementary affinity proteins allowed specific cell-cell binding between and or 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.

Minor pilin genes are involved in motility and natural competence inSynechocystissp. PCC 6803

“…Therefore, we surmised that these mutants do not form a comparable priming complex. Indeed, the minor pilin PilA4 from Synechocystis was detected within the T4P fibre (Cengic et al, 2018) and PilA2 and PilA11 localize at the cell surface (Hu et al, 2018;Cengic et al, 2018). Hu et al (2018) reported that suppression or overexpression of pilA11 expression via the antisense RNA PilR leads to smaller or thicker pili diameters, respectively.…”

“…Thus, overexpression of the asRNA led to inhibition of motility in this study. In another study it was shown that PilA4 is located within the pilus fibre if the major pilin PilA1 is present (Cengic et al, 2018). As in many other bacteria, a functional T4P system is crucial for natural competence of Synechocystis (Yoshihara et al, 2001).…”

The involvement of type IV pili and phytochrome in gliding motility, lateral motility and phototaxis of the cyanobacteriumPhormidium lacuna

“…In Synechocystis PCC 6803, PilA1 is required for motility as gene knockouts result in a nonmotile phenotype. There are 9 PilA homologs in Synechocystis PCC 6803 with either indirect roles in motility or unknown functions [17,18].…”

“…In Synechocystis PCC 6803, PilA1 is required for motility as gene knockouts result in a nonmotile phenotype. There are 9 PilA homologs in Synechocystis PCC 6803 with either indirect roles in motility or unknown functions [17,18]. Knockouts of pilC, pilD and pilT1 also resulted in a nonmotile phenotype, whereas knockouts of pilA2 and pilT2 were still motile [9].…”

Expression and secretion of a lytic polysaccharide monooxygenase by a fast-growing cyanobacterium

“…To develop a novel production chassis, one important aspect is to be able to express heterologous proteins at high titers and to target them to a desired intra- or extracellular location. Targeting to the outer membrane has been successfully demonstrated with several reports of surface display systems established in cyanobacteria [17–20]. However, in some cases, it is useful to secrete the protein out of the cell into the culture medium.…”