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. 2021 Jun 1;10:e67789. doi: 10.7554/eLife.67789

Figure 1. Scheme for site-specific fluorescent labeling of SPI-2 effectors.

Figure 1.

(A) Chemical structure of 4-azidophenylalanine (AzF), 7-(Hydroxy-coumarin-4-yl) ethylglycine (Hco), trans-Cyclooct-2-en – L - Lysine (TCO*Lys), BDP-DBCO, and SiR-tetrazine. (B) Incorporation of a ncAA in a SPI-2 effector is schematically represented. A plasmid carrying the gene for an effector of interest (green) and an orthogonal suppressor tRNA (dark blue)/aminoacyl synthetase (orange) pair are introduced into the bacterial cell by transformation. The amber stop codon (TAG, red) replaces a native codon at a permissive site within the sequence of the effector gene. The ncAA (red stars) is supplemented in the growth medium. Inside the cell, the orthogonal synthetase specifically charges the orthogonal suppressor tRNA with the desired ncAA through a catalytic reaction driven by ATP. The ncAA-acylated tRNA which contains a CUA anticodon, then enters the ribosomal machinery and incorporates the attached ncAA into the effector in response to the complementary amber codon on the effector mRNA (black). Once released from the ribosome, the tRNA can be further reused for ncAA amino-acylation by the cognate synthetase. The full-length polypeptide chain of the effector site-specifically carries the ncAA and undergoes folding and assembly into a functioning effector protein. The newly formed effector is translocated into the host cell through the T3SS. The secreted SPI-2 effectors incorporated with ncAA can be labeled by an externally added fluorophore. (C) Reaction scheme of effector labeling with a genetically encoded azide-containing protein. An azide-containing amino acid (AzF) is genetically incorporated into a protein (SifA) and the azido group reacts with a conjugation reagent containing dibenzocyclooctyne (BDP-DBCO) through SPAAC. (D) Reaction scheme for the copper-free click reaction with a fluorogenic tetrazine dye. An ncAA with a strained alkene group (e.g. TCO-Lys, as shown here) incorporated into an effector protein (SsaP) reacts with tetrazine‐coupled dyes (SiR-Tz) through SPIEDAC click reaction. Dyes coupled to tetrazine are only fluorescent (magenta) after successful labeling.