Stereoisomer-specific reprogramming of a bacterial flagellin sialyltransferase (2022)
Figure 7

Figure 7. Reconstitution of FljKBs glycosylation by Leg in C. crescentus (C.c.) and rewiring of FlmG

(A) Schematic of the legionaminic acid biosynthetic pathway as it has been described in C. jejuni and elucidated in this study. The pathway requires the addition of GDP into α-D-glucosamine-1-phosphate (GlcN-1P) by Bresu_3267 (in blue). Then, the different steps are catalyzed by Bresu_3266, Bresu_0765, Bresu_0506, Bresu_3264, Bresu_0507 and Bresu_3265 to ultimately produced CMP-legionaminic acid (in red). The activated legionaminic acid is then transferred to the flagellins by the glycosyltransferase FlmG.

(B) Anti-FljKCc immunoblot analyses of whole cell lysates (cells) from C. crescentus (C.c.) mutant cultures expressing fljKBssyn (codon optimized for E. coli) and flmGBs from the replicative pMT463 plasmid, in the presence or absence of a compatible integrative plasmid carrying a legBs synthetic operon (pXGFP4-legBs) and in the presence or absence of an additional compatible replicative plasmid carrying Bresu_3267 (pMT375). In the presence of pXGFP4-legBs (integrated at the xylX locus) and pMT375-Bresu_3267, FljKBssyn migration is shifted toward higher molecular mass, indicative of glycosylation. Note that in the C.c. Δfljx6 background, the six flagellin-encoding genes have been deleted and the protein detected by the antibodies only corresponds to FljKBssyn. The legBs synthetic operon (in red) is composed of Bresu_3266, Bresu_0765, Bresu_0506, Bresu_3264, Bresu_0507 (legI) and Bresu_3265. Molecular masses are indicated in kDa by the blue lines. Empty carets indicate the position of modified (glycosylated) flagellin, whereas filled carets mark unmodified flagellin. Asterisk indicates a modification of flagellin by FlmGBs that does not require Pse or Leg. Immunoblots are representative of at least two independent experiments.

(C) Anti-FljKCc immunoblot analyses of whole cell lysates from C. crescentus mutant cultures expressing FlmGCc, FlmGBs and FlmGCc-Bschim chimera from the replicative pSRK-Gm plasmid (Plac) in the presence of a compatible integrative plasmid carrying a legBs synthetic operon (pXGFP4-legBs) and in the presence or absence of an additional compatible replicative plasmid carrying Bresu_3267 (pMT375). Molecular masses are indicated in kDa by the blue lines. Empty carets indicate the position of modified (glycosylated) flagellin, whereas filled carets mark unmodified flagellin. Immunoblots are representative of at least two independent experiments.

(D) Table showing the glycopeptide LC-MS/MS analyses. Only the two major modifications (when possible) are listed in the table with the number of peptide spectra matching indicated in bracket. Full results are available in Dataset EV2. In red and underlined are the threonines for which the MS/MS data did not contain sufficient evidence to indicate which one was modified. In green are indicated the threonine for which the MS/MS data contained sufficient evidence to indicate which one was modified. Of note, the two threonines in green are also the ones that have been shown to harbor post-translational modification (Montemayor et al., 2021). The threonine highlighted in yellow has been show to harbor post-translational modification (Montemayor et al., 2021) in a FljK mutant strain but does not seem to be modified in our glycopeptide analyses.

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