These DAPT nmr amino acids were changed into either a phenylalanine (F) residue that cannot become phosphorylated or an aspartate (D) residue to mimic a modification resulting in an additional negative charge. All constructs were functionally active, i.e. AI-2 was still produced by these modified proteins (data not shown). Total protein lysates of S. Typhimurium luxS mutant strains containing one of these point mutated LuxS constructs, were analyzed with 2D gel electrophoresis (2DE). As shown in Figure 2D-F, all strains with Y to

F mutations still possess two LuxS spots. This rules out any of the tyrosine residues as target sites for modification. Furthermore, the pI shift seen in the Y to D mutation strains (Figure 2G-I) confirms the charge difference on the modified LuxS form. This result also illustrates that the interpretation of proteomic results has to be done with great care. Posttranslational modifications all correspond to a specific shift in pI and/or molecular weight. In this respect, we suggest that the postulated phosphorylation of LuxS in Bifidobacterium longum proposed by Yuan et al. should be re-investigated [22]. Figure 2 2DE analysis of Salmonella Typhimurium luxS mutants. (A) Total gel image of wildtype S. Typhimurium proteins. The two LuxS forms are indicated with an arrow. Based

on pI calculations, the right spot corresponds to native LuxS and the left spot carries a posttranslational modification. this website (B-J) Close-up view of the area of the LuxS spots in a luxS mutant carrying different LuxS complementation constructs. (B) negative control – empty vector; (C) wildtype LuxS; (D) LuxS-Y88F; (E) LuxS-Y126F; (F) LuxS-Y131F; (G) LuxS-Y88D; (H) LuxS-Y126D; (I) LuxS-Y131D; (J) LuxS-C83A. Remark that in theory, on the gels from which panels Phospholipase D1 G-I are taken, an additional modified LuxS spot is expected, accumulating the Y to D mutation and the cysteine modification.

For Bacillus subtilis LuxS, oxidation of C84 has previously been reported with purified LuxS protein in studies to reveal the reaction mechanism of the synthase [23–25]. This oxidation is irreversible and adds one negative charge to the protein [23], which makes it a good candidate for the LuxS modification we detected in the S. Typhimurium proteome. Analogous to the tyrosine mutant constructs, we made a point mutation of the corresponding cysteine residue in S. Typhimurium to an alanine residue (C83A) which can no longer be oxidized and subsequently analyzed this strain by 2DE. As shown in Figure 2J the C83A luxS strain lacks the acid shifted LuxS spot confirming C83 as the target for posttranslational modification. As this cysteine residue is required for LuxS catalytic activity [26], the LuxSC83A mutant strain failed to produce AI-2 as revealed by the use of the AI-2 bioassay [27] (data not shown).