Table 1.
Characteristics of bacterial strains, plasmids and primers used in this study
Table 2.
Oligonucleotides used for the site-directed mutagenesis in this study.
Table 3.
Crysal structure parameters, data collection and refinement statistics.
Fig 1.
X-ray crystallographic structure of S. suis LuxS.
(A) The overall structure of the LuxS. Four LuxS monomers are in one asymmetry unit, the different color coding represents different monomers. Zn is represented as a large sphere. (B) The structural comparisons of S. suis LuxS with four known structures of LuxS. The four known structures of LuxS proteins are sampled from Protein Data Bank. The different color coding as the LuxS structure of Heamophilus influenzae (1JOE, yellow),Deinococcus radiodurans (1VH2, green), Helicobacter subtilis (1J6X, cyan), Bacillus subtilis (2FQO, grey). (C) Residue phe80 and his87 were shown, the Met and Tyr were from 1JOE and 1J6X. HYI was compound (2S)-2-amino-4-[(2R,3R)-2,3-dihydroxy-3-N-hydroxycarbamoyl-propylmercapto]butyric acid from structure 2FQO. Four structures were shown, including 1VH2 (green), 1JOE (yellow), 1J6X (cyan) and 2FQO (grey).
Fig 2.
Structure-based multiple sequence alignment of LuxS homologues.
Multiple alignments of LuxS homologue from S. suis HA9801 with the related LuxS proteins. The multiple alignment was conducted using ClustalW2 (http://www.ebi.ac.uk/Tools/clustalw2/index.html), and the figure was generated with the program ESPript 2.2 (http://espript.ibcp.fr/ESPript/cgi-bin/ESP ript.cgi). Sequence alignment was performed for the various LuxS sequences, which included S.suis, E.facecium, H.pylori, V.cholerae, V.harveyi, E.coli, S.enterica, N.meningitidis, A.pleuropneumoniae, H.parasuis, H.influenzae, C.gracilis, C.jejuni, B.cereus, B.subtilis. The secondary structure of the SS LuxS protein is shown in top. α: α-helix; β: β-sheet; T: β-turns/coils. Three conserved zinc-binding sites (His57, His61, and Cys127) are indicated with black triangles. A critical amino acid G81 was also very conserved (highlighted with an arrow), which was recently found to be one of the important residues in the active site for AI-2 production in Campylobacter jejuni (Plummer et al., 2011). The Cys82 is the catalytic active residue of the LuxS protein. The distinct residues are indicated by labeling with black stars.
Table 4.
Kinetic parameters for SAH, wild-type and mutant enzymes, measured at pH7.2
Fig 3.
Measurement of AI-2 activity of the different complementation of ΔluxS with luxS variants.
S.suis ΔluxS cells were transformed with pSET2 carrying luxS variants. AI-2 activity of cell-free culture fluids in the stationary exponential phase was measured using the V. harveyi bioluminescence assay. AI-2 activity determination is expressed as relative light units and compared with the level of luminescence produced by the positive control (V. harveyi BB120). AI-2 activity of S. suis HA9801 was set to 100% activity for normalization. Sterile THB medium served as a negative control. Values are means from three independent experiments. Error bars indicate standard deviations (p < 0.05).
Fig 4.
Quantitative determination of biofilm formation of the different complementation of ΔluxS with luxS variants.
All strains were cultured in THB medium supplemented with 1% fibrinogen. Each strain was tested in 8 wells of a 96-well microtiter plate. HA9801, F80M, H80Y, C80A and F80M/H80Y refer to the WT strain and the complementation of ΔluxS with F80M, H80Y, C80A and F80M/H80Y point or double mutantion luxS variants. Negative control (NC) wells contained broth only. The columns represent the means and standard deviations of four or more experiments. The asterisk showed significant difference (p < 0.05).