Table 1.
Bacterial strains and plasmids used in this study.
Table 2.
Primers used in this study.
Figure 1.
Autoinducer-2 activity of strains used in study.
A: Bioluminescence activity of 81116AI2- and AI2-(pLuxS) compared to the positive control strains Campylobacter jejuni 11168 and Vibrio harveyi BB152. Each bar represents the average of three replicates with standard deviation. MH both is shown as a negative control. BB152 – positive control B: AI-2 production of 81116 variants and conjugates. 81116AI2-, a laboratory strain with the G92D mutation in LuxS (AI-2 negative), AI2-(p256G), a 81116 conjugate with a shuttle vector carrying a luxS gene in which the G92D mutation is reverted; 81116AI2+, a 81116 variant with no mutation in luxS (AI-2 positive); and MH Broth, culture media serving as a negative control for AI-2 activity.
Figure 2.
AI-2 concentration curves for the two recombinant proteins as a function of time and measured by FRET.
Figure 3.
Sequence alignment of luxS genes including 81116.
Highlighted amino acids are mutated in the 81116 (bottom row) compared to the published 11168 sequence (2nd from bottom). “X” denotes the site of amino acid substitutions. Proteins used in the sequence alignment are as follows (Protein Information Resource database ID numbers are shown in parentheses, with GenBank ID numbers given for ST, VH, and BH): HP, Helicobacter pylori (C71973); DR, Deinococcus radiodurans (D75280); HI, Haemophilus influenzae (G64008); BB, Borrelia burgdorferi (BB0377); CP, Clostridium perfringens (T43793); NM, Neisseria meningitides (G81963); ST, Salmonella typhimurium (AAF73475.1); VH, Vibrio harveyi (AAD17292.1); EC, Escherichia coli (H65048); VC, Vibrio cholerae (F82309); BS, Bacillus subtillis (A69994); and BH, Bacillus halodurans (BAB07072.1).11168, Campylobacter jejuni ATCC 11168; 81116AI2-, Campylobacter jejuni Strain 81116 from this study; 81116AI2+, Campylobacter jejuni Strain 81116 from this study.
Figure 4.
The purity and activity of recombinant LuxS proteins synthesized in this paper.
A: SDS-PAGE gel of recombinant LuxS proteins from 81116AI2- and 81116AI2+ Lane 1: rLuxS- (expected MW = 18.1 kD), the recombinant 81116AI2- 6HIS-tagged LuxS Protein, Lane 2: rLuxS+ (expected MW = 18.1 kD), the 81116AI2+ 6HIS-tagged LuxS Protein, Molecular weight markers as shown. B: Enzymatic activity of recombinant LuxS protein measured by Vibrio harveyi bioluminescence assay. SRH alone with no enzyme provides a negative control. BB152 is cell-free supernatent of V. harveyi BB152 as a positive control.
Figure 5.
Circular dichroism spectra of the rLuxS+ and rLuxS- recombinant proteins.
The two protein spectra essentially overlay each other identically throughout the spectra. The buffer is also shown as a control.
Figure 6.
Recombinant protein activity of the AI2+ and AI2- strains used in this paper.
A. Paired homocysteine and AI-2 data from the LuxS recombinant proteins. rLuxS+ is the recombinant LuxS protein from 81116AI2+ and rLuxS- is the recombinant protein from 81116AI2-. Black bars represent the homocysteine levels measured using the DNTB reaction measured by Absorbance at 412 nm (right axis). The grey bars represent the AI-2 values measured by V. harveyi bioluminescence (left axis). Values for each sample were measured on split samples.
Figure 7.
AI-2 and SRH levels of various cultures.
The first 6 columns depict the cell-free supernatent AI-2 concentrations of cultures as a reference for endogenous LuxS activity. The four columns labeled boiled are cell-free supernatent post-boiling which destroys AI-2 activity. Finally the last 4 columns show the AI-2 activity produced in the presence of exogenous rLuxS+ recombinant protein which provides a semi-quantitative measure of extracellular SRH levels.