Many bacteria produce extracellular molecules which function in cell-to-cell communication. One of these molecules, autoinducer 2 (AI-2), was first described as an extracellular signal produced by Vibrio harveyi to control luciferase expression. Subsequently, a number of bacteria have been shown to possess AI-2 activity in their culture supernatants, and bear the luxS gene product, which is required for AI-2 synthesis. In Porphyromonas gingivalis, luxS and pfs, encoding a 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTA/SAH'ase), form an operon, suggesting that Sadenosylhomocysteine (SAH) or 5'-methylthioadenosine (MTA) serves as a substrate for AI-2 production. Cell-free extracts of Escherichia coli MG1655, but not DH5α (which carries a luxS frame-shift mutation) were capable of generating AI-2 activity upon addition of SAH, but not MTA. S-Ribosylhomocysteine (RH) derived from SAH also served as a substrate in E. coli MG1655 extracts. RH-supplemented cell-free extracts of Pseudomonas aeruginosa, a bacterium that lacks luxS, only generated AI-2 activity following the introduction of a plasmid containing the Por. gingivalis pfs-luxS operon. In addition, defined in vitro systems consisting of the purified LuxS proteins from Por. gingivalis, E. coli, Neisseria meningitidis or Staphylococcus aureus converted RH to homocysteine and a compound that exhibits AI-2 activity. 4-Hydroxy-5-methyl-3(2H)-furanone was identified by mass spectrometry analysis as a major product formed in this in vitro reaction. In E. coli MG1655, expression of T3SH [the bacteriophage T3 S-adenosylmethionine (SAM) hydrolase] significantly reduced AI-2 activity in culture supernatants, suggesting that AI-2 production is limited by the amount of SAH produced in SAM-dependent transmethylase reactions. The authors suggest that the LuxS protein has an important metabolic function in the recycling of SAH. They also show that Ps. aeruginosa is capable of removing AI-2 activity, implying that this molecule may act as a nutrient. In many bacteria AI-2 may in fact represent not a signal molecule but a metabolite which is released early and metabolized in the later stages of growth.

Porphyromonas gingivalis is a Gram-negative black-pigmented obligate anaerobe implicated in the aetiology of human periodontal disease. The virulence of P. gingivalis is associated with the elaboration of the cysteine proteases Arg-gingipain (Rgp) and Lys-gingipain (Kgp), which are produced at high bacterial cell densities. To determine whether quorum sensing plays a role in the regulation of Rgp and Kgp, biosensors capable of detecting either Nacylhomoserine lactone (AHLs) or the luxS-dependent autoinducer (AI-2) quorum-sensing signalling molecules in spent culture supernatants were first employed. While no AHLs could be detected, the Vibrio harveyi BB170 biosensor was activated by spent P. gingivalis W50 culture supernatants. The P. gingivalis luxS gene was cloned and demonstrated to restore AI-2 production in the Escherichia coli luxS mutant DH5α. Mutation of luxS abolished AI-2 production in P. gingivalis. Western blotting using antibodies raised against the recombinant protein revealed that LuxS levels increased throughout growth even though AI-2 activity was only maximally detected at the midexponential phase of growth and disappeared by the onset of stationary phase. Similar results were obtained with E. coli DH5α transformed with luxS, suggesting that AI-2 production is not limited by a lack of LuxS protein. Analysis of Rgp and Kgp protease activities revealed that the P. gingivalis luxS mutant produced around 45 % less Rgp and 30 % less Kgp activity than the parent strain. In addition, the luxS mutant exhibited a fourfold reduction in haemagglutinin titre. However, these reductions in virulence determinant levels were insufficient to attenuate the luxS mutant in a murine lesion model of P. gingivalis infection.