LuxS: its role in central metabolism and the in vitro synthesis of 4-hydroxy-5-methyl-3(2H)-furanone

Many bacteria produce extracellular molecules which function in cell-to-cell communication. One of these molecules, autoinducer 2 (Al-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 Al-2 activity in their culture supernatants, and bear the luxS gene product, which is required for Al-2 synthesis. In Porphyromonas gingivalis, luxS and pfs, encoding a 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase (MTA/SAH'ase), form an operon, suggesting that S-adenosylhomocysteine (SAH) or 5'-methylthioadenosine (MTA) serves as a substrate for Al-2 production. Cell-free extracts of Escherichia coli MG1655, but not DH5alpha (which carries a luxS frame-shift mutation) were capable of generating Al-2 activity upon addition of SAH, but not MTA. S-Ribosyl-homocysteline (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 Al-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 Al-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 Al-2 activity in culture supernatants, suggesting that Al-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 Al-2 activity, implying that this molecule may act as a nutrient. In many bacteria Al-2 may in fact represent not a signal molecule but a metabolite which is released early and metabolized in the later stages of growth.